Method for treating autoimmune disease using cd4 t-cells with engineered stabilization of expression of endogenous foxp3 gene

Abstract

This invention discloses a method for preparing genetically engineered cells expressing FOXP3 and a treatment method. In some embodiments, the method provides a first nucleotide sequence comprising a coding strand containing one or more regulatory elements and a portion thereof of the FOXP3 gene; provides a nuclease; and performs a gene editing process on the first nucleotide sequence, the gene editing process editing the one or more regulatory elements, and, where appropriate, editing the FOXP3 gene or a portion thereof. Methods for treating individuals with autoimmune diseases and individuals suffering from organ transplant effects are also provided.

Description

Technical Field

[0001] This article describes methods for treating autoimmune diseases and inducing tolerance to transplanted organs. These methods may also be used for cell therapy of severe autoimmune diseases and organ transplantation. Prior Art

[0002] Immune self-tolerance in the periphery can be achieved through negative regulation of immune responses by a variety of cells, the best-characterized of which are regulatory T cells (T regs). T regs mediate self-tolerance and tolerance to alloantigens by suppressing the activation of effector T cells (T eff) and exerting anti-inflammatory activity. For example, CD4+CD25+Foxp3+ T regs are among the best-studied and characterized T cells in autoimmune diseases and organ transplantation. Tregs are also known as "suppressor T cells." Specifically, it is described that CD4+ and CD25+ regulatory T cells can exhibit regulatory functions both in vitro and in vivo, and that they suppress the proliferation of CD4+ and CD8+ Teffs in vitro. These cells have also been shown to play an important role in autoimmunity, allergies, inflammation, maintaining maternal-fetal tolerance, infection, and cancer. Tregs are known to reduce immune responses. In cancer, excessive Treg cell activity can prevent the immune system from destroying cancer cells. In autoimmune diseases, a lack of regulatory T cell activity can enable other autoimmune cells to attack the body's own tissues. T reg cells can also include natural (n) T regs generated in the thymus and inducible (iT regs) generated in the periphery. nT regs arise in the thymus and express the forkhead / winged helix transcription factor FOXP3, which in turn controls nT reg differentiation. iT regs emerge in the periphery from memory and naive CD4+ T cells following stimulation with self- or alloantigens in the presence of IL-4, IL-10, TGF-β, and IL-2. FOXP3 is a protein involved in immune system responses and is encoded by the FOXP3 gene, which contains 11 coding exons, the first of which is designated exon 2 in the revised nomenclature. FOXP3 has been characterized as a major regulator of regulatory pathways in the development and function of regulatory T cells. In animal studies, T regs expressing FOXP3 have been shown to be crucial in the transfer of immune tolerance, such as self-tolerance. Furthermore, induction or administration of FOXP3-positive cells has been shown to reduce the severity of autoimmune diseases in models of diabetes, multiple sclerosis, asthma, inflammatory bowel disease, thyroiditis, and kidney disease. The use of Tregs has been previously proposed for treating diseases or suppressing the immune system in the setting of organ transplantation. However, one of the greatest obstacles to using tTregs or pTregs to treat autoimmune diseases is the epigenetic regulation of FOXP3 expression. In tTregs, an upstream region of the FOXP3 gene, known as the "thymus-specific demethylation region," is completely demethylated, a state believed to stabilize FOXP3 expression. Generally, complete demethylation is not observed in pTregs. In inflammatory conditions, FOXP3 can be epigenetically silenced in pTregs, and possibly in tTregs (although some researchers believe tTregs are completely stable), potentially leading to the conversion of pTregs into pro-inflammatory CD4+ T cells. The lack of stability in pTregs is a significant concern, as the infusion of pTregs that have reverted to an inflammatory phenotype can exacerbate autoimmune symptoms. Therefore, safe methods for inducing FOXP3 in Tregs are needed. Summary of the Invention

[0003] Due to their potential to induce antigen-specific tolerance, many groups have focused on utilizing regulatory T cells to treat autoimmune diseases. There are many forms of regulatory T cells ("T regs"), and current nomenclature divides T regs into those generated in the thymus during T cell development, designated thymic regulatory T cells or "tT regs," and those induced peripherally, designated peripheral regulatory T cells or "pT regs." A key aspect of regulatory T cell biology is the expression of the transcription factor FOXP3. FOXP3 is believed to be essential for specifying the regulatory T cell lineage. This concept is based on the observation that humans lacking FOXP3 develop severe autoimmune diseases beginning in the neonatal period. However, a major obstacle to using tT regs or pT regs to treat autoimmune diseases is that FOXP3 expression is epigenetically regulated. In tT regs, an upstream region of the FOXP3 gene, known as the "thymus-specific demethylation region," is completely demethylated, a state believed to stabilize FOXP3 expression. Generally, complete demethylation is not observed in pT regs. In inflammatory conditions, FOXP3 can be epigenetically silenced in pT regs, and possibly in tT regs. This can lead to the conversion of pT regs into pro-inflammatory CD4 T cells. The lack of stability in pT regs is a significant concern, as the infusion of pT regs that have reverted to an inflammatory phenotype can exacerbate autoimmune symptoms. The proposed method for FOXP3 expression in mixed CD4 T cell populations represents an improvement over other methods for isolating naturally occurring regulatory T cell populations because it provides the ability to capture the TCR repertoire present in inflammatory T cell populations. In patients with autoimmune diseases or organ transplant rejection, the endogenous TCR repertoire within the inflammatory T cell population includes TCRs with the correct binding specificity to recognize inflamed or allogeneic tissue within the organ. These T cells are believed to mediate autologous inflammatory responses or organ rejection. By converting a portion of the mixed CD4 T cell population to a regulatory phenotype, TCR specificities present in the pro-inflammatory population will be expressed in the therapeutic cell population. This is an improvement over therapies based on thymic regulatory T cells, which are believed to have a distinct and non-overlapping TCR repertoire with inflammatory T cells. Furthermore, it is hypothesized that in patients with autoimmune diseases or organ rejection, existing tTreg populations fail to develop the tolerance necessary to avoid inflammation. Therefore, several aspects of the invention described herein concern methods for treating or ameliorating autoimmune diseases using CD4 T cells that have been engineered to stably express their endogenous FOXP3 gene. Several methods described herein utilize engineering approaches, for example, to stabilize FOXP3 expression in CD4 T cells and allow for the generation of expanded populations of potential suppressor T cells that are no longer susceptible to epigenetic modifications of their suppressive function. Consequently, these cells possess improved properties for therapeutic applications. [] In a first aspect, a method for preparing a nucleic acid for expressing FOXP3 is provided, wherein the method comprises providing a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof; providing a nuclease; and subjecting the first nucleotide sequence to a gene editing process that edits the one or more regulatory elements and, optionally, the FOXP3 gene or portion thereof. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus comprises a native first coding exon. In some alternatives, completing the gene editing process produces the first coding exon in the targeted locus after the gene editing process. In some alternatives, the targeted locus is at one or more regulatory elements. In some alternatives, the targeted locus is at the FOXP3 gene or portion thereof. In some alternatives, the FOXP3 gene or portion thereof comprises the first native coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the gene editing process is a knock-in procedure for inserting a heterologous promoter, a heterologous transcriptional enhancer domain, or both. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the promoter is a heterologous weak promoter (e.g., one that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the promoter is an inducible promoter. In some alternatives, an inducible promoter can be induced by a drug or steroid. In some alternatives, the method further comprises inserting a heterologous transcriptional enhancer domain, wherein the enhancer domain is upstream or downstream of one or more regulatory sequences. In some alternatives, the method further comprises inserting a heterologous transcriptional activation domain. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE). In some alternatives, the method further comprises inserting an inducible effector. In some alternatives, the inducible effector can be induced by a steroid or drug. In some alternatives, a heterologous promoter is inserted, wherein insertion of the heterologous promoter generates the first coding exon, wherein the heterologous promoter is located anywhere upstream of the first coding exon on the coding strand, and wherein the coding strand further comprises the start codon of the FOXP3 gene. In some alternatives, the heterologous promoter is inserted anywhere upstream of the first native exon on the coding strand, and wherein the coding strand further comprises the start codon of the FOXP3 gene. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements. In a second aspect, a nucleic acid for FOXP3 expression is provided, produced by any method of any alternative described herein. The method comprises providing a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof; providing a nuclease; and subjecting the first nucleotide sequence to a gene editing process that edits the one or more regulatory elements and, optionally, the FOXP3 gene or portion thereof. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus comprises a native first coding exon. In some alternatives, completing the gene editing process results in the first coding exon in the targeted locus after the gene editing process. In some alternatives, the targeted locus is located at one or more regulatory elements. In some alternatives, the targeted locus is located at a FOXP3 gene or portion thereof. In some alternatives, the FOXP3 gene or portion thereof comprises the first native coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the gene editing process is a knock-in procedure for inserting a heterologous promoter, a heterologous transcriptional enhancer domain, or both. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the promoter is a heterologous weak promoter (e.g., one that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the promoter is an inducible promoter. In some alternatives, an inducible promoter can be induced by a drug or steroid. In some alternatives, the method further comprises inserting a heterologous transcriptional enhancer domain, wherein the enhancer domain is upstream or downstream of one or more regulatory sequences. In some alternatives, the method further comprises inserting a heterologous transcriptional activation domain. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE). In some alternatives, the method further comprises inserting an inducible effector. In some alternatives, the inducible effector can be induced by a steroid or drug. In some alternatives, a heterologous promoter is inserted, wherein the insertion of the heterologous promoter generates the first coding exon, wherein the heterologous promoter is located anywhere upstream of the first coding exon on the coding strand, and wherein the coding strand further comprises the start codon of the FOXP3 gene. In some alternatives, the heterologous promoter is inserted anywhere upstream of the first native exon on the coding strand, and wherein the coding strand further comprises the start codon of the FOXP3 gene. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements. In a third aspect, a nucleic acid is provided, comprising a coding strand comprising a heterologous regulatory element and a heterologous promoter, wherein the heterologous regulatory element and heterologous promoter are operably linked to a FOXP3 gene or portion thereof, wherein the FOXP3 gene or portion thereof comprises the first coding exon. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the constitutive promoter is an EF1α promoter, a PGK promoter, or an MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., one that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the promoter is an inducible promoter. In some alternatives, the inducible promoter is induced by a drug or steroid. In some alternatives, the heterologous promoter is located upstream of the first coding exon. In some alternatives, the nucleic acid further comprises a heterologous transcription enhancer domain. In some alternatives, the nucleic acid further comprises a heterologous transcription activation domain. In some alternatives, the nucleic acid further comprises a ubiquitous chromatin opening element (UCOE). In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the one or more regulatory elements are genetically modified. In a fourth aspect, a method for preparing a genetically engineered cell is provided, comprising providing a cell, wherein the cell comprises a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements and the FOXP3 gene or portion thereof; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; and introducing the second oligonucleotide or protein nuclease into the cell for a gene editing process. In some alternatives, the gene editing process is a knock-in process in which a heterologous promoter is inserted into the first nucleotide sequence. In some alternatives, the coding strand comprises a first coding exon. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus comprises a native coding exon or a first coding exon. In some alternatives, completing the gene editing process results in the generation of the first coding exon in the targeted locus after the gene editing process. In some alternatives, the targeted locus is within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110 base pairs upstream of the first coding exon, or any number of base pairs within a range defined by any two of the aforementioned values. In some alternatives, the gene editing process further comprises generating a synthetic first coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the heterologous promoter is an EF1α promoter, a PGK promoter, or an MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the method further comprises inserting a heterologous enhancer domain (e.g., a TCRα enhancer) into the first nucleotide sequence, wherein the enhancer domain is upstream or downstream of one or more regulatory sequences. In some alternatives, the method further comprises inserting a heterologous transcriptional activation domain into the first nucleotide sequence. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE) into the first nucleotide sequence. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon on the coding strand during genetic modification, or the genetic modification results in a synthetic first coding exon produced by completing the gene editing process, wherein the heterologous promoter is upstream of the synthetic first coding exon. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell.In some alternatives, the cell line represents a CD4+ cell. In some alternatives, the cell line represents a CD8+ cell. In some alternatives, the cell line represents a regulatory T cell. In a fifth aspect, a genetically engineered cell for expressing FOXP3 is provided, produced by the method of any of the alternatives described herein. A method for preparing a genetically engineered cell is provided, comprising providing a cell, wherein the cell comprises a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; and introducing the second oligonucleotide or protein nuclease into the cell for a gene editing process. In some alternatives, the gene editing process is a knock-in process in which a heterologous promoter is inserted into the first nucleotide sequence. In some alternatives, the coding strand comprises a first coding exon. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus comprises a native coding exon or a first coding exon. In some alternatives, completing the gene editing process results in the first coding exon being produced in the targeted locus after the gene editing process. In some alternatives, the targeted locus is within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110 base pairs upstream of the first coding exon, or any number of base pairs within a range defined by any two of the aforementioned values. In some alternatives, the gene editing process further comprises generating a synthetic first coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the heterologous promoter is an EF1α promoter, a PGK promoter, or an MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the method further comprises inserting a heterologous enhancer domain (e.g., a TCR alpha enhancer) into the first nucleotide sequence, wherein the enhancer domain is upstream or downstream of one or more regulatory sequences. In some alternatives, the method further comprises inserting a heterologous transcriptional activation domain into the first nucleotide sequence. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE) into the first nucleotide sequence. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon on the coding strand during genetic modification, or the genetic modification results in a synthetic first coding exon generated by completing the gene editing process, wherein the heterologous promoter is upstream of the synthetic first coding exon. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements.In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line represents a CD4+ cell. In some alternatives, the cell line represents a CD8+ cell. In some alternatives, the cell line is a regulatory T cell. In a sixth aspect, a genetically engineered cell for expressing FOXP3 is provided, wherein the genetically engineered cell comprises a nucleic acid comprising a coding strand comprising one or more regulatory elements operably linked to a FOXP3 gene and a heterologous promoter, wherein the FOXP3 gene comprises the first coding exon. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the constitutive promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter that generates less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter is inducible by a drug or steroid. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon. In some alternatives, the nucleic acid further comprises a heterologous transcription enhancer domain. In some alternatives, the nucleic acid further comprises a heterologous transcription activation domain. In some alternatives, the nucleic acid further comprises a ubiquitous chromatin opening element (UCOE). In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the one or more regulatory elements are genetically modified. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line is a regulatory T cell. In a seventh aspect, a composition is provided comprising any one or more genetically engineered cells selected from any of the alternatives herein and a pharmaceutical excipient. Genetically engineered cells expressing FOXP3 can be produced by the methods provided in any of the alternatives herein. A method for preparing a genetically engineered cell is provided, comprising providing a cell comprising a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; and introducing the second oligonucleotide or protein nuclease into the cell for a gene editing procedure. In some alternatives, the gene editing procedure is a knock-in procedure in which a heterologous promoter is inserted into the first nucleotide sequence. In some alternatives, the coding strand comprises a first coding exon. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus comprises a native coding exon or the first coding exon. In some alternatives, completing the gene editing process generates a first coding exon in the targeted locus after the gene editing process. In some alternatives, the targeted locus is within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110 base pairs upstream of the first coding exon, or any number of base pairs within a range defined by any two of the aforementioned values. In some alternatives, the gene editing process further comprises generating a synthetic first coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the heterologous promoter is an EF1α promoter, a PGK promoter, or an MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the method further comprises inserting a heterologous enhancer domain (e.g., a TCR alpha enhancer) into the first nucleotide sequence, wherein the enhancer domain is upstream or downstream of one or more regulatory sequences. In some alternatives, the method further comprises inserting a heterologous transcriptional activation domain into the first nucleotide sequence. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE) into the first nucleotide sequence. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon on the coding strand during genetic modification, or the genetic modification results in a synthetic first coding exon generated by completing a gene editing process, wherein the heterologous promoter is upstream of the synthetic first coding exon. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand.In some alternatives, the method further comprises genetically modifying the one or more regulatory elements. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line is a regulatory T cell. In some alternatives, the genetically engineered cell for expressing FOXP3 comprises a nucleic acid, wherein the nucleic acid comprises a coding strand comprising one or more regulatory elements operably linked to a FOXP3 gene and a heterologous promoter, wherein the FOXP3 gene comprises the first coding exon. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the constitutive promoter is an EF1α promoter, a PGK promoter, or an MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by drugs or steroids. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon. In some alternatives, the nucleic acid further comprises a heterologous transcriptional enhancer domain. In some alternatives, the nucleic acid further comprises a heterologous transcriptional activation domain. In some alternatives, the nucleic acid further comprises a ubiquitous chromatin opening element (UCOE). In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the one or more regulatory elements are genetically modified. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line is a regulatory T cell. In an eighth aspect, a method for treating, suppressing, or ameliorating an autoimmune disorder in a subject is provided, comprising: administering to the subject a genetically modified cell comprising any one or more cells of any of the alternatives described herein or a composition provided by any of the alternatives. Compositions comprising any one or more genetically engineered cells of any of the alternatives described herein and a pharmaceutical excipient are provided. Genetically engineered cells expressing FOXP3 can be produced by the methods provided by any of the alternatives described herein. Methods for preparing genetically engineered cells are provided, comprising providing a cell comprising a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; and introducing the second oligonucleotide or protein nuclease into the cell for a gene editing procedure. In some alternatives, the gene editing procedure is a knock-in procedure in which a heterologous promoter is inserted into the first nucleotide sequence. In some alternatives, the coding strand comprises a first coding exon. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus comprises a native coding exon or the first coding exon. In some alternatives, completion of the gene editing process produces the first coding exon in the targeted locus after the gene editing process. In some alternatives, the targeted locus is within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110 base pairs upstream of the first coding exon, or any number of base pairs within a range defined by any two of the above values. In some alternatives, the gene editing process further comprises producing a synthetic first coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the heterologous promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the method further comprises inserting a heterologous enhancer domain (e.g., a TCRα enhancer) into the first nucleotide sequence, wherein the enhancer domain is upstream or downstream of one or more regulatory sequences. In some alternatives, the method further comprises inserting a heterologous transcriptional activation domain into the first nucleotide sequence. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE) into the first nucleotide sequence.In some alternatives, the heterologous promoter is inserted upstream of the first coding exon on the coding strand during genetic modification, or the genetic modification results in a synthetic first coding exon produced by completing the gene editing process, wherein the heterologous promoter is upstream of the synthetic first coding exon. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line is a cell expressing CD4+. In some alternatives, the cell line is a cell expressing CD8+. In some alternatives, the cell line is a regulatory T cell. In some alternatives, the genetically engineered cell for expressing FOXP3 comprises a nucleic acid, wherein the nucleic acid comprises a coding strand, wherein the coding strand comprises one or more regulatory elements operably linked to a FOXP3 gene and a heterologous promoter, wherein the FOXP3 gene comprises the first coding exon. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the constitutive promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon. In some alternatives, the nucleic acid further comprises a heterologous transcription enhancer domain. In some alternatives, the nucleic acid further comprises a heterologous transcription activation domain. In some alternatives, the nucleic acid further comprises a ubiquitous chromatin opening element (UCOE). In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the one or more regulatory elements are genetically modified. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line is a regulatory T cell. In some alternatives, the autoimmune disease is rheumatoid arthritis, diabetes, inflammatory bowel disease, IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome), Crohn's disease, multiple sclerosis, idiopathic or systemic lupus erythematosus. In some alternatives, an individual is identified or selected to receive therapy for the autoimmune disease. In a ninth aspect, a method for treating, inhibiting, or ameliorating the side effects of organ transplantation in a subject is provided, comprising: administering to the subject a genetically modified cell comprising any one or more cells of any of the alternatives described herein or a composition provided by any of the alternatives. Compositions comprising any one or more genetically engineered cells of any of the alternatives described herein and a pharmaceutical excipient are provided. Genetically engineered cells expressing FOXP3 can be produced by the methods provided by any of the alternatives described herein. A method for preparing a genetically engineered cell is provided, comprising providing a cell comprising a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; and introducing the second oligonucleotide or protein nuclease into the cell for a gene editing procedure. In some alternatives, the gene editing procedure is a knock-in procedure in which a heterologous promoter is inserted into the first nucleotide sequence. In some alternatives, the coding strand comprises a first coding exon. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus comprises a native coding exon or the first coding exon. In some alternatives, completion of the gene editing process produces the first coding exon in the targeted locus after the gene editing process. In some alternatives, the targeted locus is within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110 base pairs upstream of the first coding exon, or any number of base pairs within a range defined by any two of the above values. In some alternatives, the gene editing process further comprises producing a synthetic first coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the heterologous promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the method further comprises inserting a heterologous enhancer domain (e.g., a TCRα enhancer) into the first nucleotide sequence, wherein the enhancer domain is upstream or downstream of one or more regulatory sequences. In some alternatives, the method further comprises inserting a heterologous transcriptional activation domain into the first nucleotide sequence. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE) into the first nucleotide sequence.In some alternatives, the heterologous promoter is inserted upstream of the first coding exon on the coding strand during genetic modification, or the genetic modification results in a synthetic first coding exon produced by completing the gene editing process, wherein the heterologous promoter is upstream of the synthetic first coding exon. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line is a cell expressing CD4+. In some alternatives, the cell line is a cell expressing CD8+. In some alternatives, the cell line is a regulatory T cell. In some alternatives, the genetically engineered cell for expressing FOXP3 comprises a nucleic acid, wherein the nucleic acid comprises a coding strand, wherein the coding strand comprises one or more regulatory elements operably linked to a FOXP3 gene and a heterologous promoter, wherein the FOXP3 gene comprises the first coding exon. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the constitutive promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon. In some alternatives, the nucleic acid further comprises a heterologous transcription enhancer domain. In some alternatives, the nucleic acid further comprises a heterologous transcription activation domain. In some alternatives, the nucleic acid further comprises a ubiquitous chromatin opening element (UCOE). In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the one or more regulatory elements are genetically modified. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line represents a CD4+ cell. In some alternatives, the cell line represents a CD8+ cell. In some alternatives, the cell line is a regulatory T cell. In a tenth aspect, a method for treating, inhibiting, or ameliorating a side effect of organ transplantation (e.g., organ rejection) in a subject is provided, comprising: administering to the subject a genetically modified cell comprising any one or more cells of any of the alternatives described herein or a composition provided by any of the alternatives. Compositions comprising any one or more genetically engineered cells of any of the alternatives described herein and a pharmaceutical excipient are provided. Genetically engineered cells expressing FOXP3 can be produced by the methods provided by any of the alternatives described herein. Methods for preparing genetically engineered cells are provided, comprising providing a cell comprising a first nucleotide sequence comprising a coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; and introducing the second oligonucleotide or protein nuclease into the cell for a gene editing process. In some alternatives, the gene editing process is a knock-in process in which a heterologous promoter is inserted into the first nucleotide sequence. In some alternatives, the coding strand comprises a first coding exon. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus comprises a native coding exon or a first coding exon. In some alternatives, completion of the gene editing process generates the first coding exon in the targeted locus after the gene editing process. In some alternatives, the targeted locus is within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110 base pairs upstream of the first coding exon, or any number of base pairs within a range defined by any two of the above values. In some alternatives, the gene editing process further comprises generating a synthetic first coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the heterologous promoter is an EF1α promoter, a PGK promoter, or an MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the method further comprises inserting a heterologous enhancer domain (e.g., a TCRα enhancer) into the first nucleotide sequence, wherein the enhancer domain is upstream or downstream of one or more regulatory sequences. In some alternatives, the method further comprises inserting a heterologous transcriptional activation domain into the first nucleotide sequence. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE) into the first nucleotide sequence.In some alternatives, the heterologous promoter is inserted upstream of the first coding exon on the coding strand during genetic modification, or the genetic modification results in a synthetic first coding exon produced by completing the gene editing process, wherein the heterologous promoter is upstream of the synthetic first coding exon. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line is a cell expressing CD4+. In some alternatives, the cell line is a cell expressing CD8+. In some alternatives, the cell line is a regulatory T cell. In some alternatives, the genetically engineered cell for expressing FOXP3 comprises a nucleic acid, wherein the nucleic acid comprises a coding strand, wherein the coding strand comprises one or more regulatory elements operably linked to a FOXP3 gene and a heterologous promoter, wherein the FOXP3 gene comprises the first coding exon. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the constitutive promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon. In some alternatives, the nucleic acid further comprises a heterologous transcription enhancer domain. In some alternatives, the nucleic acid further comprises a heterologous transcription activation domain. In some alternatives, the nucleic acid further comprises a ubiquitous chromatin opening element (UCOE). In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the one or more regulatory elements are genetically modified. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line is a regulatory T cell. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line is a regulatory T cell. In an eleventh aspect, a method for treating, suppressing, or ameliorating an autoimmune disorder in a subject is provided. The method comprises removing cells from a subject in need thereof, wherein the cells comprise a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements, a FOXP3 gene or portion thereof, wherein the FOXP3 gene or portion thereof comprises a first coding exon; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; introducing the second nucleotide sequence or protein nuclease into the cell for genetic modification, wherein the genetic modification is a knock-in procedure that inserts a promoter into the first nucleotide sequence; and introducing the cell into the subject for treatment, suppression, or amelioration. In some alternatives, the autoimmune disorder is rheumatoid arthritis, diabetes, inflammatory bowel disease, IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome), Crohn's disease, multiple sclerosis, idiopathic or systemic lupus erythematosus. In some alternatives, the subject is identified or selected to receive therapy for the autoimmune disorder. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line represents a CD4+ cell. In some alternatives, the cell line represents a CD8+ cell. In some alternatives, the cell line is a regulatory T cell. In a twelfth aspect, a method for treating, suppressing, or ameliorating the side effects of organ transplantation in an individual is provided. The method comprises removing cells from an individual in need thereof, wherein the cells comprise a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements, a FOXP3 gene or portion thereof, wherein the FOXP3 gene or portion thereof comprises a first coding exon; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; introducing the second nucleotide sequence or protein nuclease into the cells for genetic modification, wherein the genetic modification is a knock-in procedure that inserts a promoter into the first nucleotide sequence; and introducing the cells into the individual for treatment, suppression, or amelioration. In some alternatives, the cells are precursor stem cells. In some alternatives, the cells are hematopoietic stem cells. In some alternatives, the cells express CD4+ cells. In some alternatives, the cells express CD8+ cells. In some alternatives, the cells are regulatory T cells. In a thirteenth aspect, a method for treating, suppressing, or ameliorating a side effect of organ transplantation (e.g., organ rejection) in an individual is provided. The method comprises removing cells from an individual in need thereof, wherein the cells comprise a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements, a FOXP3 gene or portion thereof, wherein the FOXP3 gene or portion thereof comprises a first coding exon; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; introducing the second nucleotide sequence or protein nuclease into the cell for genetic modification, wherein the genetic modification is a knock-in procedure that inserts a promoter into the first nucleotide sequence; and introducing the cell into the individual for treatment, suppression, or amelioration. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line is a regulatory T cell. Simple diagram description

[0004] [picture] [1]: FOXP3 promotes the T reg lineage. As shown in the figure, naive T cells express IL-12, IL-18, IFNγ, IL-6, TGF-β, IL-4 and IL-2, which in turn regulate TH1, TH17, TH2 and iTreg. T reg plays a key role in a variety of autoimmune diseases (IPEX, T1D, SLE, RA, EAE, etc.). Methods to increase the number or function of human Treg are currently under investigation, including low-dose IL-2 and adoptive transfer of autologous expanded Treg. The efficacy of IL-2 therapy is limited by its multi-directional activity and potential "off-target" effects that can increase inflammation. Adoptive Treg therapy may be limited by the in vivo stability and survival of the expanded Treg and the lack of relevant antigen specificity. [picture] [2] [:] T cell editing tools. As shown in the figure, efficient methods for co-delivery of nucleases and repair templates are already in place for the alternative methods presented herein. As shown, T cells are activated and expanded. T cells are stimulated with CD3 / CD28 beads, which are then removed. TALEN delivery is then performed via mRNA electroporation. The editing template is delivered via adeno-associated virus. [picture] [3] FOXP3 expression is performed via gene editing. As described in this article's alternative approach, FOXP3 expression via gene editing can be used to bypass epigenetic regulation. The design of a stable, site-specifically integrated promoter upstream of the FOXP3 coding region can surpass traditional controls. Foxp3 expression is traditionally controlled by epigenetic regulation within the intronic region (intron 1) upstream of the first coding exon (exon 2). In resting naive T cells, the FOXP3 locus is epigenetically "off" (FOXP3-). Natural thymic Tregs have an "open" locus (FOXP3+). Induced Tregs (iTregs) have a partially open locus (FOXP3+ --- unstable). [picture] [4] HDR targeting was performed to engineer FOXP3+ stabilized cells. As shown in the figure, epigenetic regulatory sites of the FOXP3 gene are located upstream of the FOXP3 gene. To engineer FOXP3+ stabilized T cells, T cells were first activated using CD3 / CD28. The editing template was delivered via adeno-associated virus, and mRNA encoding the nuclease was introduced into the T cells to engineer gene-edited T cells. [picture] [5] Gene editing template design is highly flexible. As shown in the figure, five constructs were constructed: GOXP3 [GFP-ex2], FOXP3 [MND-GFP-ex2], FOXP3 [MND-EGFRt.t2A-ex2], FOXP3 [MND-IL10.t2A.GFP-ex2], and FOXP3 [deletion-intron1.GFP]. [picture] [6] showed that gene editing effectively targets the FOXP3 gene and drives high expression of the transgene. As shown in the figure, cells engineered to express GFP fused to the N-terminus of the endogenous FOXP3 gene under the control of the MND promoter showed high levels of GFP-FOXP3 fusion protein expression compared to cells without the introduction of TALEN nucleases into the T cells. [picture] [7] [:] Edited cells demonstrate seamless HR-mediated integration at the targeted site. PCR was performed using the indicated primer sets (i.e., corresponding to the arrow positions in the figure), and the resulting PCR products were analyzed by agarose gel electrophoresis. PCR using one primer external to the targeting template (primer 1 or primer 2) and one internal primer (primer 3 or primer 4) will only reveal bands of the correct size if precise targeted integration occurs. The gel fragment on the left demonstrates the lack of targeted integration in mock-edited cells, while the gel fragment on the right shows the presence of a band of the correct size in edited cells. [picture] [8] [:] GFP / FOXP3 fusions produce consistently high levels of Foxp3 expression. As shown in the figure, GFP / FOXP3 fusions induce FOXP3 expression under the control of the MND promoter relative to mock-edited cells. Shown is flow cytometric analysis of FOXP3 expression in mock-edited versus edited cells, with FOXP3 expression shown on the Y-axis and forward scatter on the x-axis. [picture] [9] [:] GFP / FOXP3 fusions produce consistently high levels of Foxp3 expression. As shown in the figure, GFP-FOXP3 fusions induce FOXP3 expression under the control of the MND promoter relative to mock-edited cells. Shown are flow cytometric analyses of FOXP3 expression and GFP expression in mock-edited cells, with FOXP3 expression shown on the Y-axis of each graph and GFP on the x-axis. [picture]

[10] [:] Sketch of Treg cells illustrating surface markers and cytokine phenotype. [] [picture]

[11] Edited cell surface phenotype. As shown in the figure, flow cytometry analysis of CD25, CD127, CTLA4, and LAG3 expression in engineered T cells stably expressing FOXP3 was performed. Similar to natural regulatory T cells, the engineered cells expressed high CD25, low CD127, and high CTLA4 and LAG3 levels. [picture]

[12] [:]Cytokine expression profile of edited cells. As shown in the figure, compared to mock-edited T cells, engineered cells displayed a T reg-like profile of intracellular cytokines, with lower levels of IL2, IL4, and IFN-g. [picture]

[13] [:] Cytokine expression profile of edited cells. As shown in the bar graph, engineered T cells stably expressing FOXP3 showed elevated IL-10 expression relative to mock-edited cells. [picture]

[14] [:] IL2 / STAT5 Signaling Sensitivity. Natural Tregs are highly sensitive to IL2 due to their high CD25 expression. The sensitivity of mock-edited and edited cells to IL2 signaling was compared by ex vivo exposure to varying concentrations of IL2. The response to IL2 was measured based on the level of STAT5 phosphorylation. As can be seen, STAT5 phosphorylation in edited cells, indicative of IL2 signaling, occurred at significantly lower IL2 concentrations relative to mock-edited cells. Profiles from top to bottom are: 0.1 ng / ml IL / 2, 1 ng / ml IL / 2, 10 ng / ml IL / 2, 100 ng / ml IL / 2, and no stimulation. [picture]

[15] [:] IL2 / STAT5 Signaling Sensitivity (FOXP3+ Subset). Natural Tregs are highly sensitive to IL2 due to their high CD25 expression. The sensitivity of mock-edited and edited cells to IL2 signaling was compared by ex vivo exposure to varying concentrations of IL2. Response to IL2 was measured based on the level of STAT5 phosphorylation. As can be seen, STAT5 phosphorylation in edited cells, indicative of IL2 signaling, occurred at significantly lower IL2 concentrations relative to mock-edited cells. Profiles from top to bottom are: 0.1 ng / ml IL / 2, 1 ng / ml IL / 2, 10 ng / ml IL / 2, 100 ng / ml IL / 2, and no stimulation. [picture]

[16] [:] Schematic diagram of an assay for reading suppressive activity of regulatory T cells. CFSE-labeled responder cells were mixed with edited Tregs or mock-edited cells and stimulated with beads. The assay was read based on the degree of CFSE dilution at 96 hours. [picture]

[17] Edited T cells can suppress T eff proliferation. Responder cells cultured with mock-edited cells were able to significantly dilute the CFSE label through their proliferation, while responder cells cultured with engineered GFP+ cells remained largely undivided (retaining a high level of labeling). [picture]

[18] Functional Activity of Edited Cells: Edited T cells from IPEX individuals lacked suppression of Teff proliferation. Using the same assay as Figure 17, normal patient control cells or IPEX patient cells were mock-edited or edited to replicate native FOXP3 gene expression. Incubation of edited healthy control cells with responding cells suppressed CFSE dilution compared to mock-edited cells, while incubation of edited IPEX patient cells did not suppress proliferation compared to mock-edited cells. [picture]

[19] Generation of antigen-specific T reg-type cells via FOXP3 gene editing. As demonstrated, this editing technology can be applied to pathogenic T cell clones that are present in a variety of autoimmune diseases. Editing of a T cell clone isolated using tetramers specific for the Flu antigen successfully generated T cells that stably expressed FOXP3. [picture]

[20] [:] Phenotype of edited T cells. As shown in the figure, T cells stably expressing FOXP3 express CTLA4 and LAG3. [picture] [twenty one] Phenotype and function of edited GFP-FOXP3 T cells. As shown in the figure, T cells stably expressing FOXP3 exhibited phenotypic changes (CD25+, CD45RO+, CCR7, and CD38 / CTLA-4 / LAP). These T cells stably expressing FOXP3 also displayed a T reg-like profile of intracellular cytokine expression (IL-2, IFN-γ, and IL-4). pSTAT5 signaling was also sensitive to IL-2 when administered at 10 ng to 100 ng. [] [picture] [twenty two] In vivo GvHD experiment. Shown is a timeline for preparing for an in vivo GvHD experiment. First, mice were irradiated to destroy myeloid cells. Engineered Treg cells stably expressing FOXP3 were then infused into the mice, followed by an infusion of Teff cells. Between 1 and 50 days after infusion, the mice's body weights were monitored, and T cell percentages in peripheral blood and various organs were assessed. [picture] [twenty three] In vivo GvHD experiment. As shown, the graph shows the survival percentage of mice receiving effector cells (ET, circles), effector cells and mock-edited T cells (squares), and effector cells and edited FOXP3-stabilized T-regs (triangles). Edited FOXP3-stabilized T-regs generated a substantial survival advantage, indicating their ability to ameliorate xenogeneic GvHD caused by effector cells. [] [picture] [twenty four] [:] TSDR CpG methylation in edited T cells. Schematic diagram showing how methylation of the regulatory region upstream of the first coding exon (actually exon 2) regulates the FOXP3 gene. [picture]

[25] [:] Methylation analysis of clones. Each black dot indicates a methylated CpG dinucleotide in a PCR fragment spanning the TSDR of a T cell clone. As can be seen, CD25-negative cells have highly methylated TSDRs, indicating a closed, inactive, and unexpressed FOXP3 locus, while CD25-high cells (natural Tregs) display almost uniformly completely demethylated TSDRs. [picture]

[26] [:] TSDR methylation in edited T cells. Despite known high levels of FOXP3 expression, edited T cells displayed fully methylated TSDR, indicating that integrating a promoter downstream of TSDR effectively bypassed normal epigenetic regulation of the FOXP3 locus. Implementation Method

[0005] Detailed description of preferred embodiments This article describes several methods for treating or ameliorating autoimmune diseases using engineered CD4 T cells that stably express their endogenous FOXP3 gene. These methods can also be used to ameliorate the effects of graft-versus-host disease (GVHD). [definition] [ , , ] As used herein, "nucleic acid" or "nucleic acid molecule" refers to a polynucleotide or oligonucleotide, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), an oligonucleotide, fragments generated by polymerase chain reaction (PCR), and fragments generated by ligation, cleavage, endo-nuclease action, exo-nuclease action, and synthesis. Nucleic acid molecules can be composed of monomers that are natural nucleotides (e.g., DNA and RNA) or analogs of natural nucleotides (e.g., mirror image isomeric forms of natural nucleotides), or a combination of both. Modified nucleotides can have alterations in the sugar moiety and / or in the pyrimidine or purine base moiety. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or functionalization of sugars as ethers or esters. Furthermore, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as azasugars and carbocyclic sugar analogs. Examples of modifications in the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutions. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of these linkers. Analogs of phosphodiester linkers include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, anilinothioates, anilinophosphates, or phosphamidates. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids," which comprise natural or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be single-stranded or double-stranded. As used herein, the "coding strand" is the DNA strand that has the same base sequence as the RNA transcript produced (but with uracil replacing thymine). This strand contains codons, while the non-coding strand contains anticodons. As used herein, a "regulatory element" refers to a segment of a nucleic acid molecule that is capable of increasing or decreasing the expression of a specific gene in an organism. Regulation of gene expression is a fundamental characteristic of all living organisms and viruses. Examples of regulatory elements, without limitation, include CAAT boxes, CCAAT boxes, Pribnow boxes, TATA boxes, SECIS elements, mRNA polyadenylation signals, A boxes, Z boxes, C boxes, E boxes, G boxes, hormone response elements, such as insulin gene regulatory sequences, DNA binding domains, activation domains, and / or enhancer domains. As described herein, "FOXP3" is a protein involved in immune system responses. The FOXP3 gene contains 11 coding exons. Foxp3 is a specific marker for natural regulatory T cells (nT regs, T cell lineage) and adaptive / induced regulatory T cells (a / iT regs). In animal studies, induction or administration of Foxp3-positive T cells has been shown to significantly reduce the severity of (autoimmune) diseases in models of diabetes, multiple sclerosis, asthma, inflammatory bowel disease, thyroiditis, and kidney disease. However, T cells have been shown to exhibit plasticity in research. Therefore, the use of regulatory T cells in therapy can be risky because regulatory T cells transferred into patients can become pro-inflammatory T helper 17 (Th17) cells, which are pro-inflammatory rather than regulatory cells. Therefore, methods are provided herein to avoid the risk of this pro-inflammatory transformation of regulatory cells. As shown in Figure 1, FOXP3 expressed by iT regs serves as a master regulator of the immune system and contributes to tolerance and immunosuppression. Tregs play a key role in various autoimmune diseases, including IPEX, T1D, SLE, RA, and EAE. Methods to increase the number or function of human Tregs are currently under investigation, including low-dose IL-2 and adoptive transfer of autologous expanded Tregs. The efficacy of IL-2 therapy is limited by its multitropic activity and potential off-target effects that can increase inflammation. Adoptive Treg therapy may be limited by the in vivo stability and survival of expanded Tregs and their lack of antigen specificity. As used herein, "nucleases" are proteins or enzymes capable of cleaving phosphodiester bonds between nucleotide subunits of nucleic acids. Nucleases described herein are used in "gene editing," a type of genetic engineering that uses a nuclease or one or more engineered nucleases to insert, delete, or replace DNA in the genome of a living organism. Without limitation, the nuclease may be a nuclease of the CRISPR / CAS9 system, a zinc finger nuclease, or a TALEN nuclease. Nucleases can be used to target a locus or a targeted locus within a nucleic acid sequence. As used herein, "coding exon" refers to any portion of a gene that encodes a portion of the final mature RNA produced from the gene after introns are removed by RNA splicing. The term exon refers to both the DNA sequence within a gene and the corresponding sequence in the RNA transcript. During RNA splicing, introns are removed and exons are covalently joined to one another as part of generating the mature messenger RNA. "Cas9," as described herein, is an RNA-guided DNA endonuclease associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity system. As described herein, "zinc finger nucleases" are artificial restriction enzymes created by fusing zinc finger DNA binding domains to DNA cleavage domains. The zinc finger domains can be engineered to target specific desired DNA sequences, enabling zinc finger nucleases to target unique sequences within complex genomes. As described herein, "TALENs," or "transcription activator-like effector nucleases," are restriction enzymes that can be engineered to cleave specific DNA sequences. They are created by fusing the TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease that cuts DNA strands). Transcription activator-like effectors (TALEs) can be engineered to bind to virtually any desired DNA sequence, thereby enabling DNA cleavage at a specific location when combined with a nuclease. Restriction enzymes can be introduced into cells for gene editing or for in situ genome editing, a technique known as genome editing using engineered nucleases. In addition to zinc finger nucleases and CRISPR / Cas9, TALENs are also prominent tools in the field of genome editing. As used herein, "knock-in" refers to a genetic engineering method involving a one-to-one replacement of DNA sequence information with the wild-type copy of a genetic locus or the insertion of sequence information not found at the locus. A "promoter" is a nucleotide sequence that directs transcription of a structural gene. In some alternatives, a promoter is located in the 5' noncoding region of a gene, near the transcription start site of the structural gene. Sequence elements within a promoter that play a role in initiating transcription are typically characterized by a consensus nucleotide sequence. It is a region of DNA that initiates transcription of a specific gene. A promoter is located near the transcription start site of a gene, on the same strand of DNA and upstream (toward the 5' region of the sense strand). A promoter can be approximately 100, 200, 300, 400, 500, 600, 700, 800, or 1000 base pairs in length, or within a range defined by any two of the aforementioned lengths. As used herein, a promoter can be constitutively active, repressible, or inducible. If a promoter is inducible, the transcription rate increases in response to an inducing agent. In contrast, if a promoter is constitutive, the transcription rate is not regulated by an inducing agent. Repressible promoters are also known. Without limitation, examples of promoters include constitutive promoters, heterologous weak promoters (e.g., promoters that produce less expression than endogenous promoters and / or constitutive promoters), or inducible promoters. Examples include the EF1α promoter, PGK promoter, MND promoter, KI promoter, Ki-67 gene promoter, and / or promoters inducible by drugs (e.g., tamoxifen and / or its metabolites). Commonly used constitutive promoters include, but are not limited to, SV40, CMV, UBC, EF1A, PGK, and / or CAGG, which are used in mammalian systems. If a weak promoter and a strong promoter both drive expression of the same coding sequence, the weak promoter will produce less mRNA than the stronger promoter. This can be compared by analyzing, for example, agarose gels. An example of a promoter that is regulated by proximal chromatin is the short EF1α promoter, which is highly active in some loci but almost inactive in others (Eyquem, Biotechnol Bioeng. 2013 Aug;110(8):2225-35. doi: 10.1002 / bit.24892). As used herein, a "transcription enhancer domain" refers to a short (50-1500 bp) region of DNA that can be bound by proteins (activators) to increase the likelihood that transcription of a specific gene will occur, or to promote or enhance the level of transcription that occurs. These activator proteins are often referred to as transcription factors. Enhancers are typically cis-acting and located up to 1 Mbp (1,000,000 bp) away from the gene, either upstream or downstream of the start site, and in either the forward or reverse direction. Enhancers can be located upstream or downstream of the gene they regulate. In some embodiments, multiple enhancer domains may be used to generate more transcription; for example, multimeric activator binding domains may be used to further enhance or increase the level of transcription. Furthermore, enhancers do not need to be located near the transcription start site to affect transcription, as some enhancers have been found hundreds of thousands of base pairs upstream or downstream of the start site. Enhancers do not act on the promoter region itself but are bound by activator proteins. These activator proteins interact with the mediator complex, which recruits polymerase II and general transcription factors, which then initiate gene transcription. Enhancers can also be found within introns. The orientation of an enhancer can even be reversed without affecting its function. Furthermore, enhancers can be excised and inserted elsewhere in the chromosome and still affect gene transcription. In some alternatives, enhancers are used to silence inhibitory mechanisms that prevent FOXP3 gene transcription. An example of an enhancer binding domain is the TCR α enhancer. In some alternatives, the enhancer domain in the alternatives described herein is the TCR α enhancer. In some alternatives, the enhancer binding domain is placed upstream of the promoter so that it activates the promoter to increase protein transcription. In some alternatives, the enhancer binding domain is placed upstream of the promoter to activate the promoter to increase transcription of the FOXP3 gene. As used herein, a "transcriptional activator domain" or "transcription activation domain" refers to a specific DNA sequence that can be bound by a transcription factor, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA. Specific transcription factors may include, but are not limited to, SP1, AP1, C / EBP, heat shock factor, ATF / CREB, c-Myc, Oct-1, and NF-1. In some alternative approaches, the activator domain is used to silence inhibitory mechanisms that prevent transcription of the FOXP3 gene. As described herein, "ubiquitous chromatin opening elements" (UCOEs) are elements characterized by unmethylated CpG islands that span the dually divergently transcribed promoters of housekeeping genes. UCOEs represent a promising tool for avoiding silencing and maintaining transgene expression in a variety of cell models, including cell lines, pluripotent hematopoietic stem cells, and PSCs and their differentiated progenies. As used herein, "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, such that the heterologous nucleic acid sequence is expressed. As used herein, "autoimmune disorders" refer to abnormally underactive or overactive immune systems. In cases of overactive immune systems, the body attacks and damages its own tissues (autoimmune diseases). Immunodeficiency disorders reduce the body's ability to fight invaders, making it more susceptible to infection. Examples of autoimmune disorders or autoimmune diseases can include, for example, without limitation, systemic lupus, scleroderma, hemolytic anemia, vasculitis, type I diabetes, Graves' disease, rheumatoid arthritis, multiple sclerosis, Goodpasture's syndrome, myopathy, severe combined immunodeficiency, DiGeorge syndrome, hyperimmunoglobulin E syndrome, common variable immunodeficiency, chronic granulomatous disease, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, hyper IgM syndrome, leukocyte adhesion deficiency, essential regulator of NF-κB (NEMO) mutation, selective immunoglobulin A deficiency, X-linked agammaglobulinemia, X-linked lymphoproliferative disease, IPEX, and the like. (immune disorders, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome) and / or ataxia telangiectasia. Immune disorders can be analyzed, for example, by examining the profile of neural-specific autoantibodies or other biomarkers when detected in the patient's serum or cerebrospinal fluid. In some alternative methods provided herein, these methods are used to treat, ameliorate, or suppress autoimmune disorders. In some alternatives, the autoimmune disorder is systemic lupus, scleroderma, hemolytic anemia, vasculitis, type I diabetes mellitus, Graves' disease, rheumatoid arthritis, multiple sclerosis, Goodpasture's syndrome, myopathy, severe combined immunodeficiency, DiGeorge syndrome, hyperimmunoglobulin E syndrome, common variable immunodeficiency, chronic granulomatous disease, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, hyper-IgM syndrome, leukocyte adhesion defect, essential regulator of NF-κB (NEMO) mutation, selective immunoglobulin A deficiency, X-linked agammaglobulinemia, X-linked lymphoproliferative disorder, IPEX (Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-Linked (IPEX) Syndrome) and / or ataxia telangiectasia. As used herein, "organ transplantation" refers to the transfer of an organ from one body to another, or from a donor site to another location within the body, to replace a damaged or missing organ in the recipient. Organs and / or tissues transplanted within the same person are called autologous transplants. More recently, transplants between two individuals of the same species are called allografts. Allografts can be derived from living or cadaveric sources. Some of the alternatives described herein provide methods for treating, inhibiting, or ameliorating the side effects of organ transplantation in a subject, such as organ rejection. Examples of organs that can be transplanted include the heart, kidney, liver, lung, pancreas, intestine, and thymus. Examples of tissues used for transplantation include bone, tendon (both referred to as musculoskeletal grafts), cornea, skin, heart valves, nerves, and veins. Kidneys, livers, and hearts are the most commonly transplanted organs. Corneas and musculoskeletal grafts are the most common transplanted tissues. In some alternatives described herein, methods are provided for treating, inhibiting, or ameliorating a side effect of organ transplantation (e.g., organ rejection) in a subject. In some alternatives, the subject is selected to receive an anti-rejection agent. In some alternatives, the anti-rejection agent comprises Prednisone, Imuran (azathioprine), Cellcept (mycophenolate mofetil, or MMF), Myfortic (mycophenolic acid), Rapamune (sirolimus), Neoral (cyclosporine), or Prograf (tacrolimus). In some alternatives, individuals are selected for inhibition, amelioration, or treatment using the engineered cells of the alternatives described herein. In some alternatives, the individual has side effects from anti-inflammatory or anti-rejection drugs. Therefore, the selected individual is provided with the replacement cells or compositions provided herein. Side effects from anti-rejection drugs can include interactions with other medications that can increase or decrease tacrolimus levels in the blood, kidney toxicity, hypertension, neurotoxicity (tremors, headaches, tingling, and insomnia), diabetes (high blood sugar), diarrhea, nausea, hair loss, and / or high potassium. Therefore, patients are selected for the methods of treatment, inhibition, or amelioration described herein. "Organ rejection" or "transplant rejection" as used herein is when the transplanted tissue is rejected by the recipient's immune system, which destroys the transplanted tissue. As described herein, "graft-versus-host disease" (GVHD) refers to a medical complication following the receipt of transplanted tissue from a genetically different person. GVHD is often associated with stem cell or bone marrow transplants, but the term also applies to other forms of tissue transplants. Immune cells in the donor tissue recognize the recipient as foreign and not "self." In some alternatives described herein, the methods provided can be used to prevent or ameliorate complications that can arise from GVHD. As used herein, a "pharmaceutical excipient" is an inert substance into which the cells in the composition are provided. As used herein, "chimeric antigen receptors" (CARs) (also known as chimeric T cell receptors) refer to artificial T cell receptors or genetically engineered receptors that transfer a desired specificity to immune effector cells. For example, these receptors can be used to transfer the specificity of a monoclonal antibody or its binding portion to a T cell. In some alternatives herein, the genetically engineered cells further comprise a sequence encoding a chimeric antigen receptor. In some alternatives, the chimeric antigen receptor is specific for a molecule on tumor cells. The chimeric antigen receptor or engineered cells expressing the T cell receptor can be used to target specific tissues in need of FOXP3. In some alternatives herein, methods are included for targeting specific tissues for the provision and delivery of FOXP3. In some alternatives, the tissue is a transplanted tissue. In some alternatives, the chimeric antigen receptor is specific for a target molecule on the transplanted tissue. As described herein, the genetically engineered cells are engineered to express FOXP3, and therefore are also described herein in an alternative embodiment as "Treg-phenotype" cells. [Detailed explanation] [] Due to the potential of regulatory T cells to induce antigen-specific tolerance, many groups have focused on using these cells to treat autoimmune diseases. There are many forms of regulatory T cells ("T regs"), and current nomenclature divides T regs into the following: those generated in the thymus during T cell development, designated thymic regulatory T cells or "tT regs," and those induced peripherally, designated peripheral regulatory T cells or "pT regs." A key aspect of regulatory T cell biology is the expression of the transcription factor FOXP3. FOXP3 is believed to be essential for specifying the regulatory T cell lineage. This concept is based on the observation that humans lacking FOXP3 develop severe autoimmune disease beginning in the neonatal period. One of the greatest obstacles to using tT regs or pT regs to treat autoimmune diseases is that FOXP3 expression is epigenetically regulated. In tT regs, an upstream region of the FOXP3 gene, known as the "thymus-specific demethylation region," is completely demethylated, a state believed to stabilize FOXP3 expression. Generally, complete demethylation is not observed in pT regs. Under inflammatory conditions, FOXP3 can be epigenetically silenced in pT regs, and possibly in tT regs (although some researchers believe tT regs are completely stable), potentially leading to the conversion of pT regs into pro-inflammatory CD4 T cells. The lack of stability of pT regs is a significant issue, as infusion of pT regs that have reverted to an inflammatory phenotype can lead to exacerbation of autoimmune symptoms. Here, we provide evidence that an engineering approach to stabilize FOXP3 expression in CD4 T cells allows the generation of expanded populations of latent suppressor T cells that are no longer susceptible to epigenetic modifications of their suppressive function. Thus, these cells may have improved properties for therapeutic applications. In the alternative approach described herein, cells for therapeutic applications are engineered to have stable FOXP3 expression by using gene-editing nucleases to modify regulatory elements of the FOXP3 locus to provide stable FOXP3 expression. In the exemplary data provided, a constitutive promoter (examples of constitutive promoters include, among others, the EF1α promoter, the PGK promoter, and / or the MND promoter) is placed upstream of the FOXP3 coding exon to drive FOXP3 expression, but a variety of approaches are contemplated for modifying regulatory elements to allow for stable FOXP3 expression. By employing several methods for modifying endogenous regulatory elements, the claimed therapeutic cells exhibit constitutive expression of the native FOXP3 gene, rendering them no longer susceptible to regulatory influences that could result in FOXP3 gene silencing and reversion to a non-repressed cellular phenotype. Thus, in the alternative approach described herein, the problem of loss of FOXP3 expression due to epigenetic effects on native regulatory sequences and promoters is addressed. The proposed method for expressing FOXP3 in mixed CD4 T cell populations is also an improvement over other methods for isolating naturally occurring regulatory T cell populations because it provides a means of capturing the TCR repertoire present in inflammatory T cell populations. In patients with autoimmune diseases or organ transplant rejection, the endogenous TCR repertoire in the inflammatory T cell population includes TCRs with the correct binding specificity to recognize inflamed or allogeneic tissue within the organ. These T cells are believed to mediate autologous inflammatory responses or organ rejection. By converting a portion of the mixed CD4 T cell population to a regulatory phenotype, the TCR specificities present in the pro-inflammatory population will be expressed in the therapeutic cell population. This is an improvement over therapies based on thymic regulatory T cells, which are believed to have a distinct and non-overlapping TCR repertoire with inflammatory T cells. Furthermore, it is hypothesized that in patients with autoimmune diseases or organ rejection, existing tT reg populations fail to develop the tolerance necessary to avoid inflammation. The methods described herein can be used to treat autoimmune diseases and to induce tolerance to transplanted organs. A significant disadvantage is the need to use a gene editing tool that can efficiently perform recombination at the FOXP3 locus. Therefore, the methods provided demonstrate that this reaction can be performed efficiently using TALEN nucleases, but in principle any nuclease platform will work equally well. Regulatory T cell therapy can be used for tolerance in transplantation and autoimmunity. Currently, ex vivo expanded Tregs are infused. Phase I studies have shown marginal efficacy (if any) in T1D and, in some cases, benefit in post-transplant GvHD. For next-generation engineered regulatory T cells, some alternative approaches could be natural Tregs directed by chimeric antigen receptors (CARs). Effector T cells can also be converted into Tregs through FOXP3 expression. However, there may be differences in therapeutic approaches between engineered and natural Tregs. Natural Treg therapy has been deemed safe, but insufficient numbers of natural Tregs can lead to autoimmunity. Tregs play a key role in various autoimmune diseases, including IPEX, T1D, SLE, RA, and EAE. Methods to increase the number or function of human Tregs are currently under investigation, including low-dose IL-2 and adoptive transfer of autologous expanded Tregs. The effectiveness of IL-2 therapy is limited by its multi-directional activity and potential "off-target" effects that can increase inflammation. Adoptive Treg therapy may be limited by the in vivo stability and survival of the expanded Tregs and their lack of relevant antigen specificity. The use of natural Tregs also presents potential drawbacks. For example, autoimmune patients are genetically prone to Treg instability. For example, it seems plausible that CAR-carrying nTregs could convert into CAR T effector cells. nTregs also retain the potential for epigenetic regulation of FOXP3, which can lead to inducible downregulation of FOXP3, meaning that the function or nTreg population may never be fully predictable. Furthermore, natural Tregs may not contain the correct TCR (T cell receptor) specificity. Treg function can also be linked to selectable markers, meaning that the expanded natural Treg cell population may always contain contaminating inflammatory cells. Therefore, the methods provided herein offer an improvement over transfer using engineered cells using natural Tregs due to the potential to link CAR expression to regulatory T cell function, thereby avoiding the potential engraftment of CAR Tregs with the potential to convert into pro-inflammatory CAR T cells. T cell editing tools for engineering T cells for protein expression are well known (e.g., see [picture] [2)] For example, T cells are activated and expanded, TALEN delivery is performed via mRNA electroporation, and the template can be edited by delivering adeno-associated virus (high MOI transient expression - recombinant AAV non-integrating). FOXP3 can then be expressed through a gene editing process in which a promoter is inserted upstream of the first coding exon and downstream of native regulatory elements, which are controlled by epigenetic control mechanisms. Controlling expression by inserting a promoter upstream of the first promoter has not been reported and surprisingly results in constitutive upregulation of FOXP3, which cannot be downregulated by epigenetic control. [picture] [3] shows the insertion of a promoter, for example the MND promoter, into the FOXP3 gene. As shown, Foxp3 expression is classically controlled by epigenetic regulation within an intronic region upstream of the first coding exon (intron 1). like [picture] As shown in [4], HDR targeting was used to engineer FOXP3+ stabilized T cells, which resulted in strong constitutive expression. [picture] [5] shows a template of the gene construct used in the alternative scheme described in this article. Methods for controlling FOXP3 expression are also provided. In some alternatives, the promoter inserted upstream of the FOXP3 coding exon is an inducible receptor. In some alternatives, the system employs a synthetic transcriptional regulator that binds to the synthetic promoter upstream of the transgene in the presence of tamoxifen to induce FOXP3 expression. In the presence of tamoxifen, TamR-tf binds to the 7×HBD / EF1αp promoter, inducing transgene expression in an "on" state. In some alternatives, this transcriptional regulator is modified to provide varying degrees of control over transgene expression. In some alternatives, cells can also be further engineered to express a chimeric antigen receptor or TCR (T cell receptor) or other targeting moiety. Cells can be engineered to express a CAR to target specific tissues or cells. In some alternatives, the CAR comprises a ligand binding domain, which is a tumor-specific molecule, viral molecule, or another molecule expressed on the target cell population. In some alternatives, the target tissue has low expression of FOXP3. Allowing T cells to express a CAR or TRC will allow the T cell to target specific tissues where it is necessary to deliver cells expressing FOXP3 to tissue-specific sites. [Alternative Solution] [1] [:Effective Targeting of Gene Editing] [FOXP3] [Genes drive high expression of transgenics.] [ , , ] As shown in Figure 6, cells engineered to express GFP fused to the N-terminus of the endogenous FOXP3 gene under the control of the MND promoter showed high levels of GFP-FOXP3 fusion protein expression compared to cells without the introduction of TALEN nucleases into T cells. As shown in Figure 7, the edited cells showed seamless HR-mediated integration at the targeted site. For the experiments in Figure 7, PCR was performed using the indicated primer sets (i.e., corresponding to the positions of the arrows on the figure), and the resulting PCR products were analyzed by agarose gel electrophoresis. If precise targeted integration occurs, PCR using one primer outside the targeting template (primer 1 or primer 2) and one internal primer (primer 3 or primer 4) will only reveal bands of the correct size. The gel fragment on the left shows the lack of targeted integration in mock-edited cells, while the gel fragment on the right shows the presence of a band of the correct size in edited cells. GFP / FOXP3 fusions produce consistently high levels of Foxp3 expression. As shown in the figure, GFP FOXP3 fusions induce FOXP3 expression under the control of the MND promoter relative to mock-edited cells. Shown is a flow cytometric analysis of FOXP3 expression in mock-edited cells versus edited cells, with FOXP3 expression shown on the Y-axis and forward scatter shown on the x-axis ( [picture] [8)]. [] GFP / FOXP3 fusions produce consistently high levels of Foxp3 expression. As shown in Figure 9, GFP FOXP3 fusions induced FOXP3 expression under the control of the MND promoter relative to mock-edited cells. Shown are flow cytometric analyses of FOXP3 expression and GFP expression in mock-edited cells, with FOXP3 expression shown on the Y-axis of each graph and GFP on the x-axis. (See [picture] [9]). [] As demonstrated in experiments with these exemplary alternatives, gene editing effectively targets the FOXP3 gene and drives high expression of the transgene. Because the experiments presented herein were performed in vitro, it is difficult to predict whether the same high level of FOXP3 expression will be maintained in in vivo tests, cells, or individuals. Therefore, it will be necessary to monitor the cells used for treatment in individuals to see whether they are maintained, increased, or decreased. Methods for examining engineered cells in individuals after administration are known to those skilled in the art. For example, Adaptive Biotech Technology's commercially available technology has systems for immunosequencing B and T cells to detect specific cells (e.g., engineered cells) to determine whether they are maintained, increased, or decreased. Because cells may have been edited at different distances upstream of the coding exons, this can affect FOXP3 expression in vivo, and therefore in vivo data do not predict the results of in vivo experiments. Furthermore, using different promoters, effector domains, and activation domains can produce different results. [Alternative Solution] [2] [:implement] [FOXP3] [Performance produces] [T , reg , ] [Surface and cytokine phenotype] [T] [cell] [] Figure 10 shows a schematic diagram of T-reg cells illustrating their surface marker and cytokine phenotypes. It was desirable to examine whether the edited cells possessed a specific surface phenotype. As shown in Figure 11 , engineered T cells stably expressing FOXP3 were analyzed by flow cytometry for expression of CD25, CD127, CTLA4, and LAG3. Similar to natural regulatory T cells, the engineered cells showed high expression of CD25, low CD127, and high expression of CTLA4 and LAG3. The cytokine expression profiles of the edited cells are also shown in Figure 12. As shown in Figure 12, compared to mock-edited T cells, the engineered cells displayed a T reg-like profile of intracellular cytokine expression, with lower expression of IL2, IL4, and IFN-g. As shown in the bar graph of Figure 13, engineered T cells stably expressing FOXP3 displayed high IL-10 expression relative to mock-edited cells. The edited cells were also shown to be sensitive to IL2 / STAT5 signaling (Figure 14). Natural Tregs are highly sensitive to IL2 due to their high CD25 expression. The sensitivity of mock-edited and edited cells to IL2 signaling was compared by exposing them to different concentrations of IL2 in vitro. The response to IL2 was measured based on the phosphorylation level of STAT5. As can be seen, STAT5 phosphorylation in edited cells indicates IL2 signaling at significantly lower IL2 concentrations than in mock-edited cells. As shown in Figure 15, natural T regs are highly sensitive to IL2 due to their high CD25 expression. By exposing mock-edited and edited cells to different concentrations of IL2 in vitro, the sensitivity to IL2 signaling was compared. The response to IL2 was measured based on the phosphorylation level of STAT5. As can be seen, STAT5 phosphorylation in edited cells indicates IL2 signaling at significantly lower IL2 concentrations than in mock-edited cells. As shown in this exemplary alternative, expression of FOXP3 results in T cells with a T reg surface and cytokine phenotype. Because some of the experiments presented herein were performed in vitro, it is difficult to predict whether the same elevated levels of FOXP3 expression in cells will be maintained in in vivo assays, cells, or individuals, and whether such elevated levels of FOXP3 expression in cells will be sufficient to ameliorate pathogenic T cell and / or B cell responses in, for example, autoimmune disorders or GVHD. It is also difficult to predict whether such cells will possess regulatory T cell characteristics in vivo. Therefore, it may be advantageous to monitor cells used for treatment, suppression, or amelioration in individuals to determine whether such cells are maintained, increased, or decreased before, during, and / or after treatment. Methods for examining engineered cells in individuals after administration are known to those skilled in the art. For example, commercially available technologies from Adaptive Biotechnologies and others are systems for sequencing B and T cells to detect specific immune cell types (e.g., engineered cells or pathogenic B or T cells) and thereby determine whether such cells are maintained, increased, or decreased. Since cells may have been edited at different distances upstream of the coding exon, which may affect the expression of FOXP3 in vivo, in vivo data are not predictive of the results of in vivo experiments. In addition, the use of different promoters, effector domains, and activation domains may have different results. In some alternatives to the treatment methods described herein, the methods further comprise monitoring the individual to determine whether the engineered cells are maintained, increased, or decreased before, during, and / or after treatment. In some alternatives, a cell sample is removed from the individual to detect the engineered cells and determine whether the engineered cells are maintained, increased, or decreased. In some alternatives, if the engineered cells decrease in the individual, the engineered cells are administered again to the individual in need. The cells in vivo will also have to be tested to see if they also express markers similar to those of regulatory T cells. [Alternative Solution] [3] [: Functional activity of edited cells can be suppressed by edited cells] [Teff] [Proliferation.] [] Figure 16 shows a schematic diagram of an assay for reading suppressive activity of regulatory T cells. CFSE-labeled responder cells were mixed with edited Tregs or mock-edited cells and stimulated with beads. The assay was read based on the degree of CFSE dilution at 96 hours. Figure 17 shows that edited T cells can suppress T eff proliferation. Responder cells cultured with mock-edited cells were able to significantly dilute the CFSE label through their proliferation, while responder cells cultured with engineered GFP+ cells remained largely undivided (retaining a large amount of labeling). Figure 18 shows the functional activity of edited cells: Lack of suppression of Teff proliferation using edited T cells from IPEX individuals. Using the same analysis as Figure 17, normal patient control cells or IPEX patient cells were mock-edited or edited to produce native FOXP3 gene expression. Incubation of edited healthy control cells with responding cells suppressed CFSE dilution of responding cells compared to mock-edited cells, while incubation of edited IPEX patient cells showed no suppression of proliferation compared to mock-edited cells. Antigen-specific T reg cells have also been generated through FOXP3 gene editing. As shown in Figure 19, this editing technology can be applied to pathogenic T cell clones that appear in various autoimmune diseases. The results show that editing of a T cell clone isolated using tetramers specific for the Flu antigen successfully generated T cells that stably expressed FOXP3. As shown in these exemplary alternatives, edited cells can suppress Teff proliferation. Because some of the experiments presented herein were performed in vitro, it is difficult to predict whether the same elevated levels of FOXP3 expression in cells will be maintained in in vivo assays, cells, or individuals, and whether such elevated levels of FOXP3 expression in cells will be sufficient to ameliorate pathogenic T cell and / or B cell responses in, for example, autoimmune disorders or GVHD. It is also difficult to predict whether such cells will possess regulatory T cell characteristics in vivo. Therefore, it may be advantageous to monitor cells used for treatment, amelioration, or suppression in individuals to determine whether such cells are maintained, increased, or decreased before, during, and / or after treatment. Methods for examining engineered cells in individuals after administration are known to those skilled in the art. For example, commercially available technologies from Adaptive Biotechnologies and others are systems for sequencing B and T cells to detect specific immune cell types (e.g., engineered cells or pathogenic B or T cells) and thereby determine whether such cells are maintained, increased, or decreased. Since cells may have been edited at different distances upstream of the coding exon, which may affect the expression of FOXP3 in vivo, in vivo data are not predictive of the results of in vivo experiments. In addition, the use of different promoters, effector domains, and activation domains may have different results. In some alternatives to the methods of treatment, improvement, or inhibition described herein, the methods further comprise monitoring the individual to determine whether the cells are maintained, increased, or decreased before, during, and / or after receiving the engineered cells. In some alternatives, a cell sample is taken from the individual to detect the engineered cells and determine whether the engineered cells are maintained, increased, or decreased. In some alternatives, if the engineered cells decrease in the individual, the engineered cells are administered again to the individual in need. The engineered cells will also have to be tested to see whether they have an effect on T effector cells in vivo, as in vitro experiments cannot be used to predict their effect on T effector cells in vivo or whether they suppress T effector cells. [Summarize] [ , , ] Gene editing in primary human T cells allows the expression of Foxp3 and the generation of a stable T reg phenotype by introducing a strong promoter. Gene editing in CD4+ T cells is effective and produces high and stable expression of Foxp3. It was also shown that gene expression is limited to endogenous loci. The edited cells displayed T reg functional properties (pSTAT5 and intracellular cytokine production), surface phenotype, and in vitro function (T effector suppression). It is feasible to use antigen-specific T cells for FOXP3 editing. The successful establishment of the GvHD mouse model will allow the functional testing of T cells edited by FOXP3 to be expanded in vivo. [Alternative Solution] [4] [Edited] [GFP FOXP3] [Cell phenotype and function] [ , , ] As shown in Figure 21, T cells stably expressing FOXP3 exhibited phenotypic changes (CD25+, CD45RO+, CCR7, CD38 / CTLA-4 / LAP). These T cells stably expressing FOXP3 also displayed a T reg-like profile of intracellular cytokine expression (IL-2, IFN-γ, and IL-4). When administered at 10 ng to 100 ng, pSTAT5 signaling was also sensitive to IL-2. Thus, T cells stably expressing FOXP3 displayed a T reg-like profile of intracellular cytokine expression. Because some of the experiments presented herein were performed in vitro, it is difficult to predict whether the same elevated levels of FOXP3 expression in cells will be maintained in in vivo assays, cells, or individuals, and whether such elevated levels of FOXP3 expression in cells will be sufficient to ameliorate pathogenic T cell and / or B cell responses in, for example, autoimmune disorders or GVHD. It is also difficult to predict whether such cells will possess regulatory T cell characteristics in vivo. Therefore, it may be advantageous to monitor cells used for treatment, suppression, or amelioration in individuals to determine whether such cells are maintained, increased, or decreased before, during, and / or after treatment. Methods for examining engineered cells in individuals after administration are known to those skilled in the art. For example, commercially available technologies from Adaptive Biotechnologies and others are systems for sequencing B and T cells to detect specific immune cell types (e.g., engineered cells or pathogenic B or T cells) and thereby determine whether such cells are maintained, increased, or decreased. Since cells may have been edited at different distances upstream of the coding exon, which may affect the expression of FOXP3 in vivo, in vivo data are not predictive of the results of in vivo experiments. In addition, the use of different promoters, effector domains, and activation domains may have different results. In some alternatives to the methods of treating, inhibiting, or ameliorating described herein, the methods further comprise monitoring the individual to determine whether the engineered cells are maintained, increased, or decreased before, during, and / or after treatment. In some alternatives, a cell sample is taken from the individual to detect the engineered cells and determine whether the engineered cells are maintained, increased, or decreased. In some alternatives, if the engineered cells decrease in the individual, the engineered cells are administered again to the individual in need. [Alternative Solution] [5] [:In vivo] [GVHD] [experiment] [ , , ] Figure 22 shows the timeline for preparing for an in vivo GvHD experiment. Mice were first irradiated to destroy myeloid cells. Engineered cells with a Treg phenotype that stably expressed FOXP3 were then infused into the mice, followed by an infusion of Teff cells. Between 1 and 50 days after infusion, the mice's body weights were monitored, and T cell percentages in peripheral blood and various organs were assessed. In Figure 23, the graph shows the survival percentage of mice that received effector cells (circles), effector cells and mock-edited T cells (squares), and effector cells and edited FOXP3-stabilized cells with a T-reg phenotype (triangles). Edited FOXP3-stabilized Treg-phenotype cells generated a substantial survival advantage, indicating their ability to improve xenogeneic GvHD caused by effector cells. [Alternative Solution] [6] [:Engineering Tolerogenicity] [T] [Cells and natural thymus] [T , reg , ] Molecular similarity [ / ] [Difference.] [ , , ] A schematic diagram of TSDR CpG methylation in edited T cells is shown in Figure 24. The FOXP3 gene is regulated by methylation of the regulatory region upstream of the first coding exon (actually exon 2). The clones were then analyzed for methylation (Figure 25). Each black dot indicates a methylated CpG dinucleotide in a PCR fragment spanning the TSDR of a T cell clone. As can be seen, CD25-negative cells have highly methylated TSDRs, indicating a closed, inactive, and non-expressed FOXP3 locus, while high CD25 cells (natural Tregs) show almost uniformly completely demethylated TSDRs. Despite known high levels of FOXP3 expression, edited T cells displayed fully methylated TSDR, indicating that integrating the promoter downstream of TSDR effectively bypassed the normal epigenetic regulation of the FOXP3 locus ( FIG. 26 ). Because some of the experiments presented herein were performed in vitro, it is difficult to predict whether the same elevated levels of FOXP3 expression in cells will be maintained in in vivo assays, cells, or individuals, and whether such elevated levels of FOXP3 expression in cells will be sufficient to ameliorate pathogenic T cell and / or B cell responses in, for example, autoimmune disorders or GVHD. It is also difficult to predict whether such cells will possess regulatory T cell characteristics in vivo. Therefore, it may be advantageous to monitor cells used for treatment, suppression, or amelioration in individuals to determine whether such cells are maintained, increased, or decreased before, during, and / or after treatment. Methods for examining engineered cells in individuals after administration are known to those skilled in the art. For example, commercially available technologies from Adaptive Biotechnologies and others are systems for sequencing B and T cells to detect specific immune cell types (e.g., engineered cells or pathogenic B or T cells) and thereby determine whether such cells are maintained, increased, or decreased. Since cells may have been edited at different distances upstream of the coding exon, which may affect the expression of FOXP3 in vivo, in vivo data are not predictive of the results of in vivo experiments. In addition, the use of different promoters, effector domains, and activation domains may have different results. [Method for preparing nucleic acid] [ , , ] In some alternatives, methods are provided for preparing nucleic acids that bypass epigenetic control of protein expression. In some alternatives, the protein is FOXP3. In some alternatives, methods are provided for preparing nucleic acids for expressing FOXP3. The method comprises providing a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof; providing a nuclease; and subjecting the first nucleotide sequence to a gene editing process that edits the one or more regulatory elements and, optionally, the FOXP3 gene or portion thereof. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the first targeted locus comprises a first coding exon. In some alternatives, after the gene editing process is completed, the targeted locus comprises a first coding exon. In some alternatives, the targeted locus is located at one or more regulatory elements. In some alternatives, the targeted locus is located at a FOXP3 gene or portion thereof, wherein the FOXP3 gene or portion thereof comprises a first coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the gene editing process is a knock-in procedure for inserting a heterologous promoter, a heterologous transcriptional enhancer domain, or both. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the promoter is an EF1α promoter, a PGK promoter, or an MND promoter. In some alternatives, the promoter is a heterologous weak promoter (e.g., one with less activity or transcriptional efficiency than an endogenous promoter and / or a constitutive promoter). In some alternatives, the promoter is an inducible promoter. In some alternatives, an inducible promoter can be induced by a drug or steroid. In some alternatives, the method further comprises inserting one or more heterologous transcriptional enhancer domains. In some alternatives, the method further comprises inserting one or more heterologous transcriptional activation domains. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE). In some alternatives, the method further comprises inserting an inducible effector. In some alternatives, the inducible effector can be induced by a steroid or drug. In some alternatives, the heterologous promoter is inserted at any position upstream of the first coding exon on the coding strand, wherein the first coding exon represents the exon after the gene editing process is completed and wherein the coding strand further comprises the start codon of the FOXP3 gene. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements. [For stable performance] [FOXP3] [Nucleic Acid] [ , , ] In some alternatives, a nucleic acid is provided for bypassing epigenetic control of protein expression. In some alternatives, the protein is FOXP3. In some alternatives, a nucleic acid is provided, wherein the nucleic acid comprises a coding strand comprising heterologous regulatory elements and a heterologous promoter, wherein the heterologous regulatory elements and heterologous promoter are operably linked to a FOXP3 gene or portion thereof, wherein the FOXP3 gene or portion thereof comprises the first coding exon. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the constitutive promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter with less activity or transcription efficiency than an endogenous promoter and / or a constitutive promoter). In some alternatives, the promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon. In some alternatives, the nucleic acid further comprises one or more heterologous transcriptional enhancer domains. In some alternatives, the nucleic acid further comprises one or more heterologous transcriptional activation domains. In some alternatives, the nucleic acid further comprises a ubiquitous chromatin opening element (UCOE). In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the one or more regulatory elements are genetically modified. [Method for preparing genetically engineered cells for stably expressing proteins] [ , , ] In some alternatives, methods for preparing genetically engineered cells that bypass epigenetic control are provided. In some alternatives, methods for preparing genetically engineered cells are provided, comprising providing a cell, wherein the cell comprises a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease and introducing the second oligonucleotide or protein nuclease into the cell for genetic modification. In some alternatives, the genetic modification is a knock-in procedure in which a heterologous promoter is inserted into the first nucleotide sequence. In some alternatives, the coding strand comprises a first coding exon. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus is at the first coding exon. In some alternatives, the targeted locus is within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110 base pairs upstream of the first coding exon, or any number of base pairs within a range defined by any two of the aforementioned values. In some alternatives, the gene editing process further comprises generating a synthetic first coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the heterologous promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter with less activity or transcriptional efficiency than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the method further comprises inserting one or more heterologous enhancer domains into the first nucleotide sequence. In some alternatives, the method further comprises inserting one or more heterologous transcriptional activation domains into the first nucleotide sequence. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE) into the first nucleotide sequence. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon on the coding strand or upstream of a synthetic first coding exon produced by completing a gene editing process. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line is a regulatory T cell. [cell] [ , , ] In some alternatives, genetically engineered cells are provided for bypassing epigenetic control of protein expression. In some alternatives, the protein is FOXP3. In some alternatives, genetically engineered cells for expressing FOXP3 are provided, produced by any of the methods described herein. The method comprises providing a cell, wherein the cell comprises a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements and the FOXP3 gene or portion thereof; providing a second nucleotide sequence encoding a nuclease or a protein nuclease and introducing the second oligonucleotide or protein nuclease into the cell for genetic modification. In some alternatives, the genetic modification is a knock-in procedure in which a heterologous promoter is inserted into the first nucleotide sequence. In some alternatives, the coding strand comprises a first coding exon. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus is at the first coding exon. In some alternatives, the targeted locus is within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110 base pairs upstream of the first coding exon, or any number of base pairs within a range defined by any two of the aforementioned values. In some alternatives, the gene editing process further comprises generating a synthetic first coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the heterologous promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter with less activity or transcriptional efficiency than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the method further comprises inserting one or more heterologous enhancer domains into the first nucleotide sequence. In some alternatives, the method further comprises inserting one or more heterologous transcriptional activation domains into the first nucleotide sequence. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE) into the first nucleotide sequence. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon on the coding strand or upstream of a synthetic first coding exon produced by completing a gene editing process. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells.In some alternatives, the cell line is a regulatory T cell. In some alternatives, a genetically engineered cell for expressing FOXP3 is provided, wherein the genetically engineered cell comprises a nucleic acid comprising a coding strand comprising one or more regulatory elements operably linked to a FOXP3 gene and a heterologous promoter, wherein the FOXP3 gene comprises the first coding exon. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the constitutive promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter with less activity or transcriptional efficiency than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon. In some alternatives, the nucleic acid further comprises one or more heterologous transcription enhancer domains. In some alternatives, the nucleic acid further comprises one or more heterologous transcriptional activation domains. In some alternatives, the nucleic acid further comprises a ubiquitous chromatin opening element (UCOE). In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the one or more regulatory elements are genetically modified. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line is a regulatory T cell. In some alternatives, the cells may be engineered to express a chimeric antigen receptor. [Composition] [ , , ] The present disclosure provides methods utilizing these compositions or methods of using these compositions to perform cellular immunotherapy in individuals suffering from a disease or condition. In some alternatives, the composition comprises any one or more genetically engineered cells of any of the alternatives described herein and a pharmaceutical excipient. The cell line is for genetically engineered cells expressing FOXP3, produced by any of the methods described herein. The method comprises providing a cell, wherein the cell comprises a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; and introducing the second oligonucleotide or protein nuclease into the cell for genetic modification. In some alternatives, the genetic modification is a knock-in procedure in which a heterologous promoter is inserted into the first nucleotide sequence. In some alternatives, the coding strand comprises a first coding exon. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus is at the first coding exon. In some alternatives, the targeted locus is within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110 base pairs upstream of the first coding exon, or any number of base pairs within a range defined by any two of the aforementioned values. In some alternatives, the gene editing process further comprises generating a synthetic first coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the heterologous promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter with less activity or transcriptional efficiency than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the method further comprises inserting one or more heterologous enhancer domains into the first nucleotide sequence. In some alternatives, the method further comprises inserting one or more heterologous transcriptional activation domains into the first nucleotide sequence. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE) into the first nucleotide sequence. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon on the coding strand or upstream of a synthetic first coding exon produced by completing a gene editing process. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell.In some alternatives, the cell line represents a CD4+ cell. In some alternatives, the cell line represents a CD8+ cell. In some alternatives, the cell line represents a regulatory T cell. In some alternatives, the genetically engineered cell comprises a nucleic acid comprising a coding strand comprising one or more regulatory elements operably linked to a FOXP3 gene and a heterologous promoter, wherein the FOXP3 gene comprises the first coding exon. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the constitutive promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., one whose activity or transcription efficiency is less than that of an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon. In some alternatives, the nucleic acid further comprises one or more heterologous transcription enhancer domains. In some alternatives, the nucleic acid further comprises one or more heterologous transcription activation domains. In some alternatives, the nucleic acid further comprises a ubiquitous chromatin opening element (UCOE). In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the one or more regulatory elements are genetically modified. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line is a regulatory T cell. In some alternatives, the cells may be engineered to express a chimeric antigen receptor. [Treatment or improvement methods] [ , , ] In some alternatives, methods are provided for treating, suppressing, or ameliorating an autoimmune disorder in a subject, comprising administering to the subject a genetically modified cell comprising any one or more of the cells of any of the alternatives provided herein, or a composition of any of the alternatives provided herein. In some alternatives, the composition comprises any one or more of the genetically engineered cells of any of the alternatives provided herein and a pharmaceutical excipient. The cell line is a genetically engineered cell for expressing FOXP3, produced by any of the methods described herein. The method comprises providing a cell comprising a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; and introducing the second oligonucleotide or protein nuclease into the cell for genetic modification. In some alternatives, the genetic modification is a knock-in procedure in which a heterologous promoter is inserted into the first nucleotide sequence. In some alternatives, the coding strand comprises a first coding exon. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus is at the first coding exon. In some alternatives, the targeted locus is within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110 base pairs upstream of the first coding exon, or any number of base pairs within a range defined by any two of the above values. In some alternatives, the gene editing process further comprises generating a synthetic first coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the heterologous promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter with less activity or transcriptional efficiency than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by drugs or steroids. In some alternatives, the method further comprises inserting one or more heterologous enhancer domains into the first nucleotide sequence. In some alternatives, the method further comprises inserting one or more heterologous transcriptional activation domains into the first nucleotide sequence. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE) into the first nucleotide sequence. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon on the coding strand or upstream of a synthetic first coding exon produced by completing a gene editing process. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements.In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line represents a CD4+ cell. In some alternatives, the cell line represents a CD8+ cell. In some alternatives, the cell line is a regulatory T cell. In some alternatives, the genetically engineered cell comprises a nucleic acid comprising a coding strand comprising one or more regulatory elements operably linked to a FOXP3 gene and a heterologous promoter, wherein the FOXP3 gene comprises the first coding exon. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the constitutive promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., one whose activity or transcription efficiency is less than that of an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon. In some alternatives, the nucleic acid further comprises one or more heterologous transcription enhancer domains. In some alternatives, the nucleic acid further comprises one or more heterologous transcription activation domains. In some alternatives, the nucleic acid further comprises a ubiquitous chromatin opening element (UCOE). In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the one or more regulatory elements are genetically modified. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line is a regulatory T cell. In some alternatives, the cells may be engineered to express a chimeric antigen receptor. In some alternatives, the autoimmune disease is rheumatoid arthritis, diabetes, inflammatory bowel disease, IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome), Crohn's disease, multiple sclerosis, idiopathic or systemic lupus erythematosus. In some alternative embodiments, the autoimmune disease is systemic lupus, scleroderma, hemolytic anemia, vasculitis, type I diabetes mellitus, Graves' disease, rheumatoid arthritis, multiple sclerosis, Goodpasture's syndrome, myopathy, severe combined immunodeficiency, DiGeorge syndrome, hyperimmunoglobulin E syndrome, common variable immunodeficiency, chronic granulomatous disease, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, hyper IgM syndrome, leukocyte adhesion defect, essential regulator of NF-κB (NEMO) mutation, selective immunoglobulin A deficiency, X-linked agammaglobulinemia, X-linked lymphoproliferative disorder, and / or ataxia telangiectasia.In some alternatives, the autoimmune disorder is systemic lupus, scleroderma, hemolytic anemia, vasculitis, type I diabetes, Graves' disease, rheumatoid arthritis, multiple sclerosis, Goodpasture's syndrome, myopathy, severe combined immunodeficiency, DiGeorge syndrome, hyperimmunoglobulin E syndrome, common variable immunodeficiency, chronic granulomatous disease, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, hyper-IgM syndrome, leukocyte adhesion deficiency, essential regulator of NF-κB (NEMO) mutation, selective immunoglobulin A deficiency, X-linked agammaglobulinemia, X-linked lymphoproliferative disease, IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome), and / or ataxia telangiectasia. In some alternatives, the individual is identified or selected to receive therapy for the autoimmune disease. In some alternative scenarios, individuals are refractory to standard therapies for autoimmune diseases or anti-inflammatory therapies. This identification or selection can be done based on a clinical or diagnostic assessment. In some alternatives, a subject is given combination therapy, wherein the subject is administered an anti-rejection drug or anti-inflammatory drug in combination with the engineered cells. In some alternatives, the subject is selected to receive an anti-rejection agent. In some alternatives, the anti-rejection agent includes Prilosec, Imuran (azathioprine), Mycophenolate Mofetil (MMF), Mycophenolic Acid, Sirolimus, Cyclosporine, or Tacrolimus. In some alternatives, the anti-inflammatory drug includes Tysabri®, steroids, immunosuppressants, NSAIDs, immunoglobulins, acetaminophen, celecoxib, indomethacin, or diclofenac. In some alternatives, the subject is monitored for changes in engineered T cells expressing FOXp3+, engineered cell populations exhibiting a Treg phenotype, or pathogenic T or B cell populations in vivo before, during, and / or after the cell administration process. In some alternatives, depending on the first response to the first cell bolus, the subject is selected to receive another administration of the engineered cells. In some alternatives, the subject is administered another dose of cells within 1 week, 2 weeks, or a month from the initial dose. In some alternatives to the treatment methods described herein, the methods further comprise monitoring the individual to determine whether the engineered cells are maintained, increased, or decreased before, during, and / or after treatment. In some alternatives, a cell sample is removed from the individual to detect the engineered cells and determine whether the engineered cells are maintained, increased, or decreased. In some alternatives, if the engineered cells decrease in the individual, the engineered cells are administered again to the individual in need. In some alternatives, a method for treating, inhibiting, or ameliorating the side effects of organ transplantation in a subject is provided, comprising administering to the subject a genetically modified cell comprising any one or more of the cells described in any of the alternatives herein or a composition described in any of the alternatives herein. In some alternatives, the composition comprises any one or more of the genetically engineered cells described in any of the alternatives herein and a pharmaceutical excipient. The cell line is a genetically engineered cell expressing FOXP3, produced by any of the methods described herein. The method comprises providing a cell comprising a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; and introducing the second oligonucleotide or protein nuclease into the cell for genetic modification. In some alternatives, the genetic modification is a knock-in procedure in which a heterologous promoter is inserted into the first nucleotide sequence. In some alternatives, the coding strand comprises a first coding exon. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus is at the first coding exon. In some alternatives, the targeted locus is within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110 base pairs upstream of the first coding exon, or any number of base pairs within a range defined by any two of the above values. In some alternatives, the gene editing process further comprises generating a synthetic first coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the heterologous promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter with less activity or transcriptional efficiency than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by drugs or steroids. In some alternatives, the method further comprises inserting one or more heterologous enhancer domains into the first nucleotide sequence. In some alternatives, the method further comprises inserting one or more heterologous transcriptional activation domains into the first nucleotide sequence. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE) into the first nucleotide sequence. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon on the coding strand or upstream of a synthetic first coding exon produced by completing a gene editing process. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements.In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line expresses regulatory T cells. In some alternatives, the individual is refractory to standard therapies for autoimmune diseases or standard therapies for anti-inflammatory therapies. In some alternatives, the individual is refractory to standard anti-rejection agents or standard anti-rejection therapies. Therefore, in some alternatives, the individual is selected to receive genetically engineered cells expressing FOXP3. In some alternatives, the genetically engineered cell comprises a nucleic acid comprising a coding strand comprising one or more regulatory elements operably linked to a FOXP3 gene and a heterologous promoter, wherein the FOXP3 gene comprises the first coding exon. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the constitutive promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., one whose activity or transcription efficiency is less than that of an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon. In some alternatives, the nucleic acid further comprises one or more heterologous transcription enhancer domains. In some alternatives, the nucleic acid further comprises one or more heterologous transcription activation domains. In some alternatives, the nucleic acid further comprises a ubiquitous chromatin opening element (UCOE). In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the one or more regulatory elements are genetically modified. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line is a regulatory T cell. In some alternatives, methods are provided for treating, inhibiting, or ameliorating side effects of organ transplantation (e.g., organ rejection) in a subject, comprising administering to the subject a genetically modified cell of any one or more of the cells described in any of the alternatives herein or a composition described in any of the alternatives herein. In some alternatives, the composition comprises any one or more of the genetically engineered cells described in any of the alternatives herein and a pharmaceutical excipient. The cell line is a genetically engineered cell expressing FOXP3, produced by any of the methods described herein. The method comprises providing a cell, wherein the cell comprises a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; and introducing the second oligonucleotide or protein nuclease into the cell to perform the genetic modification. In some alternatives, the genetic modification is a knock-in procedure in which a heterologous promoter is inserted into the first nucleotide sequence. In some alternatives, the coding strand comprises a first coding exon. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus is in the first coding exon. In some alternatives, the targeted locus is within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110 base pairs upstream of the first coding exon, or any number of base pairs within a range defined by any two of the above values. In some alternatives, the gene editing process further comprises generating a synthetic first coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the heterologous promoter is an EF1α promoter, a PGK promoter, or an MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter with less activity or transcriptional efficiency than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by drugs or steroids. In some alternatives, the method further comprises inserting one or more heterologous enhancer domains into the first nucleotide sequence. In some alternatives, the method further comprises inserting one or more heterologous transcriptional activation domains into the first nucleotide sequence. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE) into the first nucleotide sequence. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon on the coding strand or upstream of a synthetic first coding exon produced by completing a gene editing process. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements.In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line expresses regulatory T cells. In some alternatives, the individual is refractory to standard therapies for autoimmune diseases or standard therapies for anti-inflammatory therapies. In some alternatives, the individual is refractory to standard anti-rejection agents or standard anti-rejection therapies. Therefore, in some alternatives, the individual is selected to receive genetically engineered cells expressing FOXP3. In some alternatives, the genetically engineered cell comprises a nucleic acid comprising a coding strand comprising one or more regulatory elements operably linked to a FOXP3 gene and a heterologous promoter, wherein the FOXP3 gene comprises the first coding exon. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the constitutive promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., one whose activity or transcription efficiency is less than that of an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon. In some alternatives, the nucleic acid further comprises one or more heterologous transcription enhancer domains. In some alternatives, the nucleic acid further comprises one or more heterologous transcription activation domains. In some alternatives, the nucleic acid further comprises a ubiquitous chromatin opening element (UCOE). In some alternatives, a heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the one or more regulatory elements are genetically modified. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line is a regulatory T cell. In some alternatives of the methods for treating, inhibiting, or ameliorating described herein, the methods further comprise monitoring the individual to determine whether the cells are maintained, increased, or decreased before, during, and / or after receiving the engineered cells. In some alternatives, a cell sample is removed from the individual to detect the engineered cells and determine whether the engineered cells are maintained, increased, or decreased. In some alternatives, if the engineered cells decrease in the individual, the engineered cells are re-administered to the individual in need. In some alternatives, methods are provided for treating, suppressing, or ameliorating an autoimmune disorder in a subject, the methods comprising removing cells from a subject in need thereof, wherein the cells comprise a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements, a FOXP3 gene or portion thereof, wherein the FOXP3 gene or portion thereof comprises a first coding exon; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; introducing the second nucleotide sequence or protein nuclease into the cell for genetic modification, wherein the genetic modification is a knock-in procedure that inserts a promoter into the first nucleotide sequence; and introducing the cell into the subject for treatment, suppression, or amelioration. In some alternatives, the autoimmune disorder is rheumatoid arthritis, diabetes, inflammatory bowel disease, IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome), Crohn's disease, multiple sclerosis, idiopathic or systemic lupus erythematosus. In some alternatives, the autoimmune disease is systemic lupus, scleroderma, hemolytic anemia, vasculitis, type I diabetes, Graves' disease, rheumatoid arthritis, multiple sclerosis, Goodpasture's syndrome, myopathy, severe combined immunodeficiency disorder, DiGeorge syndrome, hyperimmunoglobulin E syndrome, common variable immunodeficiency, chronic granulomatous disease, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, hyper-IgM syndrome, leukocyte adhesion deficiency, essential regulator of NF-κB (NEMO) mutation, selective immunoglobulin A deficiency, X-linked agammaglobulinemia, X-linked lymphoproliferative disorder, and / or ataxia telangiectasia. In some alternatives, the individual is identified or selected to receive therapy for the autoimmune disease. In some alternatives, the autoimmune disease is rheumatoid arthritis, diabetes, inflammatory bowel disease, IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome), Crohn's disease, multiple sclerosis, idiopathic or systemic lupus erythematosus. This identification or selection can be accomplished through diagnostic and / or clinical evaluation. In some alternatives, the individual is refractory to standard therapies for the autoimmune disease or standard therapies for anti-inflammatory therapy. In some alternatives, the individual is refractory to standard anti-rejection agents or standard anti-rejection therapies. Therefore, in some alternatives, the individual is selected to receive genetically engineered cells expressing FOXP3. In some alternatives, methods for treating, inhibiting, or ameliorating the side effects of organ transplantation in an individual are provided, the methods comprising removing cells from an individual in need thereof, wherein the cells comprise a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements, a FOXP3 gene or portion thereof, wherein the FOXP3 gene or portion thereof comprises a first coding exon; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; introducing the second nucleotide sequence or protein nuclease into the cell for genetic modification, wherein the genetic modification is a knock-in process that inserts a promoter into the first nucleotide sequence; and introducing the cell into the individual for treatment, inhibition, or amelioration. In some alternatives of the methods for treating, inhibiting, or ameliorating described herein, the methods further comprise monitoring the individual to determine whether the cells are maintained, increased, or decreased before, during, and / or after receiving the engineered cells. In some alternatives, a cell sample is removed from the individual to detect the engineered cells and determine whether the engineered cells are maintained, increased, or decreased. In some alternatives, if the engineered cells decrease in a subject, the engineered cells are administered again to a subject in need. In some alternatives, the subject is refractory to standard therapies for autoimmune diseases or standard therapies for anti-inflammatory therapies. In some alternatives, the subject is refractory to standard anti-rejection agents or standard anti-rejection therapies. Therefore, in some alternatives, the subject is selected to receive genetically engineered cells expressing FOXP3. In some alternatives, methods are provided for treating, inhibiting, or ameliorating side effects of organ transplantation (e.g., organ rejection) in an individual. The methods comprise removing cells from an individual in need thereof, wherein the cells comprise a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements, a FOXP3 gene or portion thereof, wherein the FOXP3 gene or portion thereof comprises a first coding exon; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; introducing the second nucleotide sequence or protein nuclease into the cells for genetic modification, wherein the genetic modification is a knock-in procedure that inserts a promoter into the first nucleotide sequence; and introducing the cells into the individual for treatment. In some alternatives, the cells are precursor stem cells. In some alternatives, the cells are hematopoietic stem cells. In some alternatives, the cells express CD4+ cells. In some alternatives, the cells express CD8+ cells. In some alternatives, the cells express regulatory T cells. In some alternatives, the individual is refractory to standard therapies for autoimmune diseases or standard therapies for anti-inflammatory therapies. In some alternatives, the subject is refractory to standard anti-rejection agents or standard anti-rejection therapy. Thus, in some alternatives, the subject is selected to receive genetically engineered cells expressing FOXP3. [More Alternatives] [ , , ] In some alternatives, methods are provided for preparing a nucleic acid for expressing FOXP3, wherein the method comprises providing a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof; providing a nuclease; and subjecting the first nucleotide sequence to a gene editing process that edits the one or more regulatory elements and, optionally, the FOXP3 gene or portion thereof. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus comprises a native first coding exon. In some alternatives, completing the gene editing process produces the first coding exon in the targeted locus after the gene editing process. In some alternatives, the targeted locus is at one or more regulatory elements. In some alternatives, the targeted locus is at the FOXP3 gene or portion thereof. In some alternatives, the FOXP3 gene or portion thereof comprises the first native coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the gene editing process is a knock-in procedure for inserting a heterologous promoter, a heterologous transcriptional enhancer domain, or both. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the promoter is a heterologous weak promoter (e.g., one that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the promoter is an inducible promoter. In some alternatives, an inducible promoter can be induced by a drug or steroid. In some alternatives, the method further comprises inserting a heterologous transcriptional enhancer domain, wherein the enhancer domain is upstream or downstream of one or more regulatory sequences. In some alternatives, the method further comprises inserting a heterologous transcriptional activation domain. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE). In some alternatives, the method further comprises inserting an inducible effector. In some alternatives, the inducible effector can be induced by a steroid or drug. In some alternatives, a heterologous promoter is inserted, wherein the insertion of the heterologous promoter generates the first coding exon, wherein the heterologous promoter is located anywhere upstream of the first coding exon on the coding strand, and wherein the coding strand further comprises the start codon of the FOXP3 gene. In some alternatives, the heterologous promoter is inserted anywhere upstream of the first native exon on the coding strand, and wherein the coding strand further comprises the start codon of the FOXP3 gene. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements. In some alternatives, a nucleic acid for FOXP3 expression is provided, produced by any method of any alternative described herein. The method comprises providing a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof; providing a nuclease; and subjecting the first nucleotide sequence to a gene editing process that edits the one or more regulatory elements and, optionally, the FOXP3 gene or portion thereof. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus comprises a native first coding exon. In some alternatives, completing the gene editing process produces the first coding exon in the targeted locus after the gene editing process. In some alternatives, the targeted locus is at one or more regulatory elements. In some alternatives, the targeted locus is at a FOXP3 gene or portion thereof. In some alternatives, the FOXP3 gene or portion thereof comprises the first native coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the gene editing process is a knock-in procedure for inserting a heterologous promoter, a heterologous transcriptional enhancer domain, or both. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the promoter is a heterologous weak promoter (e.g., one that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the promoter is an inducible promoter. In some alternatives, an inducible promoter can be induced by a drug or steroid. In some alternatives, the method further comprises inserting a heterologous transcriptional enhancer domain, wherein the enhancer domain is upstream or downstream of one or more regulatory sequences. In some alternatives, the method further comprises inserting a heterologous transcriptional activation domain. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE). In some alternatives, the method further comprises inserting an inducible effector. In some alternatives, the inducible effector can be induced by a steroid or drug. In some alternatives, a heterologous promoter is inserted, wherein the insertion of the heterologous promoter generates the first coding exon, wherein the heterologous promoter is located anywhere upstream of the first coding exon on the coding strand, and wherein the coding strand further comprises the start codon of the FOXP3 gene. In some alternatives, the heterologous promoter is inserted anywhere upstream of the first native exon on the coding strand, and wherein the coding strand further comprises the start codon of the FOXP3 gene. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements. In some alternatives, a nucleic acid is provided, comprising a coding strand comprising heterologous regulatory elements and a heterologous promoter, wherein the heterologous regulatory elements and heterologous promoter are operably linked to a FOXP3 gene or portion thereof, wherein the FOXP3 gene or portion thereof comprises the first coding exon. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the constitutive promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., one that generates less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the promoter is an inducible promoter. In some alternatives, the inducible promoter is induced by a drug or steroid. In some alternatives, the heterologous promoter is located upstream of the first coding exon. In some alternatives, the nucleic acid further comprises a heterologous transcription enhancer domain. In some alternatives, the nucleic acid further comprises a heterologous transcription activation domain. In some alternatives, the nucleic acid further comprises a ubiquitous chromatin opening element (UCOE). In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the one or more regulatory elements are genetically modified. In some alternatives, methods for preparing genetically engineered cells are provided, comprising providing a cell, wherein the cell comprises a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; and introducing the second oligonucleotide or protein nuclease into the cell for a gene editing process. In some alternatives, the gene editing process is a knock-in process in which a heterologous promoter is inserted into the first nucleotide sequence. In some alternatives, the coding strand comprises a first coding exon. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus comprises a native coding exon or a first coding exon. In some alternatives, completing the gene editing process results in the first coding exon being generated in the targeted locus after the gene editing process. In some alternatives, the targeted locus is within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110 base pairs upstream of the first coding exon, or any number of base pairs within a range defined by any two of the aforementioned values. In some alternatives, the gene editing process further comprises generating a synthetic first coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the heterologous promoter is an EF1α promoter, a PGK promoter, or an MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the method further comprises inserting a heterologous enhancer domain (e.g., a TCRα enhancer) into the first nucleotide sequence, wherein the enhancer domain is upstream or downstream of one or more regulatory sequences. In some alternatives, the method further comprises inserting a heterologous transcriptional activation domain into the first nucleotide sequence. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE) into the first nucleotide sequence. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon on the coding strand during genetic modification, or the genetic modification results in a synthetic first coding exon produced by completing the gene editing process, wherein the heterologous promoter is upstream of the synthetic first coding exon. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell.In some alternatives, the cell line represents a CD4+ cell. In some alternatives, the cell line represents a CD8+ cell. In some alternatives, the cell line represents a regulatory T cell. In some alternatives, genetically engineered cells for expressing FOXP3 are provided, produced by the methods of any of the alternatives herein. Methods for preparing genetically engineered cells are provided, comprising providing a cell, wherein the cell comprises a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; and introducing the second oligonucleotide or protein nuclease into the cell for a gene editing process. In some alternatives, the gene editing process is a knock-in process in which a heterologous promoter is inserted into the first nucleotide sequence. In some alternatives, the coding strand comprises a first coding exon. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus comprises a native coding exon or a first coding exon. In some alternatives, completing the gene editing process results in the first coding exon being produced in the targeted locus after the gene editing process. In some alternatives, the targeted locus is within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110 base pairs upstream of the first coding exon, or any number of base pairs within a range defined by any two of the aforementioned values. In some alternatives, the gene editing process further comprises generating a synthetic first coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the heterologous promoter is an EF1α promoter, a PGK promoter, or an MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the method further comprises inserting a heterologous enhancer domain (e.g., a TCR alpha enhancer) into the first nucleotide sequence, wherein the enhancer domain is upstream or downstream of one or more regulatory sequences. In some alternatives, the method further comprises inserting a heterologous transcriptional activation domain into the first nucleotide sequence. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE) into the first nucleotide sequence. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon on the coding strand during genetic modification, or the genetic modification results in a synthetic first coding exon generated by completing the gene editing process, wherein the heterologous promoter is upstream of the synthetic first coding exon. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements.In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line represents a CD4+ cell. In some alternatives, the cell line represents a CD8+ cell. In some alternatives, the cell line is a regulatory T cell. In some alternatives, a genetically engineered cell for expressing FOXP3 comprises a nucleic acid comprising a coding strand comprising one or more regulatory elements operably linked to a FOXP3 gene and a heterologous promoter, wherein the FOXP3 gene comprises the first coding exon. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the constitutive promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., one that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter is induced by a drug or steroid. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon. In some alternatives, the nucleic acid further comprises a heterologous transcription enhancer domain. In some alternatives, the nucleic acid further comprises a heterologous transcription activation domain. In some alternatives, the nucleic acid further comprises a ubiquitous chromatin opening element (UCOE). In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the one or more regulatory elements are genetically modified. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line is a regulatory T cell. In some alternatives, compositions are provided comprising any one or more genetically engineered cells selected from any of the alternatives herein and a pharmaceutical excipient. Genetically engineered cells expressing FOXP3 can be produced by the methods provided in any of the alternatives herein. Methods for preparing genetically engineered cells are provided, comprising providing a cell comprising a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; and introducing the second oligonucleotide or protein nuclease into the cell for gene editing. In some alternatives, the gene editing process is a knock-in procedure in which a heterologous promoter is inserted into the first nucleotide sequence. In some alternatives, the coding strand comprises a first coding exon. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus comprises a native coding exon or the first coding exon. In some alternatives, completing the gene editing process generates a first coding exon in the targeted locus after the gene editing process. In some alternatives, the targeted locus is within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110 base pairs upstream of the first coding exon, or any number of base pairs within a range defined by any two of the aforementioned values. In some alternatives, the gene editing process further comprises generating a synthetic first coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the heterologous promoter is an EF1α promoter, a PGK promoter, or an MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the method further comprises inserting a heterologous enhancer domain (e.g., a TCRα enhancer) into the first nucleotide sequence, wherein the enhancer domain is upstream or downstream of one or more regulatory sequences. In some alternatives, the method further comprises inserting a heterologous transcriptional activation domain into the first nucleotide sequence. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE) into the first nucleotide sequence. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon on the coding strand during genetic modification, or the genetic modification results in a synthetic first coding exon generated by completing a gene editing process, wherein the heterologous promoter is upstream of the synthetic first coding exon.In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line is a regulatory T cell. In some alternatives, the genetically engineered cell for expressing FOXP3 comprises a nucleic acid comprising a coding strand comprising one or more regulatory elements operably linked to a FOXP3 gene and a heterologous promoter, wherein the FOXP3 gene comprises the first coding exon. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the constitutive promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by drugs or steroids. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon. In some alternatives, the nucleic acid further comprises a heterologous transcription enhancer domain. In some alternatives, the nucleic acid further comprises a heterologous transcription activation domain. In some alternatives, the nucleic acid further comprises a ubiquitous chromatin opening element (UCOE). In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the one or more regulatory elements are genetically modified. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line represents CD4+ cells. In some alternatives, the cell line represents CD8+ cells. In some alternatives, the cell line is a regulatory T cell. In some alternatives, methods for treating, suppressing, or ameliorating an autoimmune disorder in a subject are provided, comprising: administering to the subject a genetically modified cell comprising any one or more of the cells described in any of the alternatives herein, or a composition provided in any of the alternatives. Compositions comprising any one or more of the genetically engineered cells described in any of the alternatives herein and a pharmaceutical excipient are provided. Genetically engineered cells expressing FOXP3 can be produced by the methods provided in any of the alternatives herein. Methods for preparing genetically engineered cells are provided, comprising providing a cell comprising a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; and introducing the second oligonucleotide or protein nuclease into the cell for a gene editing procedure. In some alternatives, the gene editing procedure is a knock-in procedure in which a heterologous promoter is inserted into the first nucleotide sequence. In some alternatives, the coding strand comprises a first coding exon. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus comprises a native coding exon or the first coding exon. In some alternatives, completion of the gene editing process produces the first coding exon in the targeted locus after the gene editing process. In some alternatives, the targeted locus is within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110 base pairs upstream of the first coding exon, or any number of base pairs within a range defined by any two of the above values. In some alternatives, the gene editing process further comprises producing a synthetic first coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the heterologous promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the method further comprises inserting a heterologous enhancer domain (e.g., a TCRα enhancer) into the first nucleotide sequence, wherein the enhancer domain is upstream or downstream of one or more regulatory sequences. In some alternatives, the method further comprises inserting a heterologous transcriptional activation domain into the first nucleotide sequence. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE) into the first nucleotide sequence.In some alternatives, the heterologous promoter is inserted upstream of the first coding exon on the coding strand during genetic modification, or the genetic modification results in a synthetic first coding exon produced by completing the gene editing process, wherein the heterologous promoter is upstream of the synthetic first coding exon. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line is a cell expressing CD4+. In some alternatives, the cell line is a cell expressing CD8+. In some alternatives, the cell line is a regulatory T cell. In some alternatives, the genetically engineered cell for expressing FOXP3 comprises a nucleic acid, wherein the nucleic acid comprises a coding strand, wherein the coding strand comprises one or more regulatory elements operably linked to a FOXP3 gene and a heterologous promoter, wherein the FOXP3 gene comprises the first coding exon. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the constitutive promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon. In some alternatives, the nucleic acid further comprises a heterologous transcription enhancer domain. In some alternatives, the nucleic acid further comprises a heterologous transcription activation domain. In some alternatives, the nucleic acid further comprises a ubiquitous chromatin opening element (UCOE). In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the one or more regulatory elements are genetically modified. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line is a regulatory T cell. In some alternatives, the autoimmune disease is rheumatoid arthritis, diabetes, inflammatory bowel disease, IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome), Crohn's disease, multiple sclerosis, idiopathic or systemic lupus erythematosus. In some alternatives, an individual is identified or selected to receive therapy for the autoimmune disease. In some alternatives, a combination therapy is administered to the individual, wherein the individual is administered a drug used to suppress the autoimmune disease in combination with the engineered cells.In some alternatives, changes in the FOXp3+ Treg phenotype cell population in vivo are monitored during the course of treatment. In some alternatives, the subject is selected for re-administration of the engineered cells. In some alternatives, the subject is given combination therapy, wherein the subject is administered an anti-rejection drug or anti-inflammatory drug in combination with the engineered cells. In some alternatives, the subject is selected for anti-rejection medication. In some alternatives, anti-rejection medications include Prilosec, Imuran (azathioprine), Mycophenolate Mofetil (MMF), Mycophenolic Acid, Sirolimus, Cyclosporine, or Tacrolimus. In some alternatives, anti-inflammatory medications include Tysabri®, steroids, immunosuppressants, NSAIDs, immunoglobulins, acetaminophen, celecoxib, indomethacin, or diclofenac. In some alternatives, changes in engineered T cells expressing FOXp3+, engineered cell populations exhibiting a Treg phenotype, or pathogenic T or B cell populations are monitored in the subject before, during, and / or after treatment, suppression, or amelioration. In some alternatives, depending on the initial response to treatment, suppression, or amelioration, the subject is selected for re-administration of the engineered cells. In some alternatives, the subject is administered another dose of cells within one week, two weeks, or one month of the initial dose. In some alternatives, the subject is refractory to standard therapies for autoimmune diseases or standard therapies for anti-inflammatory therapy. In some alternatives, the subject is refractory to standard anti-rejection agents or standard anti-rejection therapies. Therefore, in some alternatives, the subject is selected to receive genetically engineered cells expressing FOXP3. [] In some alternatives, methods are provided for treating, inhibiting, or ameliorating side effects of organ transplantation in an individual, comprising administering to the individual a genetically modified cell comprising any one or more of the cells described in any of the alternatives herein or a composition provided in any of the alternatives. Compositions are also provided comprising any one or more of the genetically engineered cells described in any of the alternatives herein and a pharmaceutical excipient. Genetically engineered cells expressing FOXP3 can be produced by the methods provided in any of the alternatives herein. Methods are also provided for preparing genetically engineered cells, comprising providing a cell comprising a first nucleotide sequence comprising a coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; and introducing the second oligonucleotide or protein nuclease into the cell for a gene editing procedure. In some alternatives, the gene editing procedure is a knock-in procedure in which a heterologous promoter is inserted into the first nucleotide sequence. In some alternatives, the coding strand comprises a first coding exon. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus comprises a native coding exon or the first coding exon. In some alternatives, completion of the gene editing process produces the first coding exon in the targeted locus after the gene editing process. In some alternatives, the targeted locus is within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110 base pairs upstream of the first coding exon, or any number of base pairs within a range defined by any two of the above values. In some alternatives, the gene editing process further comprises producing a synthetic first coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the heterologous promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the method further comprises inserting a heterologous enhancer domain (e.g., a TCRα enhancer) into the first nucleotide sequence, wherein the enhancer domain is upstream or downstream of one or more regulatory sequences. In some alternatives, the method further comprises inserting a heterologous transcriptional activation domain into the first nucleotide sequence. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE) into the first nucleotide sequence.In some alternatives, the heterologous promoter is inserted upstream of the first coding exon on the coding strand during genetic modification, or the genetic modification results in a synthetic first coding exon produced by completing the gene editing process, wherein the heterologous promoter is upstream of the synthetic first coding exon. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line is a cell expressing CD4+. In some alternatives, the cell line is a cell expressing CD8+. In some alternatives, the cell line is a regulatory T cell. In some alternatives, the genetically engineered cell for expressing FOXP3 comprises a nucleic acid, wherein the nucleic acid comprises a coding strand, wherein the coding strand comprises one or more regulatory elements operably linked to a FOXP3 gene and a heterologous promoter, wherein the FOXP3 gene comprises the first coding exon. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the constitutive promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon. In some alternatives, the nucleic acid further comprises a heterologous transcription enhancer domain. In some alternatives, the nucleic acid further comprises a heterologous transcription activation domain. In some alternatives, the nucleic acid further comprises a ubiquitous chromatin opening element (UCOE). In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the one or more regulatory elements are genetically modified. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line is a regulatory T cell. In some alternatives, a subject is administered a combination therapy, wherein the subject is administered an anti-rejection drug in combination with engineered cells. In some alternatives, the subject is selected to receive an anti-rejection agent. In some alternatives, the anti-rejection agent includes Prilosec, Imuran (azathioprine), Mycophenolate Mofetil (MMF), Mycophenolic Acid, Sirolimus, Cyclosporine, or Tacrolimus. In some alternatives, the subject's in vivo FOXp3+ Treg- phenotype cell population is monitored during the course of treatment.In some alternatives, the individual is selected to receive a second administration of the engineered cells. In some alternatives, the individual in need is selected to receive anti-rejection therapy or anti-inflammatory therapy. In some alternatives, the method further comprises determining that the engineered cells are in the individual in need by sequencing the genetically engineered cells to determine whether the engineered cells are maintained, increased, or decreased in the individual. In some alternatives, the individual is administered a combination therapy, wherein the individual is administered an anti-rejection or anti-inflammatory drug in combination with the engineered cells. In some alternatives, the individual is selected to receive an anti-rejection medication. In some alternatives, the anti-rejection medication includes Prilosec, Imuran (azathioprine), Mycophenolate Mofetil (MMF), Mycophenolic Acid, Sirolimus, Cyclosporine, or Tacrolimus. In some alternatives, the anti-inflammatory drug includes Tysabri®, steroids, immunosuppressants, NSAIDs, immunoglobulins, acetaminophen, celecoxib, indomethacin, or diclofenac. In some alternatives, the engineered T cells expressing FOXp3+, engineered cell populations exhibiting a Treg phenotype, or pathogenic T or B cell populations are monitored in the subject before, during, and / or after the treatment, suppression, or improvement process. In some alternatives, the subject is selected for re-administration of the engineered cells based on the initial response to treatment, suppression, or improvement. In some alternatives, the subject is administered another dose of cells within one week, two weeks, or one month of the initial dose. In some alternatives of the treatment methods described herein, the methods further include monitoring the subject to determine whether the engineered cells are maintained, increased, or decreased before, during, and / or after treatment. In some alternatives, a cell sample is removed from the subject to detect the engineered cells and determine whether the engineered cells are maintained, increased, or decreased. In some alternatives, if the engineered cells decrease in a subject, the engineered cells are re-administered to the subject in need. In some alternatives, the subject is refractory to standard therapies for autoimmune diseases or anti-inflammatory therapies. In some alternatives, the subject is refractory to standard anti-rejection agents or standard anti-rejection therapies. Therefore, in some alternatives, the subject is selected to receive genetically engineered cells expressing FOXP3. In some alternatives, methods are provided for treating, inhibiting, or ameliorating side effects of organ transplantation (e.g., organ rejection) in a subject, comprising administering to the subject a genetically modified cell comprising any one or more of the cells described in any of the alternatives herein or a composition provided in any of the alternatives. Compositions are also provided comprising any one or more of the genetically engineered cells described in any of the alternatives herein and a pharmaceutical excipient. Genetically engineered cells expressing FOXP3 can be produced by the methods provided in any of the alternatives herein. Methods are also provided for preparing genetically engineered cells, comprising providing a cell comprising a first nucleotide sequence comprising a coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; and introducing the second oligonucleotide or protein nuclease into the cell for a gene editing procedure. In some alternatives, the gene editing procedure is a knock-in procedure in which a heterologous promoter is inserted into the first nucleotide sequence. In some alternatives, the coding strand comprises a first coding exon. In some alternatives, the first nucleotide sequence comprises a targeted locus. In some alternatives, the targeted locus comprises a native coding exon or a first coding exon. In some alternatives, completion of the gene editing process generates the first coding exon in the targeted locus after the gene editing process. In some alternatives, the targeted locus is within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110 base pairs upstream of the first coding exon, or any number of base pairs within a range defined by any two of the above values. In some alternatives, the gene editing process further comprises generating a synthetic first coding exon. In some alternatives, the nuclease is Cas9, a zinc finger nuclease, or a TALEN. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the heterologous promoter is an EF1α promoter, a PGK promoter, or an MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the method further comprises inserting a heterologous enhancer domain (e.g., a TCRα enhancer) into the first nucleotide sequence, wherein the enhancer domain is upstream or downstream of one or more regulatory sequences. In some alternatives, the method further comprises inserting a heterologous transcriptional activation domain into the first nucleotide sequence. In some alternatives, the method further comprises inserting a ubiquitous chromatin opening element (UCOE) into the first nucleotide sequence.In some alternatives, the heterologous promoter is inserted upstream of the first coding exon on the coding strand during genetic modification, or the genetic modification results in a synthetic first coding exon produced by completing the gene editing process, wherein the heterologous promoter is upstream of the synthetic first coding exon. In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the method further comprises genetically modifying the one or more regulatory elements. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line is a cell expressing CD4+. In some alternatives, the cell line is a cell expressing CD8+. In some alternatives, the cell line is a regulatory T cell. In some alternatives, the genetically engineered cell for expressing FOXP3 comprises a nucleic acid, wherein the nucleic acid comprises a coding strand, wherein the coding strand comprises one or more regulatory elements operably linked to a FOXP3 gene and a heterologous promoter, wherein the FOXP3 gene comprises the first coding exon. In some alternatives, the heterologous promoter is a constitutive promoter. In some alternatives, the constitutive promoter is the EF1α promoter, the PGK promoter, or the MND promoter. In some alternatives, the heterologous promoter is a weak promoter (e.g., a promoter that produces less expression than an endogenous promoter and / or a constitutive promoter). In some alternatives, the heterologous promoter is an inducible promoter. In some alternatives, the inducible promoter can be induced by a drug or steroid. In some alternatives, the heterologous promoter is inserted upstream of the first coding exon. In some alternatives, the nucleic acid further comprises a heterologous transcription enhancer domain. In some alternatives, the nucleic acid further comprises a heterologous transcription activation domain. In some alternatives, the nucleic acid further comprises a ubiquitous chromatin opening element (UCOE). In some alternatives, the heterologous promoter is inserted downstream of one or more regulatory elements on the coding strand. In some alternatives, the one or more regulatory elements are genetically modified. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line is a regulatory T cell. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line is a regulatory T cell. In some alternatives, a combination therapy is administered to a subject, wherein the subject is administered an anti-rejection drug in combination with the engineered cells. In some alternatives, the subject is selected to receive the anti-rejection drug.In some alternatives, the anti-rejection agent includes Prysone, Imuran (azathioprine), Mycophenolate Mofetil (MMF), Mycophenolic Acid (mycophenolic acid), Sirolimus (sirolimus), Cyclosporine (cyclosporine), or Prograf (tacrolimus). In some alternatives, the individual is monitored for changes in the population of FOXp3+ Treg- phenotype cells in vivo during the course of treatment. In some alternatives, the individual is selected for re-administration of the engineered cells. In some alternatives, the individual in need is selected for anti-rejection therapy or anti-inflammatory therapy. In some alternatives, the method further comprises determining the presence of the engineered cells in the individual in need by sequencing the genetically engineered cells to determine whether the engineered cells are maintained, increased, or decreased in the individual. In some alternatives, the individual is selected for receiving the anti-rejection agent. In some alternatives, anti-rejection agents include Prysone, Imuran (azathioprine), Mycophenolate Mofetil (MMF), Mycophenolic Acid, Sirolimus, Cyclosporine, or Tacrolimus. In some alternatives, anti-inflammatory drugs include Tysabri®, steroids, immunosuppressants, NSAIDs, immunoglobulins, paracetamol, celecoxib, indomethacin, or diclofenac. In some alternatives, changes in engineered T cells expressing FOXp3+, engineered cell populations exhibiting a Treg phenotype, or pathogenic T or B cell populations are monitored in the subject before, during, and / or after the treatment, suppression, or improvement process. In some alternatives, depending on the initial response to treatment, suppression, or improvement, the subject is selected for re-administration of the engineered cells. In some alternatives, the subject is administered another dose of cells within one week, two weeks, or one month of the initial dose. In some alternatives to the treatment methods described herein, the methods further comprise monitoring the individual to determine whether the engineered cells are maintained, increased, or decreased before, during, and / or after the first administration of the cells. In some alternatives, a cell sample is removed from the individual to detect the engineered cells and determine whether the engineered cells are maintained, increased, or decreased. In some alternatives, if the engineered cells decrease in the individual, the engineered cells are administered again to the individual in need. In some alternatives, the individual is refractory to standard therapies for autoimmune diseases or standard therapies for anti-inflammatory therapies. In some alternatives, the individual is refractory to standard anti-rejection agents or standard anti-rejection therapies. Therefore, in some alternatives, the individual is selected to receive genetically engineered cells expressing FOXP3. In some alternatives, methods are provided for treating, suppressing, or ameliorating an autoimmune disorder in a subject. The methods comprise removing cells from a subject in need thereof, wherein the cells comprise a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements, a FOXP3 gene or portion thereof, wherein the FOXP3 gene or portion thereof comprises a first coding exon; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; introducing the second nucleotide sequence or protein nuclease into the cell for genetic modification, wherein the genetic modification is a knock-in procedure that inserts a promoter into the first nucleotide sequence; and introducing the cell into the subject for treatment, suppression, or amelioration. In some alternatives, the autoimmune disorder is rheumatoid arthritis, diabetes, inflammatory bowel disease, IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome), Crohn's disease, multiple sclerosis, idiopathic or systemic lupus erythematosus. In some alternatives, the subject is identified or selected to receive therapy for the autoimmune disorder. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line expresses CD4+ cells. In some alternatives, the cell line expresses CD8+ cells. In some alternatives, the cell line expresses regulatory T cells. In some alternatives, a subject is administered combination therapy, wherein the subject is administered a drug used to ameliorate symptoms of an autoimmune disease in combination with the engineered cells. In some alternatives, the subject is selected to receive an anti-rejection agent. In some alternatives, the anti-rejection agent includes Prilosec, Imuran (azathioprine), Mycophenolate Mofetil (MMF), Mycophenolic Acid, Sirolimus, Cyclosporine, or Tacrolimus. In some alternatives, the subject is monitored for changes in the FOXp3+ Treg- phenotype cell population during the course of treatment. In some alternatives, the subject is selected to receive a second administration of the engineered cells. In some alternatives, the subject in need is selected to receive anti-rejection therapy or anti-inflammatory therapy. In some alternatives, the method further comprises determining that the engineered cells are present in the subject in need by sequencing the genetically engineered cells to determine whether the engineered cells are maintained, increased, or decreased in the subject. In some alternatives, the subject is selected to receive an anti-rejection medication. In some alternatives, the anti-rejection medication includes Prilosec, Imuran (azathioprine), Mycophenolate Mofetil (MMF), Mycophenolic Acid, Sirolimus, Cyclosporine, or Tacrolimus. In some alternatives, the anti-inflammatory medication includes Tysabri®, steroids, immunosuppressants, NSAIDs, immune globulins, acetaminophen, celecoxib, indomethacin, or diclofenac.In some alternatives, the individual's in vivo engineered T cells expressing FOXp3+, engineered cell populations exhibiting a Treg phenotype, or pathogenic T or B cell populations are monitored before, during, and / or after the course of treatment. In some alternatives, the individual is selected for a second dose of the engineered cells, depending on the initial response to treatment. In some alternatives, the individual is administered another dose of cells within one week, two weeks, or one month of the initial dose. In some alternatives of the treatment methods described herein, the methods further comprise monitoring the individual to determine whether the engineered cells are maintained, increased, or decreased before, during, and / or after treatment. In some alternatives, a cell sample is removed from the individual to detect the engineered cells and determine whether the engineered cells are maintained, increased, or decreased. In some alternatives, if the engineered cells decrease in the individual, the engineered cells are administered again to the individual in need. In some alternatives, the individual is refractory to standard therapy for an autoimmune disease or standard therapy for an anti-inflammatory disease. In some alternatives, the individual is refractory to standard anti-rejection medications or standard anti-rejection therapy. Thus, in some alternatives, the individual is selected to receive genetically engineered cells expressing FOXP3. In some alternatives, methods are provided for treating, inhibiting, or ameliorating the side effects of organ transplantation in an individual. The methods comprise removing cells from an individual in need thereof, wherein the cells comprise a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements, a FOXP3 gene or portion thereof, wherein the FOXP3 gene or portion thereof comprises a first coding exon; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; introducing the second nucleotide sequence or protein nuclease into the cells for genetic modification, wherein the genetic modification is a knock-in procedure that inserts a promoter into the first nucleotide sequence; and introducing the cells into the individual for treatment. In some alternatives, the cells are precursor stem cells. In some alternatives, the cells are hematopoietic stem cells. In some alternatives, the cells are CD4+ expressing cells. In some alternatives, the cells are CD8+ expressing cells. In some alternatives, the cells are regulatory T cells. In some alternatives, a combination therapy is administered to the individual, wherein the individual is administered an anti-rejection drug in combination with engineered cells. In some alternatives, the subject is selected to receive an anti-rejection agent. In some alternatives, the anti-rejection agent includes Prysone, Imuran (azathioprine), Mycophenolate Mofetil (MMF), Mycophenolic Acid, Sirolimus, Cyclosporine, or Tacrolimus. In some alternatives, the subject's in vivo population of FOXp3+ Treg- phenotype cells is monitored during treatment, suppression, or improvement. In some alternatives, the subject is selected to receive a second administration of the engineered cells. In some alternatives, the subject in need is selected to receive anti-rejection therapy or anti-inflammatory therapy. In some alternatives, the method further comprises determining the presence of the engineered cells in the subject in need by sequencing the genetically engineered cells to determine whether the engineered cells are maintained, increased, or decreased in the subject. In some alternatives, the subject is selected to receive an anti-rejection agent. In some alternatives, anti-rejection agents include Prysone, Imuran (azathioprine), Mycophenolate Mofetil (MMF), Mycophenolic Acid, Sirolimus, Cyclosporine, or Tacrolimus. In some alternatives, anti-inflammatory drugs include Tysabri®, steroids, immunosuppressants, NSAIDs, immunoglobulins, paracetamol, celecoxib, indomethacin, or diclofenac. In some alternatives, changes in engineered T cells expressing FOXp3+, engineered cell populations exhibiting a Treg phenotype, or pathogenic T or B cell populations are monitored in the subject before, during, and / or after the treatment, suppression, or amelioration process. In some alternatives, depending on the initial response to treatment, the subject is selected for re-administration of the engineered cells. In some alternatives, the subject is administered another dose of cells within one week, two weeks, or one month of the initial dose.In some alternatives to the methods of treatment, inhibition, or amelioration described herein, the methods further comprise monitoring the individual to determine whether the engineered cells are maintained, increased, or decreased before, during, and / or after treatment. In some alternatives, a cell sample is removed from the individual to detect the engineered cells and determine whether the engineered cells are maintained, increased, or decreased. In some alternatives, if the engineered cells decrease in the individual, the engineered cells are re-administered to the individual in need. In some alternatives, the individual is refractory to standard therapies for autoimmune diseases or standard therapies for anti-inflammatory therapies. In some alternatives, the individual is refractory to standard anti-rejection agents or standard anti-rejection therapies. Therefore, in some alternatives, the individual is selected to receive genetically engineered cells expressing FOXP3. In some alternatives, methods are provided for treating, inhibiting, or ameliorating side effects of organ transplantation (e.g., organ rejection) in a subject. The methods comprise removing cells from a subject in need thereof, wherein the cells comprise a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand comprising one or more regulatory elements, a FOXP3 gene or portion thereof, wherein the FOXP3 gene or portion thereof comprises a first coding exon; providing a second nucleotide sequence encoding a nuclease or providing a protein nuclease; introducing the second nucleotide sequence or protein nuclease into the cell for genetic modification, wherein the genetic modification is a knock-in procedure that inserts a promoter into the first nucleotide sequence; and introducing the cell into the subject for treatment. In some alternatives, the cell line is a precursor stem cell. In some alternatives, the cell line is a hematopoietic stem cell. In some alternatives, the cell line is a cell expressing CD4+. In some alternatives, the cell line is a cell expressing CD8+. In some alternatives, the cell line is a regulatory T cell. In some alternatives, a combination therapy is administered to the subject, wherein the subject is administered an anti-rejection drug in combination with the engineered cells. In some alternatives, the individual is selected to receive an anti-rejection agent. In some alternatives, the anti-rejection agent includes Prysone, Imuran (azathioprine), Mycophenolate Mofetil (MMF), Mycophenolic Acid, Sirolimus, Cyclosporine, or Tacrolimus. In some alternatives, the individual's in vivo population of FOXp3+ Treg- phenotype cells is monitored during the course of treatment. In some alternatives, the individual is selected to receive a second administration of the engineered cells. In some alternatives, the individual in need is selected to receive anti-rejection therapy or anti-inflammatory therapy. In some alternatives, the method further comprises determining the presence of the engineered cells in the individual in need by sequencing the genetically engineered cells to determine whether the engineered cells are maintained, increased, or decreased in the individual. In some alternatives, the individual is selected to receive an anti-rejection agent. In some alternatives, anti-rejection agents include Prysone, Imuran (azathioprine), Mycophenolate Mofetil (MMF), Mycophenolic Acid, Sirolimus, Cyclosporine, or Tacrolimus. In some alternatives, anti-inflammatory drugs include Tysabri®, steroids, immunosuppressants, NSAIDs, immunoglobulins, paracetamol, celecoxib, indomethacin, or diclofenac. In some alternatives, changes in engineered T cells expressing FOXp3+, engineered cell populations exhibiting a Treg phenotype, or pathogenic T or B cell populations are monitored in the subject before, during, and / or after the treatment, suppression, or amelioration process. In some alternatives, the subject is selected for a second dose of the engineered cells based on the initial response to the first administration of the cells. In some alternatives, the subject is administered another dose of the cells within one week, two weeks, or one month of the initial dose.In some alternatives of the treatment methods described herein, the methods further include monitoring the individual to determine whether the cells maintain, increase, or decrease before, during, and / or after receiving the engineered cells. In some alternatives, a cell sample is taken from the individual to detect the engineered cells and determine whether the engineered cells maintain, increase, or decrease. In some alternatives, if the engineered cells decrease in the individual, the engineered cells are administered again to the individual in need. In some alternatives, the individual is difficult to treat with standard therapies for autoimmune diseases or standard therapies for anti-inflammatory therapies. In some alternatives, the individual is difficult to treat with standard anti-rejection agents or standard anti-rejection therapies. Therefore, in some alternatives, the individual is selected to receive genetically engineered cells expressing FOXP3.

Claims

1. A method for preparing engineered cells, the method comprising contacting the cells with: (a) a first nucleic acid containing a heterologous constitutive promoter; and (b) an endonuclease or a second nucleic acid encoding the endonuclease, wherein the endonuclease cleaves a target locus within or upstream of a first coding exon of the FOXP3 gene on the nucleic acid of the cell genome, wherein the cell is a T cell, hematopoietic stem cell, or progenitor stem cell, wherein the heterologous constitutive promoter is inserted into the nucleic acid of the cell genome downstream of a Treg-specific demethylation region (TSDR), the TSDR being located on the nucleic acid of the cell genome, wherein the inserted heterologous constitutive promoter is operatively linked to the FOXP3 gene, and wherein: (a) The heterologous constitutive promoter is inserted upstream of the natural first coding exon of the FOXP3 gene; or (b) the insertion of the heterologous constitutive promoter produces a synthetic first coding exon, wherein the heterologous constitutive promoter is upstream of the first coding exon.

2. The method of claim 1, wherein the target locus is located within 110 consecutive nucleotides upstream of the natural first coding exon.

3. The method of claim 1, wherein the target locus is located at the natural first coding exon.

4. The method of claim 1, wherein the nuclease in the DNA is a Cas9 nuclease, a zinc finger nuclease, or a TALEN.

5. The method of claim 1, wherein the first nucleic acid further comprises a heterologous transcription enhancer domain and / or a ubiquitous chromatin opening element (UCOE).

6. The method of request item 1, wherein the heterogeneous constitutive promoter is an EF1 α promoter, a PGK promoter, or an MND promoter.

7. The method of request item 1, wherein the heterogeneous compositional promoter is an MND promoter.

8. The method of request item 1, wherein the cell is a T cell.

9. The method of claim 1, wherein the cell is a CD8+ T cell or a CD4+ T cell.

10. The method of claim 1, wherein the engineered cell expresses a chimeric antigen receptor (CAR).

11. The method of claim 1, wherein the engineered cell expresses a T-cell receptor (TCR).

12. An engineered cell prepared by any one of claims 1 to 11.

13. An engineered cell comprising a heterologous constitutive promoter of a FOXP3 gene operatively linked to a nucleic acid in the cell genome, wherein the heterologous constitutive promoter is downstream of (i) a Treg-specific demethylation region (TSDR) and (ii) upstream of the first coding exon of the FOXP3 gene, wherein the engineered cell is a T cell, a hematopoietic stem cell, or a progenitor stem cell.

14. The engineered cell of claim 13, wherein the heterologous constitutive promoter is inserted into 110 consecutive nucleotides upstream of the first coding exon.

15. The engineered cell of claim 13, wherein the heterologous constitutive promoter is the EF1α promoter, the PGK promoter, or the MND promoter.

16. The engineered cell of claim 13, wherein the heterologous constitutive promoter is the MND promoter.

17. The engineered cell of claim 13, wherein the engineered cell is a T cell.

18. The engineered cells of claim 13, wherein the engineered cells are CD4+ T cells.

19. The engineered cells of claim 13, wherein the engineered cells express a chimeric antigen receptor (CAR).

20. The engineered cells of claim 13, wherein the engineered cells express the T cell receptor (TCR).

21. A pharmaceutical composition comprising engineered cells as claimed in any one of claims 13 to 20, and a pharmaceutically acceptable excipient.

22. Use of an engineered cell as claimed in any one of claims 13 to 20, for the manufacture of a pharmaceutical agent for the treatment, suppression and / or improvement of a condition in an individual, wherein the condition is selected from: autoimmune diseases, graft-versus-host disease or side effects of organ transplantation.

23. As requested in claim 22, wherein the condition is an autoimmune disease.

24. As requested in claim 23, wherein the autoimmune disease is diabetes.

25. As requested in claim 23, wherein the autoimmune disease is inflammatory bowel disease.

26. As requested in claim 23, wherein the autoimmune disease is Crohn's disease.

27. As requested in claim 23, wherein the autoimmune disease is ulcerative colitis.

28. As requested in claim 23, wherein the autoimmune disease is systemic lupus erythematosus.

29. As requested in claim 23, wherein the autoimmune disease is multiple sclerosis.

30. As requested in claim 23, wherein the autoimmune disease is rheumatoid arthritis.

31. As requested in item 22, wherein the condition is a side effect of organ transplantation.

32. As used in claim 22, wherein the disease is graft-versus-host disease.

33. As claimed in claim 22, wherein the engineered cell is an autologous cell.

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