Engineered extrachromosomal nucleic acids and methods of use thereof

Abstract

The present disclosure provides an extrachromosomal nucleic acid (e.g., DNA or RNA) tether system comprising nucleic acid (e.g., DNA or RNA) binding proteins to tether the extrachromosomal nucleic acid (e.g., DNA or RNA) to a specific site in the host chromosome. Further provided herein are methods for engineering a cell to have stable expression of a target payload without integration.

Description

[0001] DESCRIPTION

[0002] ENGINEERED EXTRACHROMOSOMAL NUCLEIC ACIDS AND METHODS OF

[0003] USE THEREOF

[0004] PRIORITY CLAIM

[0005] This application claims benefit of priority to U.S. Provisional Application Serial No. 63 / 600,876, filed November 20, 2023, the entire contents of which are hereby incorporated by reference.

[0006] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0007] This invention was made with government support under Grant Nos. R21EB030772, R35GM143532, and R01EB036003 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] SEQUENCE LISTING

[0009] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on November 20, 2024, is named RICEP0 I SOWO.xml and is 33,654 bytes in size.

[0010] BACKGROUND

[0011] 1. Field

[0012] The disclosure relates generally to the field of gene and cell therapy and molecular biology. More particularly, it concerns engineered extrachromosomal DNA and nonintegrating vectors and methods of use thereof.

[0013] 2. Related Art

[0014] Current strategies to engineer human cell-based therapeutics rely upon the delivery and subsequent genomic integration of transgenic payloads. Although these approaches have catalyzed transformative medical advances, the integration of transgenic DNA permanently disrupts natural genomic sequences and can lead to unexpected and even hazardous consequences. In addition, integrated transgenic DNA is often unpredictably expressed and is prone to epigenetic silencing over time, especially within primary human immune cells. Furthermore, existing approaches to validate large transgenic genomically-integrated DNA cargoes are inefficient and costly. These critical barriers limit the extent to which human cells can be repurposed and engineered as cell-based therapeutics, and these challenges are preventing biotechnological and clinical innovations. Non-integrating, double-stranded DNA viruses have evolved sophisticated solutions to these critical barriers, and their genomes can stably persist within human cells as circularized self-contained episomes across cellular divisions and for the lifetime of infected hosts. These viruses accomplish this remarkable persistence by tailoring their own gene expression patterns, synchronizing their genomic replication, and by reshaping the endogenous transcriptional networks of host cells. Genomic integration is the conventional solution to express exogenous or transgenic payloads in engineered cells in the long term. Current strategies to engineer human cell-based therapeutics rely on the delivery and subsequent genomic integration of transgenic payloads. Currently, non-integrating viral vectors, like adenovirus, are only capable of transient expression of transgenes. Other technologies using partially non-integrating viral vectors, like adeno-associated viral vectors (AAV), rely on limited integration into the AAVS1 and other loci for permanent expression. Thus, there is an unmet need for methods of stable transgene expression without any integration into the host genome.

[0015] SUMMARY In a first embodiment, the present disclosure provides an extrachromosomal nucleic acid (e.g., DNA or RNA) tether system comprising an episomal vector encoding (a) a first nucleic acid binding protein and a chromosomal specific target sequence; (b) a second nucleic acid-binding protein and an episomal specific target sequence; and (c) a target transgene. In certain aspects, the nucleic acid is DNA or RNA. In some aspects, the nucleic acid-binding protein is a DNA-binding protein and / or RNA-binding protein. In some aspects, the target transgene encodes a genetic circuit, kill switch, or therapeutic agent. The target transgene can be delivered to cells to engineer long-lasting, effective cell therapies. For example, the target transgene could encode an inducible expression cassette of a therapeutic molecule (e.g., IL-2 for immune cells, or BDNF for primary neurons), acting as a basic sense-and-response transcriptional gene circuit. In certain aspects, the first nucleic acid-binding protein is attached to the second nucleic acid -binding protein through a linker or fused to an inducible dimerized domain. In certain aspects, the first nucleic acid (e.g., DNA or RNA)-binding protein is attached to the second nucleic acid (e.g., DNA or RNA)-binding protein through a linker or fused to an inducible dimerized domain. In some aspects, the linker is a (G4S)ylinker, such as (G4S)2, (G4S)3, (G4S)6, (G4S)9, or (G4S)12, the Xten16, or the Xten80 linker. For example, the inducible dimerized domain may comprise rapamycin or abscisic acid. In certain aspects, the vector further comprises a human origin of DNA replication. In some aspects, the chromosomal specific target sequence is specific to a chromosomal locus (e.g., beta-globin or AAVS1) or repetitive element (e.g., Alu repeat, tandem repeat, short interspersed nuclear element (SINE)), long interspersed nuclear element (LINE), or telomeric repeat). In certain aspects, the system comprises a first nucleic acid-binding protein and / or a second nucleic acid-binding (e.g., DNA-binding and / or RNA-binding) protein selected from the group consisting of a Cas9 protein, Cas13 protein, transcription activator-like effector nuclease (TALEN), and zinc finger protein (ZFP). For example, the first DNA-binding protein is Streptococcus pyogenes deactivated Cas9 (SpdCas9) and the second DNA-binding protein is Staphylococcus aureus deactivated Cas9 (SadCas9). The first and second DNA-binding proteins may be ZFPs. In particular aspects, the chromosomal specific target sequence and episomal specific target sequence are further defined as guide RNAs. In some aspects, the guide RNAs are under the control of two or more separate promoters. In specific aspects, the promoters are RNA polymerase III (pol III) type 3 promoters, such as 7sK pol III promoter and U6 pol III promoter. In additional aspects, the vector further comprises a safety switch to control the linkage between the first nucleic acid-binding protein and second nucleic acid binding protein (e.g., dCas9), such as inducible caspase 9 (iCasp9), CD20, HSV-TK, as well as an episome-targeting inducibly expressed active Cas9. In some aspects, the vector further comprises a coding region for a detectable moiety, such as a fluorescent marker. In certain aspects, the vector further comprises a coding region for a replicon, such as LaminB2 replicon, C-Myc, or a site-specific dCas9-based replicator. In certain aspects, the episomal vector is delivered by an adenovirus, integrase deficient lentivirus, baculovirus, herpesvirus, coronavirus, or sendai virus. A further embodiment provides a method of engineering a cell to express a target transgene comprising transducing said cell with an extrachromosomal nucleic acid (e.g., DNA or RNA) tether system of the present embodiments and aspects thereof (e.g., an extrachromosomal nucleic acid (e.g., DNA or RNA) tether system comprising an episomal vector encoding (a) a first nucleic acid-binding (e.g., DNA-binding and / or RNA-binding) and a chromosomal specific target sequence; (b) a second nucleic acid-binding (e.g., DNA-binding and / or RNA-binding) and an episomal specific target sequence; and (c) a target transgene). In some aspects, the cell is an immune cell, T cell (e.g., primary T cell), NK cell, mesenchymal stem cell, or neuron. Another embodiment provides a method for delivering a gene therapy in a subject comprising introducing to said subject a plurality of cells engineered to express an extrachromosomal nucleic acid (e.g., DNA or RNA) tether system of the present embodiments and aspects thereof (e.g., an extrachromosomal nucleic acid (e.g., DNA or RNA) tether system comprising an episomal vector encoding (a) a first nucleic acid-binding (e.g., DNA-binding and / or RNA-binding) and a chromosomal specific target sequence; (b) a second nucleic acid- binding (e.g., DNA-binding and / or RNA-binding) and an episomal specific target sequence; and (c) a target transgene). Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. FIGS.1A-1F: (FIG.1A) A schematic showing the dCas9 tether. dCas9-based tethering systems that permit site-specific tethering of extrachromosomal DNA to the human genome without DSBs nor integration. The Sa. dCas9 targets the inserted episome and the Sp. dCas9 targets the host genome. The two proteins are connected by a linker, causing co-localization of the episome at the targeted genomic site and subsequent mitotic stability. (FIG.1B) Schematics of vectors used to test the binding ability of the tethering system made up of a fused Sa. dCas9 and Sp. dCas9 with an HA tag at the N terminus and a flag-tag at the C-terminus in ChIP- qPCR. A guide RNA cassette encodes gRNAs for the Sa. dCas9 under a 7sK pol III promoter and for the Sp. dCas9 under a U6 pol III promoter. (FIGS. 1C-1D) ChIP-qPCR demonstrating binding of SadCas9-SpdCas9 tethers to episomal plasmid (FIG. 1C) and genomic (FIG. 1D) human β-globulin locus) DNA, respectively. Both sides of the tether only bind when using the tether with corresponding gRNAs targeting the genome on one side and the episome on the other. HEK293T cells were transfected using lipofectamine with episomes containing a tether targeting the episome on one end and the beta-globin region of the human genome on the other. Antibodies against HA and Flag tag were used to show that the full tether bound to the episome and the beta-globin region at a similar level to HA-Sa. dCas9 and Flag-Sp. dCas9 respectively. (FIG.1E) Microscope images showing fluorescence in situ hybridization (FISH) in U2OS cells demonstrates colocalization between episomal and genomic DNA targets only in the presence of corresponding gRNAs. U2OS cells were transduced at a multiplicity of infection (MOI) of 5 with integrase-deficient lentivirus (IDLV) encoding an episome with a tether binding the episome on one end and the AAVS1 locus of the human genome on the other, or else a non- targeting tether. Fluorescence in situ hybridization (FISH) was performed using an AF-488 probe against the episome and an AF-594 probe against the AAVS1 locus to see if co- localization between the two pieces of DNA, and therefore between the green and red signals, would occur in the presence of a guide. (FIG. 1F) In nuclei containing both episome probe signal and AAVS1 probe signal, the percent of AAVS1 colocalized with episome was quantified. Increased co-localization in presence of targeting guides is quantified by counting the co-localization events in the microscopy photos. FIGS. 2A-2E: (FIG. 2A) Schematic showing the vector used in the following experiment, with a replication cassette, a gRNA cassette targeting the tether to repeat genomic elements and self-targeting the tether-encoding episome, a tethering cassette, and a cassette encoding an EGFP persistence metric. (FIG. 2B) dCas9-based tethers plus the LaminB2 replicon enables episomal maintenance out to 84 days with as few as ~20 (Msa) target sites, with a target number of approximately 3,000 (3e3) sites. (FIG. 2C) By performing FACs enrichment at day 17 for EGFP, there can be a permanent, long-term increase of the level of episomal persistence and the yield of episome-harboring cells, (FIG. 2D) which is confirmed by microscopy, which shows EGFP fade away in cells without LaminB2, but remain in cells containing LaminB2 and a tether targeting ~3,000 sites. (FIG. 2E) Persistent EGFP expression seen in C-D is not due to random episomal integration as measured using the Takara Provirus Quantitation kit. FIG. 3: The guide RNAs used to target the tether to the host genome were designed such that they target repeat regions that occur at various frequencies throughout the human genome, from once for AAVS1 to approximately 3e5 for Alu. FIG. 4: It was found that the presence of the LaminB2 human replicon in an episome, when transiently transfected into HEK293T cells, was sufficient to increase the episome retention to some extent in the dividing cell population. The C-Myc replicon did not appear to have any effect. FIG. 5: Optimization of expression of SadCas9-SpdCas9 tethering construct analyzed via Western Blot on flag and HA-tagged tethers. A panel of linkers between Sa and SpdCas9 reveals different levels of expression and the occurrence of cleavage in some constructs in HEK293T cells. For future experiments, the tether linker (G4S)3 was used as it demonstrated a high expression level and caused no visible cleavage. FIG. 6: Microscope images from the experiment in FIG. 2B revealing the retention of EGFP in cells containing LaminB2 and tether construct targeted to ~3,000 sites, but far less in cells with only LaminB2 without a tethering construct. FIG.7: Fluorescence in situ hybridization using a tether construct made with a smaller DNA binding domain fusion shows that this version of the tether is capable of causing co- localization between episome and the targeted site of the genome. This version of the tethering construct is small enough to be packaged into integrase deficient lentivirus (IDLV). FIGS. 8A-8C: Application of Extrachromosomal Genetic Technology in primary T- cells. (FIG.8A) Integration-deficient lentiviral vectors (IDLVs) were used containing integrase mutations that inhibit proviral integration and therefore leads to higher levels of circular vector episomes in the transduced cells (FIG. 8B) As IDLVs containing the LaminB2 replicon willhave limitations on the size of additional payloads, two different IDLVs (IDLV- and IDLV-were used to co-transduce primary T-cells. IDLV- harbors the compact version of the tether(SadCas9-linker-denOs) and IDLV- LaminB2. Both the IDLVs were co-transduced at 1:1 ratio in CD3 / CD28 activated primary T- cells. (FIG. 8C) After 96 hrs post co-transduction the expression levels of the surface expression marker was measured using Flow Cytometry using anti-Thy1.1 antibody. FIGS. 9A-9B: Overview of the IDLV constructs used in co-transduction. (FIG. 9A)Three different IDLV- constructs were used targeting different number of sites in the genome(gDen-Scr (n=0), gDen-AAVS1 (n=1) and gDen-Line1 (n=3000)). (FIG.9B) Two IDLV- constructs were used harboring the surface expression marker Thy1.1 (With LaminB2 and without LaminB2). A total number of 6 conditions were tested for co-transduction (gDen-Scr + No_LaminB2-Thy1.1, gDen-AAVS1 + NoLaminB2-Thy1.1, gDen-Line1 + NoLaminB2- Thy1.1, gDen-Scr + LaminB2_Thy1.1, gDen-AAVS1 + LaminB2_Thy1.1, gDen-Line1 + LaminB2_Thy1.1) and 6 conditions for control (gDen-Scr only, gDen-AAVS1 only, gDen- Line1 only, LaminB2-Thy1.1 only, NoLaminB2-Thy1.1 only, Untransduced). FIG. 10: Expression of Thy1.1 measured 6 days and 10 days post co-transduction in primary T-cells. The x-axis represents the different conditions of co-transduction and controls at Day 6 and Day 10 post co-transduction. The y-axis represents the percentage of Thy1.1 expression. It was observed that on both day 6 and day 10 post co-transduction constructs with LaminB2 (black box) and the tether have higher levels of Thy1.1 expression compared to the constructs without LaminB2. Therefore, the human replicon LaminB2 combined with the tether enables persistent expression of surface markers in primary T-cells up to 10 days in vitro. FIG. 11: Two different IDLVs (IDLV- and IDLV- were used to co-transduceprimary T-cells. IDLV- harbors the compact version of the tether (SadCas9-linker-denOs)with the surface expression marker Thy 1.1 and IDLV- an anti-CD19 CAR, thesurface expression marker NGFR, and LaminB2. Both the IDLVs were co-transduced at 1:1 ratio in CD3 / CD28 activated primary T-cells. 72 hours post co-transduction, the expression levels of the surface expression markers were measured using Flow Cytometry using anti- Thy1.1 and anti-NGFR antibodies. FIG. 12: The results of the flow cytometry experiment shown in FIG. 11 are depicted. These results indicate that 72 hours after co-transduction with IDLV, episomes expressing the anti-CD19 CAR with the LaminB2 replicon are retained at higher levels in expanding T cells compared to episomes expressing the anti-CD19 CAR without the LaminB2 replicon. FIGS. 13A-13D: Tet Repressor (TetR) Tether Design. (FIG. 13A) The SpdCas9-TetR tether in the absence (Dox-) and presence (Dox+) of doxycycline. (FIG. 13B) Vector map of the SpdCas9-TetR tether. (FIG. 13C)) FISH showing co-localization between targeted locus AAVS1 and episomal DNA (U2OS, IDLV MOI 2.5). (FIG.13D) Western blots of FLAG and HA to show both DNA-binding domains are expressed in HEK293T cells. FIGS. 14A-14B: Development of a Tether Consisting of Two Zinc Finger Proteins (ZFPs). (FIG. 14A) Proposed tether design consisting of two zinc finger proteins (ZFPs) with the EF1alpha intron-less form (EFS) promoter. One binds to a target sequence in the genome, while the other binds to an orthogonal target sequence on the episome. (FIG. 14B) General vector map of the proposed ZF-ZF tether.

[0016] DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS In comparison to previous technologies, the present compositions and methods solve the problem of achieving persistence and stability of gene regulatory network expression over long time scales without requiring genomic integration. Non-integrating and partially non- integrating approaches include plasmid transfection, adeno-associated virus (AAV) transduction, adenovirus transduction, herpesvirus transduction, and mRNA delivery of gene regulatory programs. These approaches cannot maintain stable, long-term expression of the delivered cassette. Given these are non-integrating methods of delivery, they do not divide along with the cell and will dilute over time as the foreign DNA is not transmitted equally among daughter cells. Integrating approaches include lentivirus transduction, CRISPR- mediated integration, and other viral methods of delivery (e.g., herpesvirus, adenovirus). While integrated transgenes are expressed over long periods of time, they can cause insertional mutagenesis and lead to undesired side effects, notably oncogene activation or tumor suppressor inactivation. Current episomal vector technologies derived from viral vectors (e.g., HPV, BPV, EEV, etc.) cannot control the tethering loci of episomal vectors, making it difficult to control the expression level and manipulate the episomal plasmid maintenance (e.g., copy number, ON / OFF switch). Accordingly, in certain embodiments, the present disclosure provides compositions and methods for stable long-term gene expression using non-integrating vectors which avoids unintended mutagenesis induced during gene expression. Compared to other episomal vectors derived from viral vectors or gene elements, the present compositions and methods provide site-specific programmable tethering sequences, such as via guide RNA (gRNA). The present methods can be applied to engineered human cells for diagnositic and therapeutic purposes as well as for exogenous protein expression in research and manufacturing. The present methods can enable persistent, controllable expression of genetic components across cell divisions without potentially disruptive genomic integration. This enables the application of sophisticated synthetic gene networks in human cells engineered for both therapeutic and industrial applications. The present methods can be used as a platform to engineer complex cell therapies including but not limited to sense-and-response circuits, cell differentiation control systems, and other feedback control genetic circuits that rely on multiple genetic components that either exceed the packaging capacity of current gene delivery technologies or require persistent expression of expression cassettes. I. Definitions As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods. As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one. The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more. The term “about” means, in general, within a standard deviation of the stated value as determined using a standard analytical technique for measuring the stated value. The terms can also be used by referring to plus or minus 5% of the stated value. The phrase “effective amount” or “therapeutically effective” means a dosage of a drug or agent sufficient to produce a desired result. The desired result can be subjective or objective improvement in the recipient of the dosage, increased lung growth, increased lung repair, reduced tissue edema, increased DNA repair, decreased apoptosis, a decrease in tumor size, a decrease in the rate of growth of cancer cells, a decrease in metastasis, or any combination of the above. “Subject” and “patient” refer to either a human or non-human, such as primates, mammals, and vertebrates. In particular embodiments, the subject is a human. As used herein, the terms "treat," "treatment," "treating," or "amelioration" when used in reference to a disease, disorder or medical condition, refer to therapeutic treatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a symptom or condition. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a condition is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized (i.e., not worsening) state of a tumor or malignancy, delay or slowing of tumor growth and / or metastasis, and an increased lifespan as compared to that expected in the absence of treatment. “Clustered Regularly Interspaced Short Palindromic Repeats” and “CRISPRs”, as used interchangeably herein refers to loci containing multiple short direct repeats that are found in the genomes of approximately 40% of sequenced bacteria and 90% of sequenced archaea. “Coding sequence” or “encoding nucleic acid” as used herein means the nucleic acids (RNA or DNA molecule) that comprise a nucleotide sequence which encodes a protein. The coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may be codon optimize. “Complement” or “complementary” as used herein means a nucleic acid can mean Watson-Crick (e.g., A-T / U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. “Complementarity” refers to a property shared between two nucleic acid sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary. “Endogenous gene” as used herein refers to a gene that originates from within an organism, tissue, or cell. An endogenous gene is native to a cell, which is in its normal genomic and chromatin context, and which is not heterologous to the cell. Such cellular genes include, e.g., animal genes, plant genes, bacterial genes, protozoal genes, fungal genes, mitochondrial genes, and chloroplastic genes. “Enhancer” as used herein refers to non-coding DNA sequences containing multiple activator and repressor binding sites. Enhancers range from 200 bp to 1 kb in length and may or distal, in introns of neighboring genes or intergenic regions far away from the locus. Through DNA looping, active enhancers contact the promoter dependently of the core DNA binding motif promoter specificity. For example, 4 to 5 enhancers may interact with a promoter. Similarly, enhancers may regulate more than one gene without linkage restriction and may “skip” neighboring genes to regulate more distant ones. Transcriptional regulation may involve elements located in a chromosome different to one where the promoter resides. Proximal enhancers or promoters of neighboring genes may serve as platforms to recruit more distal elements. “Genetic construct” as used herein refers to the DNA or RNA molecules that comprise a nucleotide sequence that encodes a protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered. As used herein, the term “expressible form” refers to gene constructs that contain the necessary regulatory elements operable linked to a coding sequence that encodes a protein such that when present in the cell of the individual, the coding sequence will be expressed. An “episomal vector” as used herein refers to a circular, extrachromosomal genetic construct similar to, and including, a plasmid that is able to persist within cells without genomic integration. Episomal vectors can attach to and replicate with chromosomal DNA during mitosis, avoiding dilution from cell division. Episomal vectors can consist of DNA or RNA. The stable copy number of episomal vectors within cells allows for long-term, consistent levels of expression of the encoded genetic payload. Episomal vectors can be of viral or non-viral origin. Certain types of viruses, including Epstein-Barr Virus (EBV), Hepatitis B Virus (HBV), and Adeno-Associated Virus (AAV), maintain their genomes within host cells in the form of an episomal vector. Various mechanisms of persistence, such as those based on scaffold / matrix attachment regions (S / MARs), may be used by a given episomal vector. In short, circular, extrachromosomal DNA or RNA molecules that reside in the host cell without integrating into the host cell genome. “Identical” or “identity” as used herein in the context of two or more nucleic acids or polypeptide sequences means that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0. “Nucleic acid” or “oligonucleotide” or “polynucleotide” as used herein means at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions. Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods. “Operably linked” as used herein means that expression of a gene is under the control omoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function. “Promoter” as used herein means a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and / or to alter the spatial expression and / or temporal expression of same. A promoter may also comprise distal enhancer or repressor elements, which may be located as much as several thousand base pairs from the start site of transcription. A promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter may regulate the expression of a gene component constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and the CMV IE promoter. “Target enhancer” as used herein refers to enhancer that is targeted by a gRNA and CRISPR / Cas9-based gene activation system. The target enhancer may be within the target region. “Target gene” as used herein refers to any nucleotide sequence encoding a known or putative gene product. The target gene includes the regulatory regions, such as the promoter and enhancer regions, the transcribed regions, which include the coding regions, and other function sequence regions. “Transcribed region” as used herein refers to the region of DNA that is transcribed into single-stranded RNA molecule, known as messenger RNA, resulting in the transfer of genetic information from the DNA molecule to the messenger RNA. During transcription, RNA sequence is complementary to the DNA strand. “Transcriptional Start Site” or “TSS” as used interchangeably herein refers to the first nucleotide of a transcribed DNA sequence where RNA polymerase begins synthesizing the RNA transcript. “Transgene” as used herein refers to a gene or genetic material containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may retain the ability to produce RNA or protein in the transgenic organism, or it may alter the normal function of the transgenic organism's genetic code. The introduction of a transgene has the potential to change the phenotype of an organism. “Trans-regulatory elements” as used herein refers to regions of non-coding DNA which regulate the transcription of genes distant from the gene from which they were transcribed. Trans-regulatory elements may be on the same or different chromosome from the target gene. “Variant” used herein with respect to a nucleic acid means (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto. “Variant” with respect to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variant may also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes may be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes may be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of +2 are substituted. The hydrophilicity of amino acids may also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide. Substitutions may be performed with amino acids having hydrophilicity values within +2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties. “Vector” as used herein means a nucleic acid sequence containing an origin of replication. A vector may be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be a self- replicating extrachromosomal vector, and preferably, is a DNA plasmid. “Episomal specific target sequence” as used herein refers to a sequence that is orthogonal to, or not present in, the human genome and only present in the episomal vector (e.g., a viral component of the vector) so that the nucleic acid-binding modality (e.g., DNA- binding protein or RNA-binding protein) will bind exclusively to a sequence only present within the episomal or extrachromosomal DNA. Thus, the episomal specific sequence self- targets the tether-encoding episomal vector (e.g., IDLV). “Chromosomal specific target sequence” as used herein refers to a sequence present in the host genome, such as a chromosomal locus, and not present in the episomal or extrachromosomal DNA. II. Extrachromosomal Nucleic Acid Tether System In certain embodiments, the present disclosure provides compositions and methods for stable long-term gene expression using non-integrating vectors which avoid unintended mutagenesis that avoids disruption of the genome. In some embodiments, the present artificial chromosomes are delivered by non-integrating viral vectors or by plasmid transfection, tethered to the host chromosomal nucleic acid (e.g., DNA or RNA), and self-replicated during mitosis without viral vector propagation. The delivered gene cassette can encode two dCas9 orthologs, which are either directly linked or fused to a dimerized domain regulated by chemical or environmental signals. For example, the dimerized domain may comprise cyclosporin A, FK506, FKCsA, rapamycin, gibberellin, abscisic acid, HaXS, TMP-HTag or ATB-737. One dCas9 targets the delivered gene cassette, and the other targets host nucleic acid (e.g., DNA or RNA). Each dCas9 ortholog can target a specific sequence indicated by their own gRNAs respectively. Thus, the delivered gene cassette is then tethered to the host chromosomal DNA by the two linked dCas9 orthologs. The tethered extrachromosomal nucleic acid (e.g., DNA or RNA) will replicate when host chromosomes replicate and be separated equally to the daughter cells as the host chromosomes. The number of episomal plasmids depends on the repeat number of the targeted sequences in the host genome. The present extrachromosomal nucleic acid (e.g., DNA or RNA) tether system may be used for the expression of a target payload. The target payload may comprise a transgene encoding a genetic circuit, kill switch, or therapeutic agent. A therapeutic agent may include a nucleic acid (e.g., an RNA, a DNA, or an oligonucleotide), a protein (e.g., an antibody, enzyme, cytokine, hormone, receptor), a lipid, a small molecule, a metabolic agent, an oligosaccharide, a peptide, an amino acid, an antigen. The target payload may comprise a single type of therapeutic agent, e.g., a single protein or nucleic acid, or may express more than one type of therapeutic agent, e.g., a plurality of proteins or nucleic acids. In some aspects, the extrachromosomal nucleic acid (e.g., DNA or RNA) tether system may comprise a kill switch or safety switch. The safety switch may comprise the expression of a suicide gene. The term “suicide gene” refers to a gene whose protein product converts a non- toxic prodrug into a toxic drug (e.g., an active chemotherapeutic agent), thereby killing cells that express the gene product. In some embodiments, a suicide gene is a nucleic acid which, upon administration of a prodrug, effects transition of a gene product to a compound which kills its host cell. Examples of suicide gene / prodrug combinations which may be used are Herpes Simplex Virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidilate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. The E. coli purine nucleoside phosphorylase which converts the prodrug 6- methylpurine deoxyriboside to toxic purine 6-methylpurine may also be used. In some aspects, the safety switch transgene or suicide gene is selected from the group consisting of aHSVtk gene, a cytosine deaminase gene, a nitroreductase gene, a purine nucleoside phosphorylase gene, a horseradish peroxidase gene, iCaspase9 gene, HER1 transgene, RQR8 transgene, CD20 transgene, CCR4 transgene, HER2 transgene, CD19 transgene, MUC1 transgene, EGFR transgene, GD2 transgene, PSMA transgene, CD16 transgene, and CD30 transgene. Using episomal tethering, the present composition maintains stable transgene expression without any integration into the host genome. Existing episomal cell engineering approaches like Epstein-Barr nuclear Antigen (EBNA)-based and scaffold / matrix attachment region (S / MARs)-based platforms lack inducibility and tunability; users cannot target specific parts of the genome nor specify a desired episomal copy number. Further, both EBNA1 and S / MAR-based systems can cause significant disruptions to their host system, posing concern. EBNA1 interferes with host processes in a way that contributes to the oncogenicity of EBV, while targeting S / MARs interferes with endogenous sequences as S / MARs are native elements of the human genome. By only tethering to targeted sites in the genomic and episomal DNA, a dCas9-based tether system does not significantly disrupt cellular or genetic processes. In some aspects, the use of dCas9, or other DNA binding domains, to create a tether system that maintains episomal DNA at specific host sites and at specific copy numbers is used for the maintenance of extrachromosomal DNA in replicating cells. In some aspects, non- integrating viruses and other non-extrachromosomal DNA moieties are adapted into engineered persistent artificial chromosomes (i.e., episomal vectors) to maintain and / or manipulate the expression and activity of the carried gene in long terms. These vectors may include non-integrating viruses, such as integrase-deficient lentivirus (IDLV) and high- capacity adenovirus (HC AdV), that deliver DNA encoding the tether system and replication origin necessary to tether to and replicate with chromosomal DNA. With these components, the DNA within the delivered vectors will form an engineered persistent artificial chromosome that avoids both degradation over time and dilution from cell division. Persistence of this extrachromosomal DNA allows consistent and controllable expression of its genetic payload. In some aspects, non-integrating viruses like integrase-deficient lentivirus, adenovirus, and herpes simplex virus are used to deliver engineered persistent episomal vectors. These episomal vectors can be used to maintain expression of therapeutic genetic payloads or genetic circuitry. The episomal vector may encode an engineered replication origin with two linked dead Cas9 (dCas9) orthologs or other DNA-binding proteins to tether the artificial chromosome to the host DNA at a controllable copy number. The two dCas9 orthologs or DNA-binding proteins (e.g., TALENS and zinc fingers) can be directly linked by a peptide linker (e.g., G4S12) or fused to an inducible dimerized domain. In other aspects, two DNA-binding domains, such as zinc fingers, could be used in place of the two dCas9 orthologs. One zinc finger could be designed to bind to the episomal DNA while another zinc finger could be designed to bind to specific target sites on the host DNA. These DNA-binding domains can be either directly fused to one another or inducibly dimerized as mentioned previously. This would accomplish mitotic stability and copy number specificity akin to the dCas9-based system. In some aspects, the cassette further comprises the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) which can be used to increase transgene expression from a variety of viral vectors. In certain aspects, the tether may comprise a Tet repressor (TetR) which binds to a Tet Operator site in the episome and a DNA binding protein, such as SpdCas9, which binds to the genome. In the Tet-Off expression system, a tetracycline-controlled transactivator protein (tTA), which is composed of the Tet repressor DNA binding protein (TetR) from the Tc resistance operon of Escherichia coli transposon Tn10 fused to the strong transactivating domain of VP16 from Herpes simplex virus, regulates expression of a target gene that is under transcriptional control of a tetracycline-responsive promoter element (TRE).The Tet Repressor may be turned on or off in the presence of doxycycline. The TRE is made up of Tet operator (tetO) sequence concatemers fused to a minimal promoter, (commonly the minimal promoter sequence derived from the human cytomegalovirus (hCMV) immediate-early promoter). In the absence of Tc or Dox, tTA binds to the TRE and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to the TRE, and expression from the target gene remains inactive. In some aspects, the tether system may comprise RNA-binding proteins (RBPs). Proteins with well-characterized RNA binding domains like RNA recognition motif (RRM), KH domain, or zinc finger domains may be used as they allow for specific binding to target RNA sequences within a vector, enabling precise manipulation of gene expression at the post- transcriptional level; some examples of RBPs used in this way are Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), Hu antigen R (HuR), neuro-oncological ventral antigen 1 (Nova1), and Fused in sarcoma (FUS). Similar to the dCas9-based tether system, multiple tether system variants using alternative DNA-binding modalities, including the Tet Repressor (TetR) protein (FIG. 13A) and Zinc Finger (ZF) proteins (FIG.14A) were developed. Alternative DNA-binding modality systems comprise guide RNA (gRNA) cassettes and / or Zinc Finger (ZF) or Tet Operator (TetO) binding sites, and a DNA replication (not viral propagation) cassette as a platform for delivering therapeutic payloads, like genetic circuits, to primary human cells (FIG. 13B, FIG. 14B). A fluorescent marker or safety switch component can be added to test persistence or reversibility. The tethering system comprises two DNA-binding modalities that are attached to one another (FIG.13A, FIG.14A), either through direct fusion but can also be attached through fusions with domains that can be inducibly and reversibly dimerized by administering a small molecule. Since the DNA-binding modalities used are catalytically inactive, the tether system does not cleave either the chromosomal or episomal DNA. Each of these alternative tether systems (TetR- or ZF-based) comprise one DNA-binding modality that binds exclusively to a sequence only present within the host genome (e.g. Zinc Fingers that bind to specific genomic loci or repeats) and another DNA-binding modality that will bind exclusively to a sequence only present within the episomal or extrachromosomal DNA (e.g. Zinc Fingers that target a sequence orthogonal to the human genome or a Tet Repressor that targets a set of Tet Operator, or TetO, sequences that are not present within the human genome). Orthogonality between both DNA-binding modalities ensures that each respective protein will associate with either the genomic DNA or episome or extrachromosomal DNA without cross interference. In the case of a tether system consisting of catalytically dead Sp. Cas9 (SpdCas9) and TetR, the TetR DNA-binding modality will only target the Tet Operator sites on the episome, while the SpdCas9 only targets specific site(s) on the host genome (FIG. 13A). In the case of a tether system consisting of two Zinc Fingers, the episomal Zinc Finger will target a sequence on the episome that is orthogonal to, or not present in, the human genome, while the genomic Zinc Finger only targets specific site(s) on the host genome (FIG. 14A). In all of these tether systems, attachment of the dCas9 orthologs to one another tethers the episome to the host genome, conferring mitotic stability to the episome as will replicate with and bind to the chromosomal DNA of both daughter cells in mitosis rather than only being in one. In the specific case of a tether system including the Tet Repressor (TetR, also referred to as rtTA), the addition of doxycycline (dox) can cause the TetR protein to disengage from the Tet Operator (TetO) sites present on the episomal DNA (FIG. 13A). Inability of the TetR protein to bind to the TetO sites will cause episomal dilution over time and prevent the maintenance and / or establishment of mitotic stability. In particular aspects, artificial or synthetic circular chromosomes (SCCs) can encode an engineered replication origin(s) along with two linked dead Cas9 (dCas9) orthologs, or other DNA-binding domain-based structures, to form a system that tethers the artificial chromosome to the host cell’s DNA at a specified quantity. Using this episomal tethering system, the present methods can used to successfully confer mitotic stability, kill switches, and / or other therapeutic / biotechnological payloads in any mammalian cell type, such as primary immune cells, without requiring DNA integration. The tethered synthetic episomal chromosomes can also encode means of tunable or reversible transgene expression. The present methods can be used to deliver complex gene circuits to reprogram any type of mammalian cell, such as human immune cells, while avoiding the problems inherent to DNA integration. In some aspects, the present tethering system is delivered via non-integrating viruses, such as adenovirus (e.g., high-capacity adenoviral vectors (HC AdV)), integrase deficient lentivirus, baculovirus, or herpesvirus, to any mammalian cell type, including T cells, MSCs, and other primary mammalian cell types. Integrase deficient lentiviruses (IDLV) are non- replicative lentiviruses containing mutations in the catalytic domain of the viral integrase. Each IDLV has an actionable payload limit of about 10.5 kilobases (kb), thus, two or more IDLVs could be used for larger payloads, such as larger than 10, 11, 12, 13, 14, or 14 kb. As a consequence, circular cDNA off-products of the retrotranscription named 1-LTR and 2-LTR accumulate in the cell nucleus but are not able to integrate into the host genome. In some aspects, helper-dependent adenovirus (HD-AdV) can be used to deliver large payloads up to 36 kilobases (e.g., 5-10 kb, 10-20 kb, 20-25 kb, or 25-36 kb), such as genetic circuits. One exemplary episomal vector is provided herein comprising SpdCas9 and SadCas9 with corresponding gRNAs. The exemplary episomal vector, pLentiCRISPRv2_7sk_Sa- sgRNA Scaffold_hU6_Sp-sgRNAScaffold_EFS-Promoter_HA_SV40-NLS_SadCas9_GS- Linker_SpdCas9_Nucleoplasmin-NLS_Flag_WPRE may comprise the sequence or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1 or the various components of the vector (e.g., SEQ ID NOS:2-14), such as the 7sk or EF1alpha intron-less form (EFS) promoters, SadCas9, or SpdCas9. SEQ ID NO: 1

[0017] SEQ ID NO: 2 (7sk) SEQ ID NO: 3 (Sa-sgRNA Scaffold) SEQ ID NO:4 (hU6) SEQ ID NO: 5 (Sp-sgRNA Scaffold) SEQ ID NO: 6 (EFS-Promoter) SEQ ID NO: 7 (HA) SEQ ID NO :8 (SV40-NLS) SEQ ID NO: 9 (SadCas9)

[0018] SEQ ID NO: 10 (GS-Linker) SEQ ID NO: 11 (SpdCas9)

[0019] SEQ ID NO: 12 (Nucleoplasmin-NLS) SEQ ID NO: 13 (Flag) SEQ ID NO: 14 (WPRE) SEQ ID NO: 15 (dEnOsCas12f1) Generally, non-integrating viral vectors only transiently express the transgene due to inhibition of pathogenic properties and dilution of copy number as their host cells replicate. However, the present methods overcome these issues by tethering of the engineered episomal DNA to the host genome. Through combined use with safe replication cassettes, the present system can ensure episome stability through mitosis and maintain a desired episomal copy number. In other aspects, the present system may comprise a site-specific dCas9-based replicator, which is a fusion of dCas9 with a human replication factor (e.g. Cdc6), that promotes replication of the engineered episome. In additional aspects, the present system may comprise one or more safety cassettes, such as drug-inducible kill switches (e.g., HSV-TK, inducible caspase 9 (iC9), and anti-CD20) which kill their host cells containing the engineered episomes in response to induction. In some aspects, the system may comprise an inducible active Cas9 capable of degrading the engineered episome that encodes itself. This approach has the advantage of halting transgene expression from the episome without killing the cell. In some embodiments, methods are provided for the use of the present system for the delivery and stable expression of transgenes and / or gene regulatory programs in any mammalian cell type without directly integrating into the host genome. For example, the present system may be used to program engineered immune cells that are capable of complex genetic programs. These engineered cells can be used as living diagnostics, sense-and-response cell therapies, cell differentiation systems, and other genetically encoded regulatory programs. A. Cas9 DNA Binding Proteins Certain embodiments of the present disclosure concern dCas9 binding proteins and guide RNAs for use in tethering a target payload to a site in the host chromosome. The present tether system may comprise a Cas9 protein that does not have nuclease activity, known as dead Cas9 (dCas9). d and the protein-RNA pair recognizes its genomic target by complementary base pairing the protospacer. An engineered form of the Type II effector system of Streptococcus pyogenes was shown to function in human cells for genome engineering. In this system, the Cas9 protein was directed to genomic target sites by a synthetically reconstituted “guide RNA” (“gRNA”), also used interchangeably herein as a chimeric sgRNA, which is a crRNA-tracrRNA fusion that obviates the need for RNase III and crRNA processing in general. Cas9 protein is an endonuclease that cleaves nucleic acid and is encoded by the CRISPR loci and is involved in the Type II CRISPR system. The Cas9 protein may be from any bacterial or archaea species, such as Streptococcus pyogenes, Streptococcus thermophiles, or Neisseria meningitides. The Cas9 protein may be mutated so that the nuclease activity is inactivated. In some embodiments, an inactivated Cas9 protein from Streptococcus pyogenes (also referred to as dead Cas9 or “dCas9”) may be used. As used herein, “dCas9” both refer to a Cas9 protein that has the amino acid substitutions D10A and H840A and has its nuclease activity inactivated. In some embodiments, an inactivated Cas9 protein from other species, such as Neisseria meningitides, such as NmCas9, may be used. The present tether system may include at least two gRNAs that target a nucleic acid sequence, such as the episomal target payload and a chromosomal locus. The gRNA is a fusion of two noncoding RNAs: a crRNA and a tracrRNA. The sgRNA may target any desired DNA sequence by exchanging the sequence encoding a 20 bp protospacer which confers targeting specificity through complementary base pairing with the desired DNA target. gRNA mimics the naturally occurring crRNA:tracrRNA duplex involved in the Type II Effector system. The gRNA may target and bind a target region of a target gene. The target region may be a cis-regulatory region or trans-regulatory region of a target gene. In some embodiments, the target region is a distal or proximal cis-regulatory region of the target gene. The gRNA may target and bind a cis-regulatory region or trans-regulatory region of a target gene. In some embodiments, the gRNA may target and bind an enhancer region, a promoter region, or a transcribed region of a target gene. For example, the gRNA may target and bind the target human β-globuli lnocus, distal regulatory region (DRR) of the MYOD gene, core enhancer (CE) of the MYOD gene, proximal (PE) enhancer region of the OCT4 gene, or distal (DE) enhancer region of the OCT4 gene. In some embodiments, the target region may be a viral promoter, such as an HIV promoter. The target region may include a target enhancer or a target regulatory element. In some embodiments, the target enhancer or target regulatory element controls the gene expression of several target genes. In some embodiments, the target enhancer or target regulatory element controls a cell phenotype that involves the gene expression of one or more target genes. In some embodiments, the identity of one or more of the target genes is known. In some embodiments, the identity of one or more of the target genes is unknown. The CRISPR / Cas9- based gene activation system allows the determination of the identity of these unknown genes that are involved in a cell phenotype. Examples of cell phenotypes include, but not limited to, T-cell phenotype, cell differentiation, such as hematopoietic cell differentiation, oncogenesis, immunomodulation, cell response to stimuli, cell death, cell growth, drug resistance, or drug sensitivity. The gRNA may comprise a complementary polynucleotide sequence of the target DNA sequence followed by a protospacer adjacent motif, such as NGG for Sa. Cas9. The gRNA may comprise at least a 10 base pair, at least a 11 base pair, at least a 12 base pair, at least a 13 base pair, at least a 14 base pair, at least a 15 base pair, at least a 16 base pair, at least a 17 base pair, at least a 18 base pair, at least a 19 base pair, at least a 20 base pair, at least a 21 base pair, at least a 22 base pair, at least a 23 base pair, at least a 24 base pair, at least a 25 base pair, at least a 30 base pair, or at least a 35 base pair complementary polynucleotide sequence of the target DNA sequence followed by NGG. The gRNA may target at least one of the promoter region, the enhancer region, or the transcribed region of the target gene. The present extrachromosomal DNA tether system may also include a viral delivery system. For example, the viral delivery system may include a non-integrating viral vector, such as an adenoviral vector, or an integrase deficient lentiviral vector, or partially integrating vector, such as adeno-associated virus vector. Methods of introducing a nucleic acid into a host cell are known in the art, and any known method can be used to introduce a nucleic acid (e.g., an expression construct) into a cell. Suitable methods include, viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery, and the like. In some embodiments, the composition may be delivered by mRNA delivery and ribonucleoprotein (RNP) complex delivery. The compositions, as described above, may comprise genetic constructs that encode the extrachromosomal DNA tether system, as disclosed herein. The genetic construct, such as a plasmid or expression vector, may comprise a nucleic acid that encodes the extrachromosomal DNA tether system. The compositions, as described above, may comprise genetic constructs consisting of a nucleic acid sequence that encodes the extrachromosomal DNA tether system to be delivered by a non-integrating viral vector, as disclosed herein. The genetic construct, such as an episomal vector, may comprise a nucleic acid that encodes the extrachromosomal DNA tether system. The genetic construct may be present in the cell as a functioning extrachromosomal molecule. Coding sequences may be optimized for stability and high levels of expression. In some instances, codons are selected to reduce secondary structure formation of the RNA such as that formed due to intramolecular bonding. The vector may comprise a heterologous nucleic acid encoding the extrachromosomal DNA tether system and may further comprise an initiation codon and a stop codon. The initiation and stop codon may be in frame with the extrachromosomal DNA tether system coding sequence. The vector may also comprise a promoter that is operably linked to the extrachromosomal DNA tether system coding sequence. The system may be under the light- inducible or chemically-inducible control to enable the dynamic control of gene activation in space and time. The promoter may be a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter may also be a promoter from a human gene such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metalothionein. The promoter may also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic. Examples of such promoters are described in U.S. Patent Application Publication No. US20040175727, the contents of which are incorporated herein in its entirety. The vector may also comprise a polyadenylation signal, which may be downstream of the system. The polyadenylation signal may be a SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth human β-globulin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 vector (Invitrogen, San Diego, Calif.). The vector may also comprise an enhancer upstream of the extrachromosomal DNA tether system. The enhancer may be necessary for DNA expression. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer such as one from CMV, HA, RSV or EBV. Polynucleotide function enhancers are described in U.S. Patent Nos. 5,593,972, 5,962,428, and WO94 / 016737, the contents of each are fully incorporated by reference. The vector may also comprise a mammalian origin of replication to maintain the vector extrachromosomally and produce multiple copies of the vector in a cell. The vector may also comprise a regulatory sequence, which may be well suited for gene expression in a mammalian or human cell into which the vector is administered. The vector may also comprise a reporter gene, such as green fluorescent protein (“GFP”), and / or a selectable marker, such as hygromycin (“Hygro”). The vector may be expression vectors or systems to produce protein by routine techniques and readily available starting materials including Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989), which is incorporated fully by reference. In some embodiments the vector may comprise the nucleic acid sequence encoding the CRISPR / Cas9-based gene activation system, including the nucleic acid sequence encoding the CRISPR / Cas9-based acetyltransferase and the nucleic acid sequence encoding the at least one gRNA. B. Regulatory Elements Expression cassettes included in vectors useful in the present disclosure in particular contain (in a 5'-to-3' direction) a eukaryotic transcriptional promoter operably linked to a protein-coding sequence, splice signals including intervening sequences, and a transcriptional termination / polyadenylation sequence. The promoters and enhancers that control the transcription of protein encoding genes in eukaryotic cells are composed of multiple genetic elements. The cellular machinery is able to gather and integrate the regulatory information conveyed by each element, allowing different genes to evolve distinct, often complex patterns of transcriptional regulation. A promoter used in the context of the present disclosure includes a cell-cycle dependent promoter. 1. Promoter / Enhancers A promoter generally comprises a sequence that functions to position the transcriptional start site (TSS) for RNA synthesis. The best known example of a TSS is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation. Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the transcriptional start site as well. To bring acoding sequence “under the control of” a promoter, one positions the 5 end of the transcriptioninitiation site of the transcriptional reading frame “downstream” of (i.e., 3 of) the chosenpromoter. The “upstream” promoter stimulates transcription of the DNA and promotes expression of the encoded RNA. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription. A promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. A promoter may be one naturally associated with a nucleic acid sequence, as may beobtained by isolating the 5 non-coding sequences located upstream of the coding segmentand / or exon. Such a promoter can be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus, or prokaryotic or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and / or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and / or nucleic acid amplification technology, including PCR, in connection with the compositions disclosed herein (see U.S. Patent Nos. 4,683,202 and 5,928,906, each incorporated herein by reference). Furthermore, it is contemplated that the control sequences that direct transcription and / or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well. Naturally, it will be important to employ a promoter and / or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, (see, for example Sambrook et al. 1989, incorporated herein by reference). The promoters employed may be constitutive, tissue-specific, inducible, and / or useful under the appropriate conditions to direct high-level expression of the introduced DNA segment, such as is advantageous in the large- scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous. Additionally, any promoter / enhancer combination (as per, for example, the Eukaryotic Promoter Data Base EPDB, through world wide web at epd.isb-sib.ch / ) could also be used to drive expression. Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct. In certain aspects, methods of the disclosure also concern enhancer sequences, i.e., nucleic acid sequences that increase a promoter’s activity and that have the potential to act in cis, and regardless of their orientation, even over relatively long distances (up to several kilobases away from the target promoter). However, enhancer function is not necessarily restricted to such long distances as they may also function in close proximity to a given promoter. 2. Initiation Signals and Linked Expression A specific initiation signal also may be used in the expression constructs provided in the present disclosure for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be “in-frame” with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements. In certain embodiments, internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5 methylated Cap dependent translation and begin translation at internalsites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message (see U.S. Patent Nos. 5,925,565 and 5,935,819, each herein incorporated by reference). Additionally, certain 2A sequence elements could be used to create linked- or co- expression of genes in the constructs provided in the present disclosure. For example, cleavage sequences could be used to co-express genes by linking open reading frames to form a single cistron. An exemplary cleavage sequence is the F2A (Foot-and-mouth disease virus 2A) or a “2A-like” sequence (e.g., Thosea asigna virus 2A; T2A) (Minskaia and Ryan, 2013). 3. Origins of Replication In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed “ori”), for example, a nucleic acid sequence corresponding to oriP of EBV as described above or a genetically engineered oriP with a similar or elevated function in programming, which is a specific nucleic acid sequence at which replication is initiated. Alternatively, a replication origin of other extra-chromosomally replicating virus as described above or an autonomously replicating sequence (ARS) can be employed. 4. Selection and Screenable Markers In some embodiments, cells containing a construct of the present disclosure may be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selection marker is one that confers a property that allows for selection. A positive selection marker is one in which the presence of the marker allows for its selection, while a negative selection marker is one in which its presence prevents its selection. An example of a positive selection marker is a drug resistance marker. Typically, the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selection markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated. Alternatively, screenable enzymes as negative selection markers such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selection and screenable markers are well known to one of skill in the art. C. Cells Further provided herein are methods for introducing the present extrachromosomal DNA tether system to a cell, for example, an engineered cell. A cell may be derived from any mammalian organ or tissue, including the brain, nerves, ganglia, spine, eye, heart, liver, kidney, lung, spleen, bone, thymus, lymphatic system, skin, muscle, pancreas, stomach, intestine, blood, ovary, uterus, or testes. A cell may be derived from a donor (e.g., an allogeneic cell), derived from a subject (e.g., an autologous cell), or from another species (e.g., a xenogeneic cell). In an embodiment, a cell can be grown in cell culture, or prepared from an established cell culture line, or derived from a donor (e.g., a living donor or a cadaver). In an embodiment, a cell is genetically engineered. In another embodiment, a cell is not genetically engineered. A cell may include a stem cell, such as a reprogrammed stem cell or an induced pluripotent cell. Exemplary cells include mesenchymal stem cells (MSCs), fibroblasts (e.g., primary fibroblasts), HEK cells (e.g., HEK293T), Jurkat cells, HeLa cells, retinal pigment epithelial (RPE) cells, HUVEC cells, NIH3T3 cells, CHO-K1 cells, COS-1 cells, COS-7 cells, PC-3 cells, HCT 116 cells, A549MCF-7 cells, HuH-7 cells, U-2 OS cells, HepG2 cells, Neuro-2a cells, and SF9 cells. In an embodiment, a cell engineered to express the present extrachromosomal DNA tether system may express a single type of target payload or a plurality of payloads. In an embodiment, a cell may be transduced or transfected with a lentivirus. A nucleic acid introduced into a cell (e.g., by transduction or transfection) may be incorporated into a nucleic acid delivery system, such as a plasmid, or may be delivered directly. In an embodiment, a nucleic acid introduced into a cell (e.g., as part of a plasmid) may include a region to enhance expression of the target payload and / or to direct targeting or secretion, for example, a promoter sequence, an activator sequence, or a cell-signaling peptide, or a cell export peptide. Exemplary promoters include EF-1a, CMV, Ubc, hPGK, VMD2, and CAG. D. Methods of Delivery Further provided herein are methods for delivering the present pharmaceutical formulations comprising the extrachromosomal DNA tether system. Delivery may comprise transfection or electroporation as one or more nucleic acid molecules that is expressed in the cell and delivered to the surface of the cell. The nucleic acid molecules may be electroporated using BioRad Gene Pulser Xcell or Amaxa Nucleofector IIb devices or other electroporation device. Several different buffers may be used, including BioRad electroporation solution, Sigma phosphate-buffered saline product # D8537 (PBS), Invitrogen OptiMEM I (OM), or Amaxa Nucleofector solution V (N.V.). Transfections may include a transfection reagent, such as Lipofectamine 2000. The vector encoding the extrachromosomal DNA tether system, may be delivered to the mammal by DNA injection (also referred to as DNA vaccination) with and without in vivo electroporation, liposome mediated, nanoparticle facilitated, and / or recombinant vectors. The recombinant vector may be delivered by any viral mode. The viral mode may be recombinant lentivirus, recombinant adenovirus, and / or recombinant adeno-associated virus. The polynucleotides of the present disclosure may be introduced (e.g., transfected or transduced) into a host cell by viral or non-viral methods. Vectors provided herein are designed, primarily, to express a suicide gene under the control of a cell-cycle dependent promoter. One of skill in the art would be well-equipped to construct a vector through standard recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996, both incorporated herein by reference). Vectors include but are not limited to, plasmids, cosmids, viruses (e.g., bacteriophage, animal viruses, and plant viruses), artificial chromosomes (e.g., YACs), retroviral vectors (e.g. derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV, SNV etc), lentiviral vectors (e.g. derived from HIV-1, HIV-2, SIV, BIV, FIV etc.), adenoviral (Ad) vectors including replication competent, replication deficient and gutless forms thereof, adeno-associated viral (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus vectors, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, murine mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, polio virus vectors, vesicular stomatitis virus vectors, maraba virus vectors, and group B adenovirus enadenotucirev vectors. The nucleotide encoding the extrachromosomal DNA tether system, may be administered to a mammal to induce or modulate gene expression of the target gene in a mammal. The mammal may be human, non-human primate, cow, pig, sheep, goat, antelope, bison, water buffalo, bovids, deer, hedgehogs, elephants, llama, alpaca, mice, rats, or chicken, and preferably human, cow, pig, or chicken. The extrachromosomal DNA tether system, and compositions thereof may be administered to a subject by different routes including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, intrapleurally, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal intrathecal, and intraarticular or combinations thereof. For veterinary use, the composition may be administered as a suitably acceptable formulation in accordance with normal veterinary practice. The veterinarian may readily determine the dosing regimen and route of administration that is most appropriate for a particular animal. The extrachromosomal DNA tether system, and compositions thereof may be administered by traditional syringes, needleless injection devices, “microprojectile bombardment gene guns”, or other physical methods such as electroporation (“EP”), “hydrodynamic method”, or ultrasound. The composition may be delivered to the mammal by several technologies including DNA injection (also referred to as DNA vaccination) with and without in vivo electroporation, liposome mediated, nanoparticle facilitated, recombinant vectors such as recombinant lentivirus, recombinant adenovirus, and recombinant adeno-associated virus. The extrachromosomal DNA tether system may be used with any type of cell. In some embodiments, the cell is a bacterial cell, a fungal cell, an archaea cell, a plant cell or an animal cell. In some embodiments, the cell may be an ENCODE cell line, including but not limited to, GM12878, K562, H1 human embryonic stem cells, HeLa-S3, HepG2, HUVEC, SK-N-SH, IMR90, A549, MCF7, HMEC or LHCM, CD14+, CD20+, primary heart or liver cells, differentiated H1 cells, 8988T, Adult_CD4_naive, Adult_CD4_Th0, Adult_CD4_Th1, AG04449, AG04450, AG09309, AG09319, AG10803, AoAF, AoSMC, BC_Adipose_UHN00001, BC_Adrenal_Gland_H12803N, BC_Bladder_01-11002, BC_Brain_H11058N, BC_Breast_02-03015, BC_Colon_01-11002, BC_Colon_H12817N, BC_Esophagus_01-11002, BC_Esophagus_H12817N, BC_Jejunum_H12817N, BC_Kidney_01-11002, BC_Kidney_H12817N, BC_Left_Ventricle_N41, BC_Leukocyte_UHN00204, BC_Liver_01-11002, BC_Lung_01-11002, BC_Lung_H12817N, BC_Pancreas_H12817N, BC_Penis_H12817N, BC_Pericardium_H12529N, BC_Placenta_UHN00189, BC_Prostate_Gland_H12817N, BC_Rectum_N29, BC_Skeletal_Muscle_01-11002, BC_Skeletal_Muscle_H12817N, BC_Skin_01-11002, BC_Small_Intestine_01-11002, BC_Spleen_H12817N, BC_Stomach_01-11002, BC_Stomach_H12817N, BC_Testis_N30, BC_Uterus_BN0765, BE2_C, BG02ES, BG02ES-EBD, BJ, bone_marrow_HS27a, bone_marrow_HS5, bone_marrow_MSC, Breast_OC, Caco-2, CD20+_RO01778, CD20+_RO01794, CD34+_Mobilized, CD4+_Naive_Wb11970640, CD4+_Naive_Wb78495824, Cerebellum_OC, Cerebrum_frontal_OC, Chorion, CLL, CMK, Colo829, Colon_BC, Colon_OC, Cord_CD4_naive, Cord_CD4_Th0, Cord_CD4_Th1, Decidua, Dnd41, ECC-1, Endometrium_OC, Esophagus_BC, Fibrobl, Fibrobl_GM03348, FibroP, FibroP_AG08395, FibroP_AG08396, FibroP_AG20443, Frontal_cortex_OC, GC_B_cell, Gliobla, GM04503, GM04504, GM06990, GM08714, GM10248, GM10266, GM10847, GM12801, GM12812, GM12813, GM12864, GM12865, GM12866, GM12867, GM12868, GM12869, GM12870, GM12871, GM12872, GM12873, GM12874, GM12875, GM12878-XiMat, GM12891, GM12892, GM13976, GM13977, GM15510, GM18505, GM18507, GM18526, GM18951, GM19099, GM19193, GM19238, GM19239, GM19240, GM20000, H0287, H1-neurons, H7-hESC, H9ES, H9ES- -AFP+, H9ES-CM, H9ES-E, H9ES-EB, H9ES-EBD, HAc,HAEpiC, HA-h, HAL, HAoAF, HAoAF_6090101.11, HAoAF_6111301.9, HAoEC, HAoEC_7071706.1, HAoEC_8061102.1, HA-sp, HBMEC, HBVP, HBVSMC, HCF, HCFaa, HCH, HCH_0011308.2P, HCH_8100808.2, HCM, HConF, HCPEpiC, HCT-116, Heart_OC, Heart_STL003, HEEpiC, HEK293, HEK293T, HEK293-T-REx, Hepatocytes, HFDPC, HFDPC_0100503.2, HFDPC_0102703.3, HFF,HFF-Myc, HFL11W, HFL24W, HGF, HHSEC, HIPEpiC, HL-60, HMEpC, HMEpC_6022801.3, HMF, hMNC-CB, hMNC- CB_8072802.6, hMNC-CB_9111701.6, hMNC-PB, hMNC-PB_0022330.9, hMNC- PB_0082430.9, hMSC-AT, hMSC-AT_0102604.12, hMSC-AT_9061601.12, hMSC-BM, hMSC-BM_0050602.11, hMSC-BM_0051105.11, hMSC-UC, hMSC-UC_0052501.7, hMSC-UC_0081101.7, HMVEC-dAd, HMVEC-dBl-Ad, HMVEC-dBl-Neo, HMVEC-dLy- Ad, HMVEC-dLy-Neo, HMVEC-dNeo, HMVEC-LB1, HMVEC-LLy, HNPCEpiC, HOB, HOB_0090202.1, HOB_0091301, HPAEC, HPAEpiC, HPAF, HPC-PL, HPC- PL_0032601.13, HPC-PL_0101504.13, HPDE6-E6E7, HPdLF, HPF, HPIEpC, HPIEpC_9012801.2, HPIEpC_9041503.2, HRCEpiC, HRE, HRGEC, HRPEpiC, HSaVEC, HSaVEC_0022202.16, HSaVEC_9100101.15, HSMM, HSMM_emb, HSMM_FSHD, HSMMtube, HSMMtube_emb, HSMMtube_FSHD, HT-1080, HTR8svn, Huh-7, Huh-7.5, HVMF, HVMF_6091203.3, HVMF_6100401.3, HWP, HWP_0092205, HWP8120201.5, iPS, iPS CWRU1, iPS_hFib2_iPS4, iPS_hFib2_iPS5, iPS_NIHi11, iPS_NIHi7, Ishikawa, Jurkat, Kidney_BC, Kidney_OC, LHCN-M2, LHSR, Liver_OC, Liver_STL004, Liver_STLO11, LNCaP, Loucy, Lung BC, Lung_OC, Lymphoblastoid_cell_line, M059J, MCF10A-Er-Src, MCF-7, MDA-MB-231, Medullo, Medullo_D341, Mel_2183, Melano, Monocytes-CD14+, Monocytes-CD 14+_RO01746, Monocytes-CD14+_RO01826, MRT_A204, MRT_G401, MRT_TTC549, Myometr, Naive_B_cell, NB4, NH-A, NHBE, NHBE_RA, NHDF, NHDF_0060801.3, NHDF_7071701.2, NHDF-Ad, NHDF-neo, NHEK, NHEM.f_M2, NHEM.f_M2_5071302.2, NHEM.f_M2_6022001, NHEM_M2, NHEM_M2_7011001.2, NHEM_M2_7012303, NHLF, NT2-D1, Olf_neurosphere, Osteobl, ovcar-3, PANC-1, Pancreas_OC, PanIsletD, PanIslets, PBDE, PBDEFetal, PBMC, PFSK-1, pHTE, Pons_OC, PrEC, ProgFib, Prostate, Prostate_OC, Psoas_muscle_OC, Raji, RCC_7860, RPMI-7951, RPTEC, RWPE1, SAEC, SH-SY5Y, Skeletal_Muscle_BC, SkMC, SKMC, SkMC_8121902.17, SkMC_9011302, SK-N-MC, SK-N-SH_RA, Small_intestine_OC, Spleen_OC, Stellate, Stomach_BC, T_cells_CD4+, T-47D, T98G, TBEC, Th1, Th1_Wb33676984, Th1_Wb54553204, Th17, Th2, Th2 Wb33676984, Th2_Wb54553204, Treg_Wb78495824, Treg_Wb83319432, U2OS, U87, UCH-1, Urothelia, WERI-Rb-1, and WI-38. E. Formulation and Administration The present disclosure provides pharmaceutical compositions comprising the extrachromosomal DNA tether system provided herein. The extrachromosomal DNA tether system may be in a pharmaceutical composition. The pharmaceutical composition may comprise about 1 ng to about 10 mg of DNA encoding the extrachromosomal DNA tether system. The pharmaceutical compositions according to the present invention are formulated according to the mode of administration to be used. In cases where pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen free, and particulate free. An isotonic formulation is preferably used. Generally, additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstriction agent is added to the formulation. The pharmaceutical composition containing the extrachromosomal DNA tether system may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be functional molecules such as vehicles, adjuvants, carriers, or diluents. The pharmaceutically acceptable excipient may be a transfection facilitating agent, which may include surface active agents, such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. The transfection facilitating agent is a polyanion, polycation, including poly-L- glutamate (LGS), or lipid. The transfection facilitating agent is poly-L-glutamate, and more preferably, the poly-L-glutamate is present in the pharmaceutical composition containing the extrachromosomal DNA tether system at a concentration less than 6 mg / ml. The transfection facilitating agent may also include surface active agents such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used administered in conjunction with the genetic construct. In some embodiments, the DNA vector encoding the extrachromosomal DNA tether system, may also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as a DNA-liposome mixture (see for example WO9324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. Preferably, the transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS), or lipid. Such compositions comprise a prophylactically or therapeutically effective amount of an antibody or a fragment thereof, or a peptide immunogen, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington's Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, or delivered by mechanical ventilation. Generally, the ingredients of the compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. The compositions of the disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. III. Examples The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. Example 1 – Engineered Extrachromosomal DNA System A dCas9-based (or other DNA-binding modality) tether system was developed with guide RNA (gRNA) cassettes (FIG.1A), a fluorescent persistence marker, a safety switch, and a DNA replication (not viral propagation) cassette as a platform for delivering therapeutic payloads, like genetic circuits, to primary human cells (FIG. 1B). The tethering system comprises two nuclease dead Cas9 (dCas9) orthologs that are attached to one another, either through direct fusion (FIG. 1A) or through fusions with domains that can be inducibly and reversibly dimerized by administering a small molecule. Since dCas9 is catalytically inactive, the tether system does not cleave either the chromosomal or episomal DNA. The two dCas9 orthologs come from different bacterial species, Streptococcus pyogenes (Sp. dCas9) and Staphylococcus aureus (Sa. dCas9), so they associate with different gRNAs without cross interference. This ensures that Sa. dCas9 only targets the engineered episome, while the Sp. dCas9 only targets specific site(s) on the host genome. Therefore, attachment of the dCas9 orthologs to one another tethers the episome to the host genome, conferring mitotic stability to the episome as will replicate with and bind to the chromosomal DNA of both daughter cells in mitosis rather than only being in one. An HA tag was added to the N terminus of the tether that binds the episome (Sa. dCas9) and the flag tag was added to the C terminus that binds to targeted genomic regions (Sp. dCas9) so that both ends of the tether could be assayed using antibodies. Expression of the tether was optimized by testing various linkers in a Western blot using anti-Flag and anti-HA antibodies and it was found that some linkers led to a level of cleavage between the Sa. dCas9 and the Sp. dCas9 (FIG. 5). The linker (G4S)3was selected as one that allowed a high expression level without causing visible cleavage. Anti-Flag and anti-HA antibodies were used to perform CHIP-qPCR in HEK293T cells transfected with the episome, showing that the Sa.dCas9 and the Sp. dCas9 bind to the episome and the targeted host genome respectively (FIGS. 1C-D). It was also confirmed that co-localization between the episome and the targeted region of the genome occurred with fluorescence in-situ hybridization (FISH) using probes targeting the episome and the targeted region of the genome, indicating successful tether binding (FIG. 1E- F). As non-integrating viruses generally target multiple nonspecific sites in the host genome rather than just one locus, studies were performed to test whether residency would be improved by targeting repeating elements in the host genome, such as SINEs or Alu. Since binding is to specific repeats and not randomly, this also allows control over the episome copy number by controlling the number of tether binding sites (FIG. 3). Following viral packaging and delivery, all the manufacturing steps of the present methods take place at a cellular level, where the episomal tether system will be expressed and replicated with each cell division. The episome contains a human origin of DNA replication (that will not produce replication-competent viruses) to recruit host replication machinery. This allows the episomal DNA to replicate at the same time as the host genome, maintaining a desired copy number despite the dilution caused by mitosis. LaminB2 replication was used, which was shown to retain a higher percentage of episomes in dividing HEK cells through multiple passages, as compared with other human replicon candidates (FIG. 4). It was further shown that adding human replicons to the tether system increases the percentage of episomes retained in dividing HEK293T cells transfected with a GFP-marker expressing episome and that increasing the number of target sites causes increased retention (FIGS. 2A-B). These results were confirmed with microscopy showing GFP expression (FIG 6). It was further shown that sorting cells for presence of GFP-encoding episome at an early time point increased the long-term level of episomal persistence and the yield of episome-harboring cells (FIG 2C). These results were confirmed with microscopy showing GFP expression (FIG 2D), and therefore presence of the GFP-encoding episome, persists in cells containing a human replicon combined with a tether targeting multiple sites in the genome (FIG. 2E). It was shown that alternative Cas-based tethering systems using smaller Cas proteins, such as an enhanced and deactivated version of the OsCas12f1 form (referred to herein as dEnOs Cas) from the relatively compact Cas12f family of proteins, could be built. It was also shown that in these constructs co-localization between the episome and the targeted region of the genome occurred with fluorescence in-situ hybridization (FISH) using probes targeting the episome and the targeted region of the genome, indicating successful tether binding (FIG. 7). Example 2 – Engineered Extrachromsomal DNA System with Alternative DNA Binding Proteins Similar to the dCas9-based tether system, multiple tether system variants using alternative DNA-binding modalities, including the Tet Repressor (TetR) protein (FIG. 13A) and Zinc Finger (ZF) proteins (FIG.14A) were developed. Alternative DNA-binding modality systems comprise guide RNA (gRNA) cassettes and / or Zinc Finger (ZF) or Tet Operator (TetO) binding sites, and a DNA replication (not viral propagation) cassette as a platform for delivering therapeutic payloads, like genetic circuits, to primary human cells (FIG. 13B, FIG. 14B). A fluorescent marker or safety switch component can be added to test persistence or reversibility. The tethering system comprises two DNA-binding modalities that are attached to one another (FIG.13A, FIG.14A), either through direct fusion but can also be attached through fusions with domains that can be inducibly and reversibly dimerized by administering a small molecule. Since the DNA-binding modalities used are catalytically inactive, the tether system does not cleave either the chromosomal or episomal DNA. Each of these alternative tether systems (TetR- or ZF-based) comprise one DNA-binding modality that binds exclusively to a sequence only present within the host genome (e.g. Zinc Fingers that bind to specific genomic loci or repeats) and another DNA-binding modality that will bind exclusively to a sequence only present within the episomal or extrachromosomal DNA (e.g. Zinc Fingers that target a sequence orthogonal to the human genome or a Tet Repressor that targets a set of Tet Operator, or TetO, sequences that are not present within the human genome). Orthogonality between both DNA-binding modalities ensures that each respective protein will associate with either the genomic DNA or episome or extrachromosomal DNA without cross interference. In the case of a tether system consisting of catalytically dead Sp. Cas9 (SpdCas9) and TetR, the TetR DNA-binding modality will only target the Tet Operator sites on the episome, while the SpdCas9 only targets specific site(s) on the host genome (FIG. 13A). In the case of a tether system consisting of two Zinc Fingers, the episomal Zinc Finger will target a sequence on the episome that is orthogonal to, or not present in, the human genome, while the genomic Zinc Finger only targets specific site(s) on the host genome (FIG. 14A). In all of these tether systems, attachment of the dCas9 orthologs to one another tethers the episome to the host genome, conferring mitotic stability to the episome as will replicate with and bind to the chromosomal DNA of both daughter cells in mitosis rather than only being in one. In the specific case of a tether system including the Tet Repressor (TetR, also referred to as rtTA), the addition of doxycycline (dox) can cause the TetR protein to disengage from the Tet Operator (TetO) sites present on the episomal DNA (FIG. 13A). Inability of the TetR protein to bind to the TetO sites will cause episomal dilution over time and prevent the maintenance and / or establishment of mitotic stability. Fluorescence in situ hybridization (FISH) was performed with probes targeting either the episome or the targeted genomic locus to confirm the ability of the SpdCas9-TetR tether to simultaneously engage the episomal and genomic DNA. Co-localization occurs in samples where the tether is delivered using a functional SpdCas9 gRNA, indicating successful tether binding (FIG. 13C). An HA tag was added to the N terminus of the SpdCas9-TetR tether that binds the genome (Sp. dCas9), and a FLAG tag was added to the C terminus of the SpdCas9-TetR tether that binds the targeted episomal sequence (TetR) (FIG. 13B). Stable expression of the SpdCas9-TetR tether protein is confirmed using western blots targeting the HA and FLAG tags (FIG. 13D). Presence of both HA-SpdCas9 and the full SpdCas9-TetR tether on the HA western blot confirms that attachment to TetR via a peptide linker ((G4S)3) does not impede expression of the N-terminal SpdCas9 within the SpdCas9-TetR protein. Presence of both TetR-FLAG and the full SpdCas9-TetR tether on the FLAG western blot confirms that attachment to SpdCas9 via a peptide linker ((G4S)3) does not impede expression of the C- terminal TetR within the SpdCas9-TetR protein. The same methods to confirm SpdCas9-TetR tether functionality (FISH for tether formation, Western Blot for protein expression) will be used to validate future tether systems containing alternative DNA-binding domains, such as systems consisting of a genomic-targeting Zinc Finger and episomal-targeting Zinc Finger linked together (FIG. 14D). Example 3 – Application of Extrachromosomal Technology in Therapeutically Relevant Cell Types Expanding the application extrachromosomal Genetic Technology (EGT) to primary cell types with significant clinical potential, such as primary T cells, mesenchymal stem cells (MSCs), and murine neurons, could offer valuable insights into cellular dynamics and therapeutic strategies. Integration-deficient lentiviral vectors (IDLVs) were utilized that contain integrase mutations, which inhibit proviral integration, leading to higher levels of circular vector episomes in the transduced cells. Since IDLVs containing the LaminB2 repliconhave limitations on the size of additional payloads, two different IDLVs (IDLV- and IDLV-were employed to co-transduce primary T cells. IDLV- carries a compact version of thetether (SadCas9-linker-dEnOs), while IDLV- with LaminB2. Both IDLVs were co-transduced at a 1:1 ratio into CD3 / CD28-activated primary T cells. After 96 hours post-co-transduction, the expression levels of the surface marker were measured by flow cytometry using an anti-Thy1.1 antibody (Fig 8A-8C). Threedifferent IDLV- constructs were used targeting varying numbers of genomic sites: gDen-Scr(n=0), gDen-AAVS1 (n=1), and gDen-Line1 (n=~3800). Additionally, two IDLV- were employed that harbor the surface expression marker Thy1.1, with one containing LaminB2 and the other without it. A total of six conditions for co-transduction were tested: gDen-Scr + No_LaminB2-Thy1.1, gDen-AAVS1 + No_LaminB2-Thy1.1, gDen-Line1 + No_LaminB2-Thy1.1, gDen-Scr + LaminB2_Thy1.1, gDen-AAVS1 + LaminB2_Thy1.1, and gDen-Line1 + LaminB2_Thy1.1. Six control conditions were also tested: gDen-Scr only, gDen-AAVS1 only, gDen-Line1 only, LaminB2-Thy1.1 only, NoLaminB2-Thy1.1 only, and untransduced cells (Fig 9A-9B). Thy1.1 expression was measured 6 and 10 days post co- transduction in primary T cells. The x-axis represents the different co-transduction conditions and control groups at both Day 6 and Day 10, while the y-axis shows the percentage of Thy1.1 expression (Fig 10). It was observed that, at both Day 6 and Day 10 post co-transduction, constructs containing LaminB2 (denoted by the black box) and the tether exhibit higher levels of Thy1.1 expression compared to constructs lacking LaminB2. These findings suggest that the human replicon LaminB2, in combination with the tether, enables sustained expression of surface markers in primary T cells for up to 10 days in vitro. In FIGS. 11 and 12, T cells were co-transduced with a SadCas9-dEnOs IDLV with a Thy 1.1 marker and an anti-CD19 CAR IDLV with an NGFR marker. A difference was observed by 72 hours between NGFR expression in T cells transduced with constructs lacking LaminB2 and constructs harboring LaminB2. * * * * * * * * * All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0020] REFERENCES The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. International Patent Publication No. WO94 / 016737 Kyte et al., J. Mol. Biol. 157:105-132 (1982). Macejak and Sarnow, Nature, 353:90-94, 1991. Pelletier and Sonenberg, Nature, 334(6180):320-325, 1988. Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor, 1989. U.S. Patent No. 4,683,202 U.S. Patent No. 5,593,972 U.S. Patent No. 5,925,565 U.S. Patent No. 5,928,906 U.S. Patent No. 5,935,819 U.S. Patent No. 5,962,428 U.S. Patent Publication No. US20040175727

Claims

WHAT IS CLAIMED:

1. An extrachromosomal nucleic acid tether system comprising an episomal vector encoding: (a) a first nucleic acid-binding protein and a chromosomal specific target sequence; (b) a second nucleic acid-binding protein and an episomal specific target sequence; and (c) a target transgene.

2. The extrachromosomal nucleic acid tether system of claim 1, wherein the nucleic acid is DNA or RNA.

3. The extrachromosomal nucleic acid tether system of claim 1, wherein the target transgene encodes a genetic circuit, kill switch, or therapeutic agent.

4. The extrachromosomal nucleic acid tether system of claim 1, wherein the first nucleic acid-binding protein is attached to the second DNA-binding protein through a linker or fused to an inducible dimerized domain.

5. The extrachromosomal nucleic acid tether system of claim 1, further comprising a human origin of DNA replication.

6. The extrachromosomal nucleic acid tether system of claim 1, wherein the chromosomal specific target sequence is specific to a chromosomal locus or repetitive element.

7. The extrachromosomal nucleic acid tether system of claim 6, wherein the chromosomal locus is beta-globin or AAVS1.

8. The extrachromosomal nucleic acid tether system of claim 6, wherein the repetitive element is an Alu repeat, tandem repeat, short interspersed nuclear element (SINE), long interspersed nuclear element (LINE), or telomeric repeat.

9. The extrachromosomal nucleic acid tether system of claim 2, wherein the system comprises a first DNA-binding protein and / or a second DNA-binding protein selected from the group consisting of a Cas9 protein, transcription activator-like effector nuclease (TALEN), and zinc finger protein (ZFP).

10. The extrachromosomal nucleic acid tether system of claim 9, wherein the first DNA- binding protein is Streptococcus pyogenes deactivated Cas9 (SpdCas9) and the second DNA-binding protein is Staphylococcus aureus deactivated Cas9 (SadCas9).

11. The extrachromosomal nucleic acid tether system of claim 9, wherein the chromosomal specific target sequence and episomal specific target sequence are further defined as guide RNAs.

12. The extrachromosomal nucleic acid tether system of claim 11, wherein the guide RNAs are under the control of two separate promoters.

13. The extrachromosomal nucleic acid tether system of claim 12, wherein the promoters are RNA polymerase III (pol III) type 3 promoters.

14. The extrachromosomal nucleic acid tether system of claim 1, further comprising a safety switch to control the linkage between the first nucleic acid-binding protein and second nucleic acid-binding protein.

15. The extrachromosomal nucleic acid tether system of claim 1, further comprising a coding region for a detectable moiety and / or replicon.

16. The extrachromosomal nucleic acid tether system of claim 15, wherein the replicon is LaminB2 replicon, C-Myc, or a site-specific dCas9-based replicator.

17. The extrachromosomal nucleic acid tether system of claim 1, wherein the episomal vector is comprised in an adenovirus, integrase deficient lentivirus, baculovirus, coronavirus, Sendai virus, or herpesvirus vector.

18. A method of engineering a cell to express a target transgene comprising transducing said cell with an extrachromosomal nucleic acid tether system of any one of claims 1- 17.

19. The method of claim 18, wherein the cell is an immune cell, T cell, NK cell, mesenchymal stem cell, or neuron.

20. A method for delivering a gene therapy to a subject comprising introducing to said subject a plurality of cells engineered to express an extrachromosomal nucleic acid tether system according to any one of claims 1-17.

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