miRNA pathways for regulated gene expression

Abstract

Methods, compositions, and kits suitable for use in the controlled gene quantity-independent expression of a payload protein are disclosed herein. The compositions provided herein (e.g., nucleic acid compositions, one or more cells) may comprise a first promoter sequence operably ligated to a first polynucleotide containing one or more miRNA cassettes, and a second promoter sequence operably ligated to a second polynucleotide containing a payload gene. In some embodiments, the first promoter sequence is capable of inducing transcription of the first polynucleotide to produce a first transcript, which is capable of being processed to produce the miRNA. The payload gene may comprise a miRNA target region containing one or more miRNA target sequences.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Application No. 63 / 540,300, filed on 25 September 2023, and U.S. Provisional Application No. 63 / 563,033, filed on 8 March 2024. The entire contents of these applications are expressly incorporated herein by reference.

[0002] Statement on federally supported research and development This invention was developed with government support under grant number EB030015 from the National Institutes of Health. The government has certain rights in this invention.

[0003] References to sequence listings This application is filed together with an electronic sequence listing. The sequence listing is provided as a file titled "30KJ-365875-WO," created on 24 September 2024, and its size is 86,964 bytes. The information in the electronic sequence listing is incorporated herein by reference in its entirety.

[0004] field This application generally relates to the field of polynucleotide delivery and expression. [Background technology]

[0005] Description of related technologies The ability to express transgenes at specified levels is crucial for understanding cellular behavior and for applications in gene and cell therapy. Transfection, viral vectors, and other gene delivery methods result in varying protein expression levels and limited quantitative control, while targeted knock-in and stable selection are inefficient and slow. Active compensation mechanisms can improve precision, but their widespread use has been hindered by the need for additional proteins or a lack of moduloability. Research and biotechnology require compact, synthetic miRNA-based regulatory circuits that enable moduloable, orthogonal, generalizable, dose-invariant gene expression control.

[0006] In some embodiments, a composition (e.g., a nucleic acid composition, one or more cells, a DIMMER circuit) is provided. A nucleic acid composition is disclosed herein. In some embodiments, the nucleic acid composition includes a first promoter sequence operably ligated to a first polynucleotide containing one or more miRNA cassettes. In some embodiments, the first promoter sequence is capable of inducing transcription of the first polynucleotide to produce a first transcript. In some embodiments, the first transcript is capable of being processed to produce the miRNA. In some embodiments, the nucleic acid composition includes a second promoter sequence operably ligated to a second polynucleotide containing a payload gene. In some embodiments, the payload gene includes a miRNA target region containing one or more miRNA target sequences. In some embodiments, the second promoter sequence is capable of inducing transcription of a second polynucleotide to produce a payload transcript. In some embodiments, the payload transcript is capable of being translated to produce a payload protein. In some embodiments, the first promoter sequence and the second promoter sequence are components of a bidirectional promoter. In some embodiments, the first promoter sequence and the second promoter sequence are oriented in a reverse complementary orientation with respect to each other in a bidirectional promoter.

[0007] A composition comprising one or more cells is disclosed herein. In some embodiments, one or more cells comprises a first promoter sequence operably ligated to a first polynucleotide comprising one or more miRNA cassettes. In some embodiments, the first promoter sequence is capable of inducing transcription of the first polynucleotide to produce a first transcript. In some embodiments, the first transcript is capable of being processed to produce the miRNA. In some embodiments, one or more cells comprises a second promoter sequence operably ligated to a second polynucleotide comprising a payload gene. In some embodiments, the payload gene comprises a miRNA target region comprising one or more miRNA target sequences. In some embodiments, the second promoter sequence is capable of inducing transcription of the second polynucleotide to produce a payload transcript. In some embodiments, the payload transcript is capable of being translated to produce a payload protein. In some embodiments, the first promoter sequence and the second promoter sequence are components of a bidirectional promoter. In some embodiments, the first promoter sequence and the second promoter sequence are oriented in a reverse complementary orientation with respect to each other in the bidirectional promoter.

[0008] In some embodiments, the miRNA can ligate one or more miRNA target sequences, thereby reducing the stability of the payload transcript and / or reducing the translation of the payload transcript. In some embodiments, the first polynucleotide comprises a gene quantity gene. In some embodiments, the first transcript can be translated to produce a gene quantity indicator protein. In some embodiments, the intron is located at the 3'UTR of the gene quantity gene, at the 5'UTR of the gene quantity gene, or between the exons of the gene quantity gene, and is, as appropriate, a synthetic intron. In some embodiments, the intron comprises one or more miRNA cassettes. In some embodiments, the intron comprises one or more of the following: (i) an intron insertion encoding a miRNA, (ii) a donor splice site, (iii) an acceptor splice site, (iv) a branching domain, and (v) a polypyrimidine tract. In some embodiments, miRNA, or its precursor, can be released from the intron by an intron excision mechanism selected from the group including cellular RNA splicing and / or processing mechanisms, nonsense mutation-dependent degradation (NMD) processing, or any combination thereof, and the precursor may optionally include pri-miRNA. In some embodiments, the gene dose indicator protein is detectable, and the gene dose indicator protein may optionally include green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), TagRFP, Dronpa, Padron, mApple, mCherry, mruby3, rsCherry, rsCherryRev, their derivatives, or any combination thereof.

[0009] In some embodiments, one or more cells comprise two or more cells. In some embodiments, two or more cells comprise a first cell and a second cell. In some embodiments, the first cell is a first cell type, and / or the second cell is a second cell type. In some embodiments, (i) first and second cell types and / or (ii) one or more cells include antigen-presenting cells, dendritic cells, macrophages, nerve cells, brain cells, astrocytes, microglia cells, and neurons, spleen cells, lymphoid cells, lung cells, lung epithelial cells, skin cells, keratinocytes, endothelial cells, alveolar cells, alveolar macrophages, alveolar lung cells, vascular endothelial cells, mesenchymal cells, epithelial cells, colon epithelial cells, hematopoietic cells, bone marrow cells, Claudius cells, Hensen cells, Merkel cells, Müller cells, Paneth cells, Purkinje cells, Schwann cells, Sertoli cells, eosinophilic cells, acinar cells, lipoblasts, adipocytes, brown or white alpha cells, amacrine cells, beta cells, capsule cells, cementocytes, chief cells, chondrocytes, chondrocytes, chromaffin cells, pigment-averse cells, adrenocorticotropic hormone-secreting cells, delta cells, and lan Gerhans cells, follicular dendritic cells, enterochromaffin cells, ependymal cells, epithelial cells, basal cells, squamous epithelial cells, endothelial cells, transitional epithelial cells, erythroblasts, erythrocytes, fibroblasts, fibrous cells, follicular cells, germ cells, gametes, eggs, sperm, oocytes, primary oocytes, secondary oocytes, spermatids, spermatocytes, primary spermatocytes, secondary spermatocytes, germ epithelium, giant cells, glial cells, astroblasts, astrocytes, oligodendrocytes, oligo Godendrocytes, glioblasts, goblet cells, gonadotropin-secreting cells, granulosa cells, hematoblasts, hair cells, hepatoblasts, hepatocytes, vitreous cells, stromal cells, juxtaglomerular cells, keratinocytes, corneal stromal cells, Lemmal cells, leukocytes, granulocytes, basophils, eosinophils, neutrophils, lymphoblasts, B-lymphoblasts, T-lymphoblasts, lymphocytes, B-lymphoblasts, T-lymphoblasts, helper-induced T-lymphoblasts, Th1 T-lymphoblasts, Th2T lymphocytes, natural killer cells, thymocytes, macrophages, Kupffer cells, alveolar macrophages, foam cells, histiocytes, luteal cells, lymphoid stem cells, lymphoid cells, lymphoid stem cells, macroglia, mammotrophs, mast cells, medulloblasts, megakaryoblasts, megakaryocytes, melanoblasts, melanocytes, mesangial cells, mesothelial cells, metamyelocytes, monoblasts, monocytes, myxone neck cells, myoblasts, muscle cells, muscle cells, cardiomyocytes, skeletal muscle cells, smooth muscle cells, myelocytes, myeloid cells, myeloid stem cells, myoblasts, myoepithelial cells, myofibroblasts, neuroblasts, neuroepithelial cells, neurons, The group is selected from a group including odontoblasts, osteoblasts, osteoclasts, osteocytes, acid-secreting cells, parafollicular cells, paraluteal cells, digestive cells, pericytes, peripheral blood mononuclear cells, pheochromocytes, phalangeal cells, pineal cells, pituitary cells, plasma cells, platelets, podocytes, proerythroblasts, promonocytes, promyeloblasts, promyelocytes, pronortherythroblasts, reticulocytes, retinal pigment epithelial cells, retinoblasts, small cells, growth hormone-secreting cells, stem cells, supporting cells, teloglycytic cells, enzymatic cells, or any combination thereof, and optionally, stem cells include embryonic stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem / progenitor cells (HSPCs), or any combination thereof.

[0010] In some embodiments, the first polynucleotide is transcribed at a rate at least 1.1 times faster in the first cell compared to the second cell. In some embodiments, the first polynucleotide is translated at a rate at least 1.1 times faster in the first cell compared to the second cell. In some embodiments, the first polynucleotide is present in at least 1.1 times more copies in the first cell compared to the second cell. In some embodiments, the threshold gene amount at which payload protein expression is saturated is at least 1.1 times higher in the first cell compared to the second cell. In some embodiments, the transcription rate of the second polynucleotide and / or the translation rate of the payload transcript vary between a first and second time point in a single cell, and / or vary between the first and second cells at the same time point. In some embodiments, in the absence of miRNA, the payload protein reaches an unadjusted steady-state payload protein level in one or more cells. In some embodiments, the unadjusted steady-state payload protein level falls within the range between the lower and upper unadjusted thresholds of the unadjusted expression range. In some embodiments, the steady-state gene quantity indicator protein level reflects the unadjusted steady-state payload protein level. In some embodiments, in the presence of miRNA, the payload protein reaches the adjusted steady-state payload protein level in one or more cells.

[0011] In some embodiments, the adjusted steady-state payload protein level falls within the range between the lower and upper adjusted thresholds of the adjusted expression range, where the upper adjusted threshold is the saturation expression level, as appropriate. In some embodiments, at first and second time points in a single cell, the steady-state level of the payload protein remains within the adjusted expression range. In some embodiments, at the same time points in the first and second cells, the steady-state level of the payload protein remains within the adjusted expression range. In some embodiments, the lower and / or upper adjusted thresholds of the adjusted expression range can be set by adjusting one or more of the following: (i) the number of miRNA cassettes in the first polynucleotide, (ii) the number of miRNA target sequences in the miRNA target region, (iii) the complementarity between the miRNA and one or more miRNA target sequences, and (iv) the intensity of the first and / or second promoter sequences. In some embodiments, the difference between the lower and upper unadjusted thresholds of the unadjusted expression range is greater than about two orders of magnitude. In some embodiments, the difference between the lower and upper adjusted thresholds of the adjusted expression range is less than approximately one order of magnitude. In some embodiments, the payload protein is effective at steady-state payload protein levels within the adjusted expression range. In some embodiments, the payload protein is ineffective and / or toxic at steady-state payload protein levels above and / or below the adjusted expression range. In some embodiments, the payload protein can induce an immunogenic response and / or a cytokine storm at steady-state payload protein levels outside the adjusted expression range. In some embodiments, the adjusted steady-state payload protein level includes therapeutic levels of payload protein. In some embodiments, the steady-state payload protein level remains within the adjusted expression range across multiple cell types, viral vector titers, and / or viral vector capsid types. In some embodiments, the adjusted steady-state payload protein level is robust to tissue-targeted and stochastic expression.

[0012] In some embodiments, the miRNA contains a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NOs. 75–87. In some embodiments, the miRNA cassette contains a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NOs. 1–13. In some embodiments, the miRNA has a nucleotide (nt) length of approximately 20, 21, 22, 23, or 24. In some embodiments, the miRNA sequence is orthogonal to one or more cells and, as appropriate, is a miRNA sequence that targets sea urticaria luciferase. In some embodiments, the miRNA target region is located in the 3'UTR, 5'UTR, or coding region of the payload gene. In some embodiments, the miRNA target region includes a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NOs. 14–74. In some embodiments, the miRNA target region includes at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 miRNA target sequences. In some embodiments, the payload transcript can be simultaneously bound by multiple miRNA-loaded Argonaut (Ago) complexes, optionally via TNRC6. In some embodiments, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 nucleotides (nt) of the miRNA target sequence are complementary to the miRNA, and, as appropriate, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nt at the 3' end of the miRNA are not complementary. In some embodiments, the complementarity between the miRNA and the miRNA target sequence is at least, or at most, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.In some embodiments, the miRNA contains 5 to 8 GC nucleotides, optionally 1 to 4 in the seed region and 1 to 2 in the broader region. In some embodiments, the composition comprises one or more additional payload genes and one or more additional miRNAs, wherein the additional miRNAs are sequence-different with respect to one another.

[0013] In some embodiments, the composition includes, for each separate additional miRNA, an additional first promoter sequence operably ligated to an additional first polynucleotide comprising one or more additional miRNA cassettes, wherein the additional first promoter sequence is capable of inducing transcription of the additional first polynucleotide to produce an additional first transcript, which can be processed to produce the additional miRNA; and an additional second promoter sequence operably ligated to an additional second polynucleotide comprising an additional payload gene, wherein the additional payload gene comprises an additional miRNA target region comprising one or more additional miRNA target sequences, wherein the additional second promoter sequence is capable of inducing transcription of the additional second polynucleotide to produce an additional payload transcript, which can be translated to produce an additional payload protein.

[0014] In some embodiments, the first promoter sequence and / or the second promoter sequence include a ubiquitous promoter, which may optionally be a cytomegalovirus (CMV) early promoter, a CMV promoter, a viral monkey virus 40 (SV40) (e.g., early or late), a Moloney's mouse leukemia virus (MoMLV) LTR promoter, a Roussarcoma virus (RSV) LTR, an RSV promoter, a herpes simplex virus (HSV) (thymidine kinase) promoter, or a vaccinia virus promoter. Derived H5, P7.5, and P11 promoters, elongation factor 1-alpha (EF1a) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5 (HSPA5), heat shock protein 90kDa beta, member (HSP90B1), heat shock protein 70kDa (HSP70), β-kinesin (β-KIN), human ROSA The group includes 26 loci, ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, 3-phosphoglycerate kinase promoter, cytomegalovirus enhancer, human β-actin (HBA) promoter, chicken β-actin (CBA) promoter, CAG promoter, CBH promoter, or any combination thereof. In some embodiments, the first promoter sequence and / or the second promoter sequence is an inducible promoter, which may be a tetracycline-responsive promoter, TRE promoter, Tre3G promoter, ecdysone-responsive promoter, qumate-responsive promoter, glucocorticoid-responsive promoter, and estrogen-responsive promoter, PPAR-γ promoter, or RU-486-responsive promoter. In some embodiments, the first promoter sequence and / or the second promoter sequence includes a tissue-specific promoter and / or a lineage-specific promoter.In some embodiments, the tissue-specific promoter is a liver-specific thyroxine-binding globulin (TBG) promoter, insulin promoter, glucagon promoter, somatostatin promoter, pancreatic polypeptide (PPY) promoter, synapsin-1 (Syn) promoter, creatine kinase (MCK) promoter, mammalian desmin (DES) promoter, α-myosin heavy chain (a-MHC) promoter, or cardiac troponin T (cTnT) promoter.

[0015] In some embodiments, the tissue-specific promoter is a neuron-specific promoter, and optionally, the neuron-specific promoter is the synapsin-1 (Syn) promoter, CaMKIIa promoter, calcium / calmodulin-dependent protein kinase IIa promoter, tubulin alpha I promoter, neuron-specific enolase promoter, platelet-derived growth factor beta chain promoter, TRPV1 promoter, Na v 1.7 promoter, Na v 1.8 promoter, Na v 1.9 promoter, or Advillin promoter. In some embodiments, the tissue-specific promoter is a muscle-specific promoter, and optionally, the muscle-specific promoter comprises the creatine kinase (MCK) promoter. In some embodiments, the first promoter sequence and / or the second promoter sequence is a methyl CpG binding protein 2 (MeCP2) promoter or a derivative thereof, and optionally, the MeCP2 promoter or derivative thereof comprises a MeCP2 promoter truncated to about 229 base pairs or a MeCP2 promoter truncated to about 406 base pairs. In some embodiments, the one or more cells comprise an endogenous version of the payload gene, wherein the promoter comprises the promoter of the endogenous version or is derived from the promoter of the endogenous version.

[0016] In some embodiments, the payload protein comprises a disease-related protein, where the abnormal expression of the disease-related protein correlates with the onset and / or progression of the disease. In some embodiments, the payload protein comprises a protein associated with an expression-sensitive disease or disorder. In some embodiments, the payload protein comprises methyl CpG-binding protein 2 (MeCP2), SMN, DRK1A, KAT6A, NIPBL, HDAC4, UBE3A, EHMT1, one or more genes encoded on chromosome 9q34.3, NPHP1, LIMK1, one or more genes encoded on chromosome 7q11.23, P53, TPI1, FGFR1 and related genes, RA1, SHANK3, CLN3, NF-1, TP53, PFK, CD40L, CYP19A1, PGRN, CHRNA7, PMP22, CD40LG, derivatives thereof, or any combination thereof. In some embodiments, the payload protein comprises an imaging agent, which may optionally include green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), TagRFP, Dronpa, Padron, mApple, mCherry, mruby3, rsCherry, rsCherryRev, their derivatives, or any combination thereof, and may optionally be conjugated to an endogenous or exogenous protein. In some embodiments, the payload protein comprises an endogenous or exogenous protein conjugated to a binding domain, which may optionally be configured for separation or imaging.In some embodiments, the payload protein comprises a programmable nuclease, which may optionally be SpCas9 or its derivatives; VRER, VQR, EQR SpCas9; xCas9-3.7; eSpCas9; Cas9-HF1; HypaCas9; evoCas9; HiFi Cas9; ScCas9; StCas9; NmCas9; SaCas9; CjCas9; CasX; Cas9 The group is selected from H940A nickase; Cas12 and its derivatives; dCas9-APOBEC1 fusion, BE3, and dCas9-deaminase fusion; dCas9-Krab, dCas9-VP64, dCas9-Tet1, and dCas9-transcription regulator fusion; Dcas9-fluorescent protein fusion; Cas13-fluorescent protein fusion; RCas9-fluorescent protein fusion; and Cas13-adenosine deaminase fusion. In some embodiments, the programmable nuclease includes zinc finger nucleases (ZFNs) and / or transcription activator-like effector nucleases (TALENs).In some embodiments, the programmable nuclease comprises Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), zinc finger nuclease, TAL effector nuclease, meganuclease, MegaTAL, Tev-m TALEN, MegaTev, homing endonuclease, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, C2c1, C2c3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, derivatives thereof, prime editing versions thereof, or any combination thereof. The composition can comprise (i) a targeting molecule and / or (ii) a polynucleotide encoding a donor nucleic acid. In some embodiments, the targeting molecule is capable of binding to the programmable nuclease, optionally wherein the targeting molecule comprises single-stranded DNA or single-stranded RNA, further optionally wherein the targeting molecule comprises a single guide RNA (sgRNA). In some embodiments, the payload protein is capable of modulating the expression, concentration, localization, stability, and / or activity of one or more endogenous proteins of a cell. In some embodiments, the payload protein is a therapeutic protein or a variant thereof, optionally a therapeutic protein configured to prevent or treat a disease or disorder in a subject, further optionally wherein the subject suffers from a deficiency of said therapeutic protein.

[0017] In some embodiments, the payload protein includes fluorescence activity, polymerase activity, protease activity, phosphatase activity, kinase activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, or any combination thereof. In some embodiments, the payload protein includes nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimerization activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosylase activity, acetyltransferase activity, deacetylase activity, adenylation activity, deadenylation activity, or any combination thereof. In some embodiments, the payload protein includes CRE recombinase, GCaMP, cell therapy components, knockdown gene therapy components, epitopes exposed on the cell surface, or any combination thereof. In some embodiments, the payload protein includes a bispecific T cell engager (BiTE).In some embodiments, the payload protein comprises a cytokine, and optionally the cytokine is interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-1 9, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32 , IL-33, IL-34, IL-35, interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-1 Selected from the group consisting of 0, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, granulocyte-macrophage colony-stimulating factor (GM-CSF), M-CSF, SCF, TSLP, oncostatin M, leukemia suppressor (LIF), CNTF, cardiotropin-1, NNT-1 / BSF-3, growth hormone, prolactin, erythropoietin, thrombopoietin, leptin, G-CSF, or their receptors or ligands. In some embodiments, the payload protein includes a member of the TGF-beta / BMP family selected from the group consisting of TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-3a, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-9, BMP-10, BMP-11, BMP-15, BMP-16, endometrial hemorrhage-related factor (EBAF), growth differentiation factor-1 (GDF-1), GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-12, GDF-14, Müllerian inhibitor (MIS), activin-1, activin-2, activin-3, activin-4, and activin-5.In some embodiments, the payload protein includes a cytokine member of the TNF family selected from the group consisting of TNF-alpha, TNF-beta, LT-beta, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, and 4-1 BBL. In some embodiments, the payload protein includes a cytokine member of the immunoglobulin superfamily selected from the group consisting of B7.1(CD80) and B7.2(B70). In some embodiments, the payload protein includes an interferon, which may be selected from interferon-alpha, interferon-beta, or interferon-gamma. In some embodiments, the payload protein comprises a chemokine, which may be selected from CCL1, CCL2, CCL3, CCR4, CCL5, CCL7, CCL8 / MCP-2, CCL11, CCL13 / MCP-4, HCC-1 / CCL14, CTAC / CCL17, CCL19, CCL22, CCL23, CCL24, CCL26, CCL27, VEGF, PDGF, lymphotactin (XCL1), eotaxin, FGF, EGF, IP-10, TRAIL, GCP-2 / CXCL6, NAP-2 / CXCL7, CXCL8, CXCL10, ITAC / CXCL11, CXCL12, CXCL13, or CXCL15. In some embodiments, the payload protein comprises an interleukin, which may be selected from IL-10, IL-12, IL-1, IL-6, IL-7, IL-15, IL-2, IL-18, or IL-21, as appropriate. In some embodiments, the payload protein comprises tumor necrosis factor (TNF), which may be selected from TNF-alpha, TNF-beta, TNF-gamma, CD252, CD154, CD178, CD70, CD153, or 4-1BBL, as appropriate. In some embodiments, the payload protein comprises a factor that locally downregulates the activity of endogenous immune cells. In some embodiments, the payload protein is capable of remodeling the tumor microenvironment at the target site and / or reducing immunosuppression.

[0018] In some embodiments, the payload protein comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the CAR and / or TCR comprises one or more of the following: an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a primary signaling domain, a costimulatory domain, or both a primary signaling domain and a costimulatory domain. In some embodiments, the primary signaling domain comprises a functional signaling domain of one or more proteins selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc epsilon R1b), CD79a, CD79b, Fc gamma RIIa, DAP10, and DAP12, or a functional variant thereof. In some embodiments, the co-stimulatory domain is a ligand that specifically binds to CD27, CD28, 4-1BB (CD137), OX40, CD28-OX40, CD28-4-1BB, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA- 6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244) , 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, C D100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8),The functional domain of one or more proteins selected from the group consisting of SELPLG(CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, and NKG2D, or a functional variant thereof. In some embodiments, the antigen-binding domain binds to a tumor antigen, and the tumor antigen is, as appropriate, a solid tumor antigen. In some embodiments, tumor antigens include CD19, CD123, CD22, CD30, CD171, CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), type C lectin-like molecule-1 (CLL-1 or CLECL1), CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), TNF receptor family member B cell maturation (BCMA), and Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), mesothelin, interleukin-11 receptor alpha (IL-11Ra), prostate stem cell antigen (PSCA), protease serine 21 (Testisin or PRSS21), vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor beta (PDGFR-beta), stage-specific embryonic antigen-4 (SSEA-4), CD20, folate receptor alpha, receptor tyrosine protein kinase ERBB2 (Her2 / neu), mucin 1, cell surface-related (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), prostase, prostatic acid phosphatase (PAP), elongation factor 2 mutant (ELF2M), ephrin B2, fibroblast-activating protein alpha (FAP),An oncogene fusion protein (bcr-abl) consisting of insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropain) subunit beta 9 (LMP2), glycoprotein 100 (gp100), cleavage cluster region (BCR), and Abelson mouse leukemia virus oncogene homolog 1 (Abl), tyrosinase, ephrin type A receptor 2 (EphA2), fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1 -4) bDGlcp(1-1)Cer), transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), folate receptor beta, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5 member D (GPRC5D), X chromosome open reading frame 61 (CXORF61), CD97, CD179a, undifferentiated ri Lymphoma kinase (ALK), polysialic acid, placenta-specific antigen 1 (PLAC1), hexasaccharide portion of globoH glycoceramide (GloboH), mammary gland differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), hepatitis A virus cell receptor 1 (HAVCR1), adrenergic receptor beta 3 (ADRB3), panexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex locus K9 (LY6K), olfactory receptor 51E2 (OR51E2), TCR gamma alternating leading frame protein (TARP). ), Wilms tumor protein (WT1), cancer / testicular antigen 1 (NY-ESO-1), cancer / testicular antigen 2 (LAGE-1a), melanoma-associated antigen 1 (MAGE-A1), ETS translocation mutant gene 6 located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X antigen family member 1A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie2), melanoma cancer testicular antigen-1 (MAD-CT-1), melanoma cancer testicular antigen-2 (MAD-CT-2), Fos-associated antigen 1, tumor protein p53 (p53),p53 mutant, prostain, survivorin, telomerase, prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), T cell-recognized melanoma antigen 1 (MelanA or MART1), rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoint, apoptotic melanoma inhibitor (ML-IAP), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosamine Niltransferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myelocytosis virus oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosine kinase-related protein 2 (TRP-2), cytochrome P4501B1 (CYP1B1), CCCTC binding factor (zinc finger protein)-like (BORIS or Brother) (of the Regulator of Imprinted Sites), squamous cell carcinoma antigen 3 (SART3) recognized by T cells, paired box protein Pax-5 (PAX5), proacrosin-binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A kinase anchor protein 4 (AKAP-4), synovial sarcoma X-section 2 (SSX2), advanced glycation end product receptor (RAGE-1), renal ubiquitous protein 1 (RU1), renal ubiquitous protein 2 (RU2), regmine, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), enteric carboxylesterase, heat shock protein 70-2 mutant (mut hsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR or CD89), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), glypican-3 (GPC3),The tumor antigen is selected from the group consisting of Fc receptor-like 5 (FCRL5) and immunoglobulin lambda-like polypeptide 1 (IGLL1). In some embodiments, the tumor antigen is selected from the group consisting of CD150, 5T4, ActRIIA, B7, BMCA, CA-125, CCNA1, CD123, CD126, CD138, CD14, CD148, CD15, CD19, CD20, CD200, CD21, CD22, CD23, CD24, CD25, CD26, CD261, CD262, CD30, CD33, CD362, CD37, CD38, CD4, CD40, CD40L, CD44, CD46, CD5, CD52, CD53, CD54, CD56, CD66a-d, CD74, CD8, CD80, CD92, CE7, CS-1, CSPG4, and ED-B fibronectin. EGFR, EGFRvIII, EGP-2, EGP-4, EPHa2, ErbB2, ErbB3, ErbB4, FBP, GD2, GD3, HER1-HER2 combination, HER2-HER3 combination, HERV-K, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, HLA-DR, HM1.24, HMW-MAA, Her2, Her2 / neu, IGF-1R, IL-11R-alpha, IL-13R-alpha2, IL-2, IL-22R-alpha, IL-6, IL-6R, Ia, Ii, L1-CAM, L1 cell adhesion molecule, Lewis Y, L1-CAM, MAGE A3, MAGE-A1, MART-1, MUC1, NKG2C ligand, NKG2D ligand, NY-ESO-1, OEPHa2, PIGF, PSCA, PSMA, ROR1, T101, TAC, TAG72, TIM-3, TRAIL-R1, TRAIL-R1(DR4), TRAIL-R2(DR5), VEGF, VEGFR2, WT-1, G protein-coupled receptor, alpha-fetoprotein (AFP), angiogenic factor, exogenous congenital linking molecule (ExoCBM), oncogene product, folate antagonist receptor, c-Met, carcinoembryonic antigen (CEA), cyclin (D1), ephrin B2, epithelial tumor antigen, estrogen receptor, fetal acetylcholine e) Receptor, folate-binding protein, gp100, hepatitis B surface antigen, kappa chain, kappa light chain, kdr, lambda chain, livin, melanoma-related antigen, mesothelin,Mouse double-minute 2 homolog (MDM2), mucin 16 (MUC16), mutant p53, mutant ras, necrotic antigen, carcinoembryonic antigen, ROR2, progesterone receptor, prostate-specific antigen, tEGFR, tenascin, β2-microglobulin, Fc receptor-like 5 (FcRL5), or those expressed by HIV, HCV, HBV, or other pathogens. The group is selected from the children. In some embodiments, the antigen-binding domain includes an antibody, an antibody fragment, scFv, Fv, Fab, (Fab')2, a single-domain antibody (SDAB), a VH or VL domain, a camelid VHH domain, Fab, Fab', F(ab')2, Fv, scFv, dsFv, a diabody, a triabody, a tetrabody, a polyspecific antibody formed from an antibody fragment, a single-domain antibody (sdAb), a single chain containing anti-complementary (cantiomplementary) scFv (tandem scFv) or a bispecific tandem scFv, an Fv construct, a disulfide-linked Fv, a bivariable-domain immunoglobulin (DVD-Ig) binding protein or nanobody, an aptamer, an afibody, an affin, an afitin, an affimer, an alphabody, an antikalin, an avimer, a DARPin, a finomer, a Knitz domain peptide, a monobody, or any combination thereof. In some embodiments, the antigen-binding domain is bound to the transmembrane domain by a hinge region.In some embodiments, the transmembrane domain is the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS( CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R Beta, IL2R Gamma, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD10 3, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNA M1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, The transmembrane domains of proteins selected from the group consisting of CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C, or functional variants thereof. In some embodiments, the CAR or TCR further comprises a leader peptide. In some embodiments, the TCR further comprises a constant region and / or CDR4.

[0019] In some embodiments, the payload protein is an activity regulator, which may optionally reduce T cell activity. In some embodiments, the activity regulator includes a ubiquitin ligase involved in TCR / CAR signaling, selected from the group including c-CBL, CBL-B, ITCH, RF125, RF128, WWP2, or any combination thereof. In some embodiments, the activity regulator includes a negative regulatory enzyme selected from the group including SHP1, SHP2, SHTP1, SHTP2, CD45, CSK, CD148, PTPN22, DGK alpha, DGK zeta, DRAK2, HPK1, HPK1, STS1, STS2, SLAT, or any combination thereof. In some embodiments, the activity regulator is a negative regulatory scaffold / adapter protein selected from the group including PAG, LIME, NTAL, LAX31, SIT, GAB2, GRAP, ALX, SLAP, SLAP2, DOK1, DOK2, or any combination thereof. In some embodiments, the regulatory factor is a dominant-negative version of an activated TCR signaling component selected from the group including ZAP70, LCK, FYN, NCK, VAV1, SLP76, ITK, ADAP, GADS, PLC gamma 1, LAT, p85, SOS, GRB2, NFAT, p50, p65, API, RAP1, CRKII, C3G, WAVE2, ARP2 / 3, ABL, ADAP, RIAM, SKAP55, or any combination thereof. In some embodiments, the regulatory factor includes the cytoplasmic tail of a negative co-regulating receptor selected from the group including CD5, PD1, CTLA4, BTLA, LAG3, B7-H1, B7-1, CD160, TFM3, 2B4, TIGIT, or any combination thereof. In some embodiments, the regulatory factor is targeted to the cell membrane with a targeting sequence derived from LAT, PAG, LCK, FYN, LAX, CD2, CD3, CD4, CD5, CD7, CD8a, PD1, SRC, LYN, or any combination thereof.In some embodiments, the activity regulator reduces or inhibits the function of a pathway and / or function selected from the group including Ras signaling, PKC signaling, calcium-dependent signaling, NF-kappa B signaling, NFAT signaling, cytokine secretion, T cell survival, T cell proliferation, CTL activity, degranulation, tumor cell death, differentiation, or any combination thereof.

[0020] In some embodiments, the payload protein is a cell reprogramming factor capable of converting at least partially differentiated cells into less differentiated cells, and is, as appropriate, Oct-3, Oct-4, Sox2, c-Myc, Klf4, Nanog, Lin28, ASCL1, MYT1L, TBX3b, SV40 Large T, hTERT, miR-291, miR-294, miR-295, or any combination thereof. In some embodiments, the payload protein is a cell reprogramming factor capable of differentiating a given cell to a desired differentiation state, and is, as appropriate, nerve growth factor (NGF), fibroblast growth factor (FGF), interleukin-6 (IL-6), bone morphogenetic protein (BMP), neurogenin 3 (Ngn3), pancreatic and duodenal homeobox 1 (Pdx1), Mafa, or any combination thereof. In some embodiments, the payload protein includes an agonist or antagonist antibody specific to a checkpoint inhibitor or checkpoint stimulant molecule, or an antigen-binding fragment thereof, optionally containing PD1, PD-L1, PD-L2, CD27, CD28, CD40, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, PD-1, and / or TIM-3. In some embodiments, the payload protein includes a constitutive signal peptide for proteolysis, optionally containing PEST. In some embodiments, the payload protein includes a nuclear localization signal (NLS) or nuclear export signal (NES). In some embodiments, the payload protein comprises components of a synthetic protein circuit, which may optionally include transcription factors, proteases, kinases, phosphatases, synthetic Notch (SynNotch) receptors, modular extracellular sensor architecture (MESA) receptors, Tango, or dCas9-synR.In some embodiments, the payload protein includes a degron and a protease cleavage site capable of cleaving the degron, where the exposed degron of the payload protein destabilizes the payload protein, and the degron may optionally include N-degron, dihydrofolate reductase (DHFR) degron, FKB protein (FKBP) degron, their derivatives, or any combination thereof. In some embodiments, the payload protein includes a protease or split protease, and the activation level of the protease may optionally be related to one or more input signals, and further optionally the protease may include tobacco etch virus (TEV) protease, tobacco vein spot virus (TVMV) protease, hepatitis C virus protease (HCVP), their derivatives, or any combination thereof.

[0021] In some embodiments, the payload protein is associated with a desired agricultural trait selected from the group consisting of increased yield, increased abiotic stress tolerance, increased drought tolerance, increased flood tolerance, increased heat tolerance, increased cold and frost tolerance, increased salt tolerance, increased heavy metal tolerance, increased low nitrogen tolerance, increased disease resistance, increased pest resistance, increased herbicide resistance, increased biomass production, male sterility, or any combination thereof. In some embodiments, the payload protein is associated with a biological manufacturing process selected from the group consisting of fermentation, distillation, biofuel production, compound production, polypeptide production, or any combination thereof.

[0022] In some embodiments, the payload protein includes a synthetic receptor, optionally a synthetic Notch (SynNotch) receptor, a modular extracellular sensor architecture (MESA) receptor, Tango, dCas9-synR, or any combination thereof. In some embodiments, the payload protein includes components of an RNA transport system, a lipid envelope nanoparticle (LN) production system, or a virus-like particle (VLP) production system, optionally an RNA transporter protein or a fusogen. In some embodiments, the nucleic acid composition includes approximately 1.0 kb, 1.5 kb, 2.0 kb, 2.5 kb, 3.0 kb, 3.5 kb, 4.0 kb, 4.5 kb, 5.0 kb, 5.5 kb, 6.0 kb, 6.5 kb, 7.0 kb, 7.5 kb, 8.0 kb, 8.5 kb, 9.0 kb, 9.5 kb, 10 kb, 12 kb, 15 kb, 20 kb, 25 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, or less than 100 kb. In some embodiments, miRNA expression disrupts the endogenous gene expression of one or more cells by less than approximately 10%. The method may include one or more nucleic acids encoding TNRC6, GW182, one or more miRNA biogenesis components, Exportin-5, Dicer, and / or one or more Argonaut proteins. In some embodiments, the first promoter sequence and the first polynucleotide are not operably ligated in the absence of a recombination event, and the first promoter sequence and the first polynucleotide are operably ligated after a recombination event so that the first promoter sequence can induce transcription of the first polynucleotide to produce a first transcript. In some embodiments, the first polynucleotide and the second polynucleotide are integrated into the genome of one or more cells.

[0023] In some embodiments, the composition is a vector, a ribonucleoprotein (RNP) complex, a liposome, nanoparticles, an exosome, a microvesicle, or any combination thereof. In some embodiments, the nucleic acid composition is complexed or bound to one or more lipid or lipid-based carriers to form liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes, which optionally encapsulate the nucleic acid composition. In some embodiments, the composition is one or more vectors, comprises one or more vectors, or further comprises one or more vectors. In some embodiments, at least one of the one or more vectors is a viral vector, a plasmid, a translocation element, a naked DNA vector, lipid nanoparticles (LNPs), or any combination thereof. In some embodiments, the viral vector is an AAV vector, lentiviral vector, retroviral vector, adenovirus vector, herpesvirus vector, herpes simplex virus vector, cytomegalovirus vector, vaccinia virus vector, MVA vector, baculovirus vector, vesicular stomatitis virus vector, human papillomavirus vector, avipoxvirus vector, Sindbisvirus vector, VEE vector, measles virus vector, influenza virus vector, hepatitis B virus vector, integration-deficient lentivirus (IDLV) vector, or any combination thereof. In some embodiments, the transposing element is a piggyback transposon or a sleeping beauty transposon. In some embodiments, the first polynucleotide and the second polynucleotide are included in one or more vectors. In some embodiments, the first polynucleotide and the second polynucleotide are included in the same vector and / or different vectors. In some embodiments, the first polynucleotide and the second polynucleotide are located on the same nucleic acid and / or different nucleic acids.In some embodiments, the viral vector is an AAV vector, a lentiviral vector, a retroviral vector, or an integration-deficient lentiviral (IDLV) vector. In some embodiments, the AAV vector includes a single-stranded AAV (ssAAV) vector or a self-complementary AAV (scAAV) vector. In some embodiments, the AAV vector includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, their derivatives, or any combination thereof. In some embodiments, the AAV vector includes an AAV9 variant engineered for systemic administration, optionally AAV-PHP.B, AAV-PHP.eB, or AAV-PHP.S. In some embodiments, the vector is a neurotropic viral vector, which, as appropriate, includes or is derived from the Herpesviridae family, varicella-zoster virus, pseudorabies virus, cytomegalovirus, Epstein-Barr virus, encephalitis virus, poliovirus, coxsackievirus, echovirus, mumps virus, measles virus, rabies virus, or any combination thereof.

[0024] Methods for treating a disease or disorder in a subject are disclosed herein. Such methods may include administering an effective amount of one or more cells disclosed herein to a subject to treat or prevent a disease or disorder in the subject.

[0025] Methods for treating a disease or disorder in a subject are disclosed herein. In some embodiments, the method includes introducing one or more cells with a nucleic acid composition disclosed herein, and administering an effective amount of the resulting cells to a subject to treat or prevent a disease or disorder in the subject. The method may include isolating one or more cells from the subject prior to the introduction step.

[0026] A method for treating a disease or disorder in a subject is disclosed herein. The method involves administering an effective amount of the nucleic acid composition disclosed herein to the subject to treat or prevent a disease or disorder in the subject.

[0027] Methods relating to the controlled gene dose-independent expression of a payload protein in one or more cells are disclosed herein. In some embodiments, such methods include introducing one or more cells into a nucleic acid composition disclosed herein.

[0028] In some embodiments, the transduction process is carried out in vivo, in vitro, and / or ex vivo. In some embodiments, the transduction process includes calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, electronuclear transport, chemotransduction, electrotransduction, lipofectamine-mediated transfection, effectene-mediated transfection, lipid nanoparticle (LNP)-mediated transfection, or any combination thereof. In some embodiments, one or more cells comprise one or more cells of the subject, which, as appropriate, suffer from a disease or disorder. In some embodiments, one or more cells comprise neurons, which, as appropriate, are associated with a neurological disease or disorder. In some embodiments, administration includes systemic administration, which, as appropriate, is intravenous, intramuscular, intraperitoneal, or intraarticular. In some embodiments, administration includes intrathecal administration, intracranial injection, aerosol delivery, nasal delivery, transvaginal delivery, rectal delivery, oral delivery, intraocular delivery, local delivery, local delivery, intracisional delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous injection, subcutaneous injection, intra-lymph node injection, intratumor injection, intraperitoneal injection, intradermal injection, or any combination thereof. In some embodiments, administration includes injection into a brain region, and further, as appropriate, administration includes direct administration into the brain parenchyma.

[0029] The method may include introducing an inducible promoter inducer into one or more cells, the inducer optionally comprising doxycycline, and the introduction step may further optionally include administering an initial dose of the inducer, followed by one or more lower maintenance doses of the inducer. In some embodiments, the disease or disorder includes MECP2-related disorders selected from the group including typical Rett syndrome, MECP2-associated severe neonatal encephalopathy, PPM-X syndrome, or any combination thereof. In some embodiments, the disease or disorder is a hematological disorder, an immunological disorder, cancer, an infection, a genetic disorder, a disorder caused by abnormal mtDNA, a metabolic disorder, a disorder caused by abnormal cell cycle, a disorder caused by abnormal angiogenesis, a disorder caused by abnormal DNA damage repair, or any combination thereof.

[0030] In some embodiments, the disease or disorder includes neurological diseases or disorders, and as appropriate, the neurological disease or disorder may be Alzheimer's disease, Creutzfeldt-Jakob syndrome / disease, bovine spongiform encephalopathy (BSE), prion-related infections, diseases with mitochondrial dysfunction, diseases with β-amyloid and / or tauopathy, Down syndrome, hepatic encephalopathy, Huntington's disease, motor neuron disease, amyotrophic lateral sclerosis (ALS), olivopontocerebellar atrophy, postoperative cognitive impairment (POCD), systemic lupus erythematosus, systemic sclerosis (systemicClerosis, Sjögren's syndrome, ceroid lipofuscinosis, neurodegenerative cerebellar ataxia, Parkinson's disease, Parkinson's dementia, mild cognitive impairment, cognitive deficits in various forms of mild cognitive impairment, cognitive deficits in various forms of dementia, Boxer dementia, vascular and frontal dementia, cognitive impairment, learning disability, eye injury, eye disease, eye disorder, glaucoma, retinopathy, macular degeneration, head or brain or spinal cord injury, head or brain or spinal cord trauma, convulsions, epileptic seizures , epilepsy, temporal lobe epilepsy, myoclonus epilepsy, tinnitus, dyskinesia, chorea, Huntington's chorea, athetosis, dystonia, stereotypic disorder, ballism, tardive dyskinesia, tic disorder, spasmodic torticollis, blepharospasm, focal and generalized dystonia, nystagmus, hereditary cerebellar ataxia, corticobasal degeneration, tremor, essential tremor, addiction, anxiety disorder, panic disorder, social anxiety disorder (SAD), attention deficit hyperactivity disorder (ADHD), attention deficit syndrome (ADS), le Stress Legs Syndrome (RLS), hyperactivity in children, autism, dementia, dementia associated with Alzheimer's disease, dementia associated with Korsakoff syndrome, Korsakoff syndrome, vascular dementia, dementia associated with HIV infection, HIV-1 encephalopathy, AIDS encephalopathy, AIDS-dementia complex, AIDS-associated dementia, major depressive disorder, major depression, depression, memory loss, stress, bipolar disorder, drug tolerance, drug tolerance to opioids, motor impairment, fragile X syndrome, irritable bowel syndrome (I) This includes BS), migraines, multiple sclerosis (MS), muscle spasms, pain, chronic pain, acute pain, inflammatory pain, neuropathic pain, post-traumatic stress disorder (PTSD), schizophrenia, spasticity, Tourette syndrome, eating disorders, food addiction, bulimia, agoraphobia, generalized anxiety disorder, obsessive-compulsive disorder, panic disorder, social phobia, phobias, substance-induced anxiety disorder, delusional disorder, schizoaffective disorder, schizotypal disorder, substance-induced mental disorders, hypertension, or any combination thereof.

[0031] In some embodiments, the disease or disorder is a susceptible disorder or disorder, and as appropriate, the susceptible disorder or disorder is Rett syndrome, Angelman syndrome, spinal muscular atrophy, Smith-Magenis syndrome, Phelan-McDermid syndrome, Cornelia de Lange syndrome and other NIPBL-related disorders, DRK1A, KAT6A and severe intellectual disability-related disorders, chromosome 2Q37 deletion syndrome and other HDAC4-related disorders, Angelman syndrome, Cleefstra syndrome, Joubert syndrome and other NPHP1-related disorders, and The group includes Liams syndrome, neurofibromatosis type 1, Leafraumeni syndrome and similar p53-related cancer syndromes, phosphofructokinase deficiency, X-linked hyper-IgM syndrome and similar primary immunodeficiency disorders, triose phosphate isomerase deficiency, Kallmann syndrome, aromatase deficiency, Batten disease, frontotemporal dementia and other neurodegenerative disorders associated with progranulin loss, cholinergic receptor nicotinic alpha 7 subunit-related disorders, and hereditary neuropathy prone to compression palsy.

[0032] In some embodiments, the expression-sensitive disorder or impairment is characterized by a decrease in the expression of one or more proteins, where ectopic overexpression of the one or more proteins at steady-state levels above an upper-adjusted threshold causes cytotoxicity and / or disease. In some embodiments, the expression-sensitive disorder is a neurodevelopmental syndrome, which may be selected from the group including Rett syndrome, Smith-Magenis syndrome (RA1), Phelan-McDermid syndrome (SHANK3), Cornelia de Lange syndrome (NIPBL) and other NIPBL-related disorders, DRK1A, KAT6A and severe intellectual disability-related disorders, chromosome 2Q37 deletion syndrome and other HDAC4-related disorders, Angelman syndrome, Cleefstra syndrome, Joubert syndrome and other NPHP1-related disorders, and Williams syndrome. In some embodiments, the expression-sensitive disorder is a proliferative disorder and / or cancer, which may be selected from the group including neurofibromatosis type 1 and Leif-Raumeni syndrome and similar p53-related cancer syndromes. In some embodiments, the expression sensitivity disorder is glycogen storage disease, and optionally, glycogen storage disease is phosphofructokinase deficiency. In some embodiments, the expression sensitivity disorder is hematological and / or immunological disorder, and optionally, hematological and / or immunological disorder is selected from the group including X-linked hyper-IgM syndrome and associated primary immunodeficiency disorders, and triose phosphate isomerase deficiency. In some embodiments, the expression sensitivity disorder is endocrine disorder, and optionally, endocrine disorder is selected from the group including Kallmann syndrome and aromatase deficiency. In some embodiments, the expression sensitivity disorder is neuropsychiatric disorder, and optionally, neuropsychiatric disorder is selected from the group including Batten disease, frontotemporal dementia and other neurodegenerative disorders associated with progranulin loss, cholinergic receptor nicotinic alpha 7 subunit-related disorders, and hereditary neuropathy prone to compression palsy.

[0033] In some embodiments, the payload protein comprises RA1 and the disease or disorder comprises Smith-Magenis syndrome. In some embodiments, the payload protein comprises SHANK3 and the disease or disorder comprises Phelan-McDermid syndrome. In some embodiments, the payload protein comprises CLN3 and the disease or disorder comprises Batten disease. In some embodiments, the payload protein comprises NF-1 and the disease or disorder comprises neurofibromatosis type 1. In some embodiments, the payload protein comprises TP53 and the disease or disorder comprises Leif-Raumeni syndrome. In some embodiments, the payload protein comprises PFK and the disease or disorder comprises phosphofructokinase deficiency. In some embodiments, the payload protein comprises CD40LG and the disease or disorder comprises X-linked hyper-IgM syndrome.

[0034] Research methods are disclosed herein. In some embodiments, the research methods include introducing one or more cells with the nucleic acid compositions disclosed herein and obtaining biological information of one or more cells, as appropriate, via sequencing or imaging. In some embodiments, imaging includes CRISPR imaging and / or super-resolution imaging, and, as appropriate, DNA-PAINT (Point Accumulation for Imaging in Nanoscale Topography).

[0035] A production method is disclosed herein. In some embodiments, the production method includes introducing one or more cells with the nucleic acid composition disclosed herein and isolating the payload protein or the payload conjugated thereto. [Brief explanation of the drawing]

[0036] [Figure 1A]Figures 1A–1C show non-restrictive illustrative schematic diagrams related to the miRNA incoherent feedforward circuit, which enables gene expression independent of gene quantity. (Figure 1A) An ideal gene expression system produces a uniform protein expression level, even with a variable gene quantity delivered. The blue gradient shown in the nucleus indicates the copy number of the delivered gene, where darker blue represents a higher gene quantity and lighter blue represents a lower gene quantity. Green indicates the desired uniform output protein expression level. (Figure 1B) Architecture of the incoherent feedforward loop (IFFL). The input is a gene quantity of any unit, which activates the expression of both mRNA and microRNA. The microRNA inhibits mRNA translation. The output is the resulting protein expression. (Figure 1C) The IFFL enables tunable (circle 1), orthogonal (circle 2) control of the target and also operates in multiple cellular situations (circle 3). Circle 1 shows a modeling of a gene quantity compensation system that allows for adjustment of the setpoint level. Circle 2 shows schematic flow cytometry data for two target genes regulated by an orthogonal and tunable miRNA-regulated gene dose compensation circuit. Cells are polytransfected with two independently regulated constructs. Each color represents one set of designs used in polytransfection. Each ellipse indicates the location of most cells. The dashed line indicates the centroid of expression. Circle 3 schematically shows relatively similar expression behavior across diverse cell types. The central figure illustrates the circuit architecture of the microRNA (miRNA)-based IFFL, where two arrows indicate branching promoters, the small rectangle on the left indicates miRNA, the long rectangle on the right indicates the regulated gene, and the short rectangle on the right indicates the miRNA binding site. [Figure 1B] Same as above [Figure 1C] Same as above

[0037] [Figure 2A]Figures 2A–2I show non-restrictive illustrative schematic diagrams and data related to miRNA-based IFFLs that achieve linear regulation and gene dose independence through TNRC6-mediated repression. (Figure 2A) A simplified model of miRNA inhibition was constructed to identify a parameter regime that enables gene dose compensation. Top panel, gray dashed arrows in the figure represent intrinsic dilution and degradation of mRNA or miRNA. Black double-directional arrows represent binding and dissociation of mRNA-miRNA complexes. Gray arrows represent mRNA breakdown. The rates of all reactions are shown next to the arrows. Bottom panel, results of modeling miRNA-mRNA interactions. Unregulated curves suggest expression in the absence of miRNA. (Figure 2B) A branched promoter circuit was designed to run the miRNA-based gene dose compensation system. The parameter space that produces gene dose independence was explored by changing the complementarity of target-miRNA and the number of target sites. (Figure 2C) Flow cytometry was performed to verify the behavior of the circuit shown in Figure 2A using a single, fully complementary miR-L target site versus an unregulated control lacking both miR-L and miR-L target sites. The mRuby3 fluorescent protein was used as a gene dose indicator, and the EGFP fluorescent protein was used as a target. Cells were gated and binned by mRuby3 intensity. The dots correspond to the geometric mean fluorescence intensity of mRuby3 at bin break and the median fluorescence intensity of EGFP in the bin, respectively. The shaded area shows the range from ((EGFG median) / (geometric standard deviation)) to (EGFG median)×(geometric standard deviation). The gray curve shows the mRuby3-only transfection control, which suggests bleed-through from the mRuby3 channel to the EGFP channel. (Figure 2D) Flow cytometry was performed to understand how single-target site complementarity affects regulation. A single miR-L target site with complementarity in the 17 bp–21 bp range, starting from the seed region, was used in this experiment. mRuby3 functioned as a gene dose indicator, and EGFP was the target. Cells were gated and binned according to the mRuby3 intensity. Dotted and shaded regions were calculated as shown in Figure 2C.The gray curve shows the control for transfection with mRuby3 alone. (Figure 2E) Flow cytometry was performed to examine the effect of increasing the target repeat while maintaining constant complementarity. The 17 bp complementarity site of miR-L, starting from the seed region, was repeated 1 to 4 times. Cells were gated and binned by mRuby3 intensity and co-transfected with constitutively expressed iRFP plasmid as a co-transfection marker. Dots and shaded areas were calculated as shown in Figure 2C. (Figure 2F) Enlarged view of the 4 × 17 bp construct used in Figure 2E. The pdf, probability distribution function, measured the distribution of mRuby3 fluorescence intensity. Cells were gated and binned by mRuby3 intensity. Dots and shaded areas were calculated as shown in Figure 2C. Gray rectangles (indicated by vertical dashed lines) within the 10⁵–2 × 10⁶ scope on the mRuby3 axis indicate areas where EGFP fluorescence intensity does not change. The light gray rectangle (indicated by vertical dashed lines) within the 5 × 10⁴–10⁷ scope on the mRuby3 axis indicates the range where the EGFP fluorescence intensity changes slightly (i.e., less than 5 times). The dashed lines show curves with a slope of 1 or 0, revealing linear or gene-quantity-independent dependence. (Figure 2G) Diagram of the inhibition mechanism of the T6B peptide. T6B is a short dominant-negative version of TNRC6B. T6B competes with TNRC6 by binding to the Ago2-miRNA complex. (Figure 2H) To elucidate the inhibition mechanism that leads to gene-quantity-compensatory behavior, cells were co-transfected with or without the T6B peptide and a 4 × 17 bp construct described in Figure 2F, and flow cytometry was performed. The T6B peptide was fused with the iRFP fluorescent protein, and the control group was co-transfected with constitutively expressed iRFP. Cells were gated and binned by mRuby3 intensity. The dots and shaded areas were calculated as shown in Figure 2C. As shown in the small graph located in the plot, T6B-transfected cells lost inhibition.(Figure 2I) To compare the mechanism of potent inhibition with TNRC6-dependent inhibition, cells were co-transfected with or without the T6B peptide and a fully complementary single-site construct as described in Figure 2C, and flow cytometry was performed. The experimental setup was the same as that described in Figure 2H. Cells were gated and binned by mRuby3 intensity. Dots and shaded areas were calculated as shown in Figure 2C. T6B had a slight effect on inhibition, and therefore suggested that the inhibition was TNRC6-independent, as shown in the small graph located in the plot. [Figure 2B] Same as above [Figure 2C] Same as above [Figure 2D] Same as above [Figure 2E] Same as above [Figure 2F] Same as above [Figure 2G] Same as above [Figure 2H] Same as above [Figure 2I] Same as above

[0038] [Figure 3A]Figures 3A–3E show non-limiting illustrative schematic diagrams and data related to sequence complementarity, miRNA cassette copy number, and promoter intensity modulating expression levels. (Figure 3A) The circuit shown in Figure 2B was designed and constructed to possess miR-L targeting sites with complementarity ranging from 8 bp to 21 bp, starting from the seed region, with each targeting site repeated four times. In the left panel, flow cytometry was performed on cells, with each dot representing one well. Cells were gated based on the fluorescence intensity of mRuby3. The median fluorescence intensity of EGFP in mRuby3-positive cells was plotted. In the right panel, gene dose response curves for five selected constructs (dashed boxes) from the left panel are shown. Gray rectangles indicate the gated regions in the left panel. Cells were binned by mRuby3 intensity. Dots were calculated as shown in Figure 2C. (Figure 3B) Flow cytometry was performed on cells transfected with the circuit shown in Figure 2B, which contained miR-L targeting sites with complementarity in the range of 17 bp to 19 bp starting from the seed region, and each targeting site was repeated four times. Cells were gated and binned according to the mRuby3 intensity. Dots and shaded areas were calculated as shown in Figure 2C. The solid black horizontal line indicates the construct setpoint level. (Figure 3C) Flow cytometry was performed on cells transfected with the circuit shown in Figure 2B, which contained miR-L cassettes with 1 to 3 repeats incorporated within the same intron, and the target site was a miR-L targeting site with complementarity of 17 bp starting from the seed region, and was repeated four times. Cells were gated and binned according to the mRuby3 intensity. Dots and shaded areas were calculated as shown in Figure 2C. The solid black horizontal line indicates the construct setpoint level. (Figure 3D) Flow cytometry was performed on cells transfected with the circuit shown in Figure 2B, which contained a miR-L cassette expressed from the EF1α or PGK promoter. The target site was a 17 bp complementary miR-L targeting site starting from the seed region, and this was repeated four times.Cells were gated and binned according to mRuby3 intensity. Dots and shaded areas were calculated as shown in Figure 2C. The black horizontal solid line indicates the construct setpoint level. The bar graph in the lower right corner shows the median mRuby3 fluorescence intensity of transfected cells. (Figure 3E) Top panel, 4-epi-tetracycline-inducible DIMMER design. The promoter driving EGFP is a CMV promoter possessing two TetO sites. When the construct is expressed in the TRex cell line, the TetR transcription repressor binds to TetO and blocks transcription. 4-epi-tetracycline removes TetR from TetO in a gradient-dependent manner, thus restoring transcription. Bottom panel, flow cytometry was performed on TRex cell lines transfected with a 4×19bp DIMMER construct, and EGFP was driven by the CMV-2×TetO promoter. The concentrations of 4-epitetracycline ranged from purple to yellow, at 0, 10, 33.3, 100, and 333.3 ng / mL. The gray curve represents the transfection control with mRuby alone, where the 4-epitetracycline concentration was 333.3 ng / mL. [Figure 3B] Same as above [Figure 3C] Same as above [Figure 3D] Same as above [Figure 3E] Same as above

[0039] [Figure 4A]Figures 4A–4H show non-restrictive illustrative schematics and data related to algorithm-based miRNA design that is orthogonal to each other and enables independent regulation of multiple genes. (Figure 4A) Diagram of the open-loop system. The miRNA is expressed from the intron of the 3'UTR of mTagBFP2. EGFP is the target, while mRuby3 functions as a gene dose indicator. (Figure 4B) Flow cytometry was performed on cells co-transfected with the circuit shown in (A) with or without miR-L (BFP-only control does not have 3'UTR miRNA), with the target site being a single, fully complementary miR-L site. Cells were gated by the intensities of mRuby3 and mTagBFP2 and binned by the intensity of mTagBFP2. Relative expression was defined as the median ratio of EGFP fluorescence intensity to mRuby3 fluorescence intensity in the mTagBFP2 bin. Dots and shaded areas were calculated as shown in Figure 2C. (Figure 4C) Diagram of the mature synthetic miRNA and the sequence design constraints used to design it. (Figure 4D) Flow cytometry was performed on cells co-transfected with the circuit shown in Figure 4A, which has synthetic miRNA, and the corresponding target site is a single, fully complementary synthetic miRNA site. The BFP-only control uses a target that does not have a 3'UTR miRNA and does not have any target site. Cells were gated by the intensities of mRuby3 and mTagBFP2 and binned by the intensity of mTagBFP2. Relative expression was quantified as shown in Figure 4B. Dots were calculated as shown in Figure 2C. (Figure 4E) To verify whether the synthetic miRNA and its target are orthogonal to each other, cells were polytransfected with all combinations of 10 miRNAs and 10 single, fully complementary sites (10 × 10 combinations) and flow cytometry was performed. Each grid represents one well. Cells were gated by the intensities of mRuby3 and mTagBFP2. Relative expression was first quantified in transfected cells as shown in Figure 4B, and then normalized by dividing by the smallest value greater than 1.(Figure 4F) Gallery of circuits with the architecture shown in Figure 2B, established based on various miRNAs and targets. The number of repeats and complementarity between the target and miRNA are expressed in m × nbp, where m represents the number of repeats and n represents complementarity. Cells were measured by flow cytometry and gated and binned by the intensity of mRuby3. Dots were calculated as shown in Figure 2C. (Figure 4G) Diagram of the dual IFFL reporter system. In addition to the construct shown in Figure 2B, which uses mRuby3 as the gene dose indicator and EGFP as the target, a separate construct was introduced that uses mTagBFP2 as the gene dose indicator and emiRFP670 as the target. These two reporters were designed to be established on orthogonal miRNAs and targets to perform independent regulation. (Figure 4H) Flow cytometry was performed on cells polytransfected with the dual reporter system shown in Figure 4G. Cells were gated by the intensity of mRuby3 and mTagBFP2. The designs of the dual reporter miRNAs and targets are listed in the upper right corner of the plot, with their nomenclature described in Figure 4F, where the first half shows the cassette that modulates EGFP and the second half shows the cassette that modulates iRFP. The solid contour lines indicate the range where 80% of the cell cluster is located around the centroid of expression. [Figure 4B] Same as above [Figure 4C] Same as above [Figure 4D] Same as above [Figure 4E] Same as above [Figure 4F] Same as above [Figure 4G] Same as above [Figure 4H] Same as above

[0040] [Figure 5A]Figures 5A–5F show non-restrictive illustrative schematics and data for the DIMMER circuit, which functions across various cell lines and does not disrupt the transcriptome. (Figure 5A) The circuit, described in Figure 2B, regulated by miR-L with a 4 × 17 bp target, was transfected into various cell lines and flow cytometry was performed. Cells were gated and binned by mRuby3 intensity. Dots and shaded areas were calculated as shown in Figure 2C. Gray vertical lines intersecting the x-axis indicate the mRuby3 fluorescence intensity at which the circuit began to exhibit gene-quantity independent behavior. Gray horizontal lines intersecting the y-axis suggest the EGFP fluorescence intensity at the setpoint. (Figure 5B) The values ​​at the intersections of the gray lines and axes in Figure 5A were plotted as a scatter plot. The solid black line shows the linearity between the threshold level and the saturation level, which is described as y=kx, where k was fitted by applying a linear regression model. (Figure 5C) Workflow for sorting and measuring DIMMER monoclones. (Figure 5D) Flow cytometry was performed on DIMMER monoclones exhibiting diverse mRuby distributions. Histograms of mRuby3 and EGFP intensities for each monoclone are shown above and to the right of the scatter plot, respectively. (Figure 5E) Bulk RNA sequencing was performed on cells transfected with the BFP-miRNA construct. Orange and red dots indicate transcripts from synmiR-3 expressing cells, while light blue and dark blue dots indicate transcripts from synmiR-L expressing cells. The space outside the black dashed line indicates locations where the absolute value of the log2 multiplier change was greater than 2. The space above the red dashed line indicates locations where the adjusted p-value was less than 0.05. The Venn diagram above shows the genes that are shared and significantly differentially expressed between synmiR-3 and synmiR-L, and the genes that are not shared and significantly differentially expressed between synmiR-3 and synmiR-L. (Figure 5F) The normalized transcript permillions (TPM) of miR-L-expressing cells are plotted against the TPM of synmiR-3-expressing cells. The solid line indicates where the TPMs of the two samples were equal.The dashed lines indicate locations where the total protein production (TPM) of synmiR-3-expressing cells is 10 times that of synmiR-L-expressing cells, or where the TPM of the latter is 10 times that of the former. [Figure 5B] Same as above [Figure 5C] Same as above [Figure 5D] Same as above [Figure 5E] Same as above [Figure 5F] Same as above

[0041] [Figure 6A]Figures 6A–6H show non-limiting illustrative schematics and data related to the DIMMER circuit for improving fluorescence imaging. (Figure 6A) Diagram of an EGFR membrane receptor imaging experiment. Modulated EGFR-mEGFP is expected to show a more homogeneous distribution of fluorescence signals. (Figure 6B) Representative fluorescence images of EGFR-mEGFP with (right) or without (left) the DIMMER modulated module. For convenience of cloning, a miR-L cassette was selected to construct the circuit. The nomenclature of the target sites was described in Figure 4F. The numbers on the color bar indicate fluorescence intensity measured by ImageJ. (Figure 6C) Illustration of EGFR in the cell membrane (source PDB 7SYE). EGFR is ligated to mEGFP labeled with a DNA-conjugated anti-GFP nanobody (PDB 6LR7). (Figure 6D) Diffraction-limited and DNA-PAINT images of CHO-K1 cells expressing EGFR-mEGFP. mEGFP is labeled with a DNA-conjugated anti-mEGFP nanobody. The right panel shows a magnified view of the framed region. (Figure 6E) Representative single-protein degradation DNA-PAINT images of cells transfected with EGFR-mEGFP with or without the DIMMER module. A miR-L cassette was used, and the nomenclature of the target sites was described in Figure 4F. (Figure 6F) Quantification of receptor density for cells transfected with EGFR-mEGFP with or without the DIMMER module. The inset shows a magnified view of cells transfected with EGFR-mEGFP regulated by the DIMMER circuit. (Figure 6G) Diagram of a dCas9 telomere imaging experiment. The circuit-regulated protein was switched to dCas9-EGFP. The gRNA is targeted to the telomere and expressed from the same vector as dCas9-EGFP, but driven by the U6 promoter. (Figure 6H) Top panel, representative fluorescence images of dCas9-EGFP with or without the DIMMER module. For convenience of cloning, a miR-L cassette was chosen to construct the circuit. The nomenclature for target sites was described in Figure 4F. The numbers on the color bar indicate the fluorescence intensity measured by ImageJ.Lower panel: Quantification of relative signal intensity of dots in cells transfected with or without the DIMMER module. Each line represents a single dot inside a cell. It should be noted that finding dots inside cells transfected with unmodulated dCas9-EGFP is extremely difficult; therefore, among all cells in the scope, only three dots were found and analyzed in a single cell. [Figure 6B] Same as above [Figure 6C] Same as above [Figure 6D] Same as above [Figure 6E] Same as above [Figure 6F] Same as above [Figure 6G] Same as above [Figure 6H] Same as above

[0042] [Figure 7A] Figures 7A to 7C show non-restrictive illustrative data related to IFFL modeling. (Figure 7A) Modeling of steady-state dimensionless mRNA concentration under various koff conditions while maintaining kon=2×10⁵ and kc=160. (Figure 7B) Modeling of steady-state dimensionless mRNA concentration under various kc conditions while maintaining kon=2×10⁵ and koff=4×10³. (Figure 7C) Modeling of steady-state dimensionless mRNA concentration under various Hill coefficients n while maintaining K=1. [Figure 7B] Same as above [Figure 7C] Same as above

[0043] [Figure 8A]Figures 8A–8H show non-limiting illustrative schematics and data related to the comparison between circuit configurations in single-transcript and dual-transcript designs. (Figure 8A) Diagram of the initial designs of the miR-L 4×17bp circuit (top) and the miRNA detector circuit (bottom). The miRNA detector has a miRNA target site corresponding to the 3'UTR of iRFP. (Figure 8B) Flow cytometry was performed on cells transfected with the initial design of the miR-L 4×17bp circuit shown in the upper panel of Figure 8A. Cells were gated and binned by mRuby3 intensity. Dots and shaded areas were calculated as shown in Figure 2C. The initial design shows very low EGFP expression levels. (Figure 8C) Flow cytometry was performed on cells transfected with the 4×17bp miRNA detector alone (Figure 8A, bottom) or with the initial miR-L4×17bp circuit (Figure 8A, top). Cells were gated and binned according to the intensity of mTagBFP2. Dots and shaded areas were calculated as shown in Figure 2C. The detector in the initial circuit showed very weak inhibition at very high gene levels, and this discrepancy in inhibition suggests that the low expression level of EGFP in the initial circuit is not due to miRNA inhibition. (Figure 8D) Flow cytometry was performed on cells transfected with the initial miR-L 4×17bp circuit or with the initial miR-L 4×17bp circuit along with a 4×17bp miRNA detector. Cells were gated and binned according to the intensity of mRuby3. Dots and shaded areas were calculated as shown in Figure 2C. Detector addition does not affect the response of the initial miR-L 4×17bp circuit. (Figure 8E) Flow cytometry was performed on cells transfected with the initial miR-L 4×17bp circuit or with either the dual transcript design shown in Figure 2A. Cells were gated and binned according to the intensity of mRuby3. Dots and shaded regions were calculated as shown in Figure 2C. (Figure 8F) Diagram of a novel single transcript design of the miR-L 4×17bp circuit. The intronic miR-L cassette was shifted to 5' of the target site.(Figures 8G-8H) Flow cytometry was performed on cells co-transfected with a dual transcript miR-L 4×17bp circuit and a 10×21bp miRNA detector pair as described in Figure 2A, and with a novel single transcript design of miR-L 4×17bp and a 10×21bp miRNA detector as shown in Figure 8F. The 10×21bp miRNA detector is capable of detecting fewer miRNAs compared to the 4×17bp miRNA detector. Cells were gated and binned with either mRuby3 (Figure 8G) or mTagBFP2 (Figure 8H), respectively. Dots and hatched regions were calculated as shown in Figure 2C. Unmodulated represents either the mRuby3-branched promoter-EGFP construct (Figure 8G) or the mTagBFP2-branched promoter-iRFP670 construct (Figure 8H) without a modulated cassette and target site. The novel single-transcript design exhibits several gene dose-independent ranges, but still presents some degree of miRNA-independent inhibition. [Figure 8B] Same as above [Figure 8C] Same as above [Figure 8D] Same as above [Figure 8E] Same as above [Figure 8F] Same as above [Figure 8G] Same as above [Figure 8H] Same as above

[0044] [Figure 9A]Figures 9A and 9B show non-limiting illustrative data related to the inability of biologically dead T6B to remove TNRC6-dependent inhibition of the miR-L 4×17bp circuit. Flow cytometry was performed on cells transfected with the miR-L 4×17bp circuit (Figure 9A) or the miR-L 1×21bp circuit described in Figure 2B (Figure 9B), with a negative control of fluorescent protein alone, the T6B peptide, or the catalytically dead T6B peptide (indicated as T6BMut), respectively. Cells were gated and binned by mRuby3 intensity. Dots and shaded areas were calculated as shown in Figure 2C. The design of the catalytically dead T6B peptide was consistent with previous literature. [Figure 9B] Same as above

[0045] [Figure 10] Figure 10 shows non-restrictive, illustrative data related to gene dose response curves for multiple miR-L 4×n circuits. Flow cytometry was performed on cells transfected with the miR-L regulated 4×n circuit described in Figure 3A. Gray rectangles indicate the gated region in the left panel of Figure 3A. Cells were gated and binned by mRuby3 intensity. Dots were calculated as shown in Figure 2C.

[0046] [Figure 11A] Figures 11A and 11B show non-restrictive, illustrative data related to the gene dose response curves of the inducible DIMMER circuit in TRex cells. Flow cytometry was performed on TRex cell lines transfected with 4 × 17 bp (Figure 11A) or 4 × 18 bp (Figure 11B) DIMMER constructs, and EGFP was driven by the CMV-2 × TetO promoter. 4-epitetracycline concentrations ranged from purple to yellow, at 0, 10, 33.3, 100, and 333.3 ng / mL. The gray curve represents the mRuby-only transfection control, with a 4-epitetracycline concentration of 333.3 ng / mL. [Figure 11B] Same as above

[0047] [Figure 12A] Figures 12A–12B show non-limiting illustrative data related to circuit design, where the initial designs for synmiR-2 and synmiR-3 were unsuccessful, while synmiR-6 was successful but showed sequence similarity to endogenous miRNA. Flow cytometry was performed on cells co-transfected with the circuit shown in Figure 4A, using the initial sequences of synmiR-2 (Figure 12A, left panel), synmiR-3 (Figure 12A, right panel), and synmiR-6 (Figure 12B), and their corresponding targets, each having a single, fully complementary target site. BFP-only controls lacked 3'UTRmiRNA. Cells were gated by mRuby3 and mTagBFP2 intensity and binned by mTagBFP2 intensity. Relative expression levels were quantified as described in Figure 4B. Dots and shaded areas were calculated as described in Figure 2C. [Figure 12B] Same as above

[0048] [Figure 13] Figure 13 shows non-limiting illustrative data related to the 4×17bp targets of synmiR-4 and synmiR-5, which exhibit strong regulation. Cells were transfected with the circuit architecture shown in Figure 2A, possessing either the synmiR-4 cassette and its corresponding 4×17bp target, or the synmiR-5 cassette and its corresponding 4×17bp target, and flow cytometry was performed. Cells were gated and binned by mRuby3 intensity. Dots and shaded areas were calculated as shown in Figure 2C.

[0049] [Figure 14-1]Figure 14 shows non-limiting illustrative data related to a gallery of all IFFL designs based on various miRNAs and targets. Cells were transfected with the circuit architecture in Figure 2B, which contains the miRNA cassette and corresponding target shown at the top of the plot. Dots and shaded areas were calculated as shown in Figure 2C. The nomenclature of target sites was described in Figure 4F. [Figure 14-2] Same as above

[0050] [Figure 15A] Figures 15A and 15B show non-limiting, illustrative data related to IFFL functioning across various cell lines. (Figure 15A) Flow cytometry was performed on various cell lines transfected with the circuit described in Figure 2B, regulated by miRNA and the indicated target site. Cells were gated and binned by mRuby3 intensity. Dots and shaded areas were calculated as shown in Figure 2C. Black vertical lines intersecting the x-axis suggest threshold levels of mRuby3 fluorescence intensity. Black horizontal lines intersecting the y-axis suggest saturation levels of EGFP fluorescence intensity. (Figure 15B) The values ​​at the intersection of the gray line and the axis in Figure 15A were plotted as a scatter plot. The black solid line was calculated as shown in Figure 5B. [Figure 15B] Same as above

[0051] [Figure 16-1] Figure 16 shows non-restrictive, illustrative data related to a plot of normalized transcript permillions (TPM) versus mean TPM of synthetic miRNA-expressing cells. Mean TPM was calculated by averaging the TPMs of all microRNA-expressing cell samples. Solid lines indicate locations where the TPMs of two samples were equal. Dashed lines indicate locations where the TPM of synmiR-expressing cells was 10 times the mean, or where the latter's TPM was 10 times that of the former. [Figure 16-2] Same as above

[0052] [Figure 17A]Figures 17A–17C show non-limiting, illustrative data related to IFFL for improving fluorescence imaging. Flow cytometry was performed on cells transfected with EGFR-mEGFP with and without the DIMMER module (Figure 17A) and dCas9-EGFP with and without the DIMMER module (Figure 17B), respectively. Cells were gated and binned by mRuby3 intensity. Dots and shaded areas were calculated as shown in Figure 2C. Figure 17C shows the quantification of the signal-to-noise ratio (SNR) of dots in cells transfected with and without the DIMMER module (dCas9-EGFP). Each scatter plot represents a single dot inside the cell. [Figure 17B] Same as above [Figure 17C] Same as above

[0053] [Figure 18] Figure 18 shows a schematic diagram illustrating a non-limiting example of the reaction in the model. [Modes for carrying out the invention]

[0054] In the detailed description below, the accompanying drawings are referenced and constitute part of this specification. In the drawings, unless the context indicates otherwise, similar symbols generally identify similar components. The exemplary embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be used and other modifications may be made without departing from the spirit or scope of the subject matter presented herein. The aspects of this disclosure generally described herein and shown in the drawings can be arranged, substituted, combined, separated, and designed in a wide variety of configurations, all of which are expressly assumed herein and constitute part of this disclosure.

[0055] All patents, patent application publications, other publications, and sequences from GenBank and other databases mentioned herein are incorporated by reference in their entirety with respect to the relevant technologies.

[0056] definition Unless otherwise specified, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this disclosure pertains. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994) and Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For the purposes of this disclosure, the following terms are defined below.

[0057] As used herein, the term “vector” refers to a polynucleotide construct, usually a plasmid or virus, used to deliver genetic material to a host cell (e.g., a target cell). A vector may be, for example, a virus, plasmid, cosmid, or phage. A vector may be a viral vector. As used herein, a vector may consist of either DNA or RNA. In some embodiments, a vector consists of DNA. An “expression vector” is a vector capable of inducing the expression of a protein encoded by one or more genes embodied by the vector, when it is present in a suitable environment. A vector is preferably capable of self-replication. Typically, an expression vector comprises a transcription promoter, a gene, and a transcription terminator. Gene expression is usually under the control of a promoter, and the gene is said to be “operably ligated to” the promoter.

[0058] As used herein, the term “expression vector” refers to a vector that induces the expression of RNA or polypeptides (e.g., components of the synthetic protein circuit) from nucleic acid sequences contained therein, ligated to transcriptional regulatory sequences on the vector. The sequences expressed are often, though not necessarily, heterogeneous to the cell. An expression vector may contain further elements; for example, an expression vector may have two replication systems, and thus it is possible to maintain the expression vector in two organisms, for example, in human cells for expression and in a prokaryotic host for cloning and amplification. The term “expression” refers to cellular processes involved in the production of RNA and proteins, and, where appropriate, protein secretion, including, but not limited to, transcription, transcript processing, translation and protein folding, modification and processing, for example, transcription, transcript processing, translation and protein folding, modification and processing. “Expression products” include RNA transcribed from genes and polypeptides obtained by translation of mRNA transcribed from genes. The term “gene” means a nucleic acid sequence that is transcribed (from DNA) to RNA in vitro or in vivo when operably ligated to an appropriate regulatory sequence. A gene may or may not include, for example, a 5' untranslated region (5'UTR) or "leader" sequence and a 3'UTR or "trailer" sequence, as well as intervening sequences (introns) between individual coding segments (exons).

[0059] As used herein, the term “operably linked” is used to describe a link between a regulatory element and a gene or its coding region. Typically, gene expression is under the control of one or more regulatory elements, such as, but not limited to, constitutive or inductive promoters, tissue-specific regulatory elements, and enhancers. A gene or coding region is said to be “operably linked to,” “operatically linked to,” or “operatably bound to” a regulatory element, meaning that the gene or coding region is controlled or influenced by the regulatory element. For example, if a promoter achieves the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence.

[0060] As used herein, the term "construct" refers to a recombinant nucleic acid that has been generated for the purpose of expressing a particular nucleotide sequence or that is intended to be used in the construction of other recombinant nucleotide sequences.

[0061] As used herein, the terms “nucleic acid” and “polynucleotide” are interchangeable and refer to any nucleic acid, whether composed of phosphodiester linkages, phosphotriesters, phosphoramidates, siloxanes, carbonates, carboxymethyl esters, acetamidates, carbamates, thioethers, cross-linked phosphoramidates, cross-linked methylenephosphonates, phosphorothioates, methylphosphonates, phosphorodithioates, cross-linked phosphorothioates or sultone linkages and combinations thereof. Furthermore, the terms “nucleic acid” and “polynucleotide” specifically include nucleic acids composed of bases other than the five biologically present bases (adenine, guanine, thymine, cytosine, and uracil).

[0062] The terms “regulatory element” and “expression regulatory element” are used interchangeably and refer to nucleic acid molecules that can influence the expression of a operably linked coding sequence in a particular host organism. These terms are used broadly to encompass all elements that promote or regulate transcription, including promoters, core elements required for the fundamental interactions of RNA polymerases and transcription factors, upstream elements, enhancers, and response elements (see, for example, Lewin, “Genes V” (Oxford University Press, Oxford), pages 847–873). Exemplary regulatory elements in prokaryotes include promoters, operator sequences, and ribosome binding sites. Regulatory elements used in eukaryotic cells may include, but are not limited to, transcription and translation regulatory sequences that provide and / or regulate the expression of a coding sequence and / or the production of encoded polypeptides in the host cell, such as promoters, enhancers, splicing signals, polyadenylation signals, terminators, proteolytic signals, internal ribosome entry elements (IRESs), and 2A sequences.

[0063] As used herein, a 2A sequence or element refers to a short peptide introduced as a linker between two proteins, enabling the auto-ribosome auto-processing of polyproteins (see, e.g., de Felipe. Genetic Vaccines and Ther. 2:13 (2004), de Felipe et al. Traffic 5:616-626 (2004)). These short peptides enable the simultaneous expression of multiple proteins from a single vector. Many 2A elements are known in the art. Examples of 2A sequences that can be used in the methods and systems disclosed herein include, but are not limited to, 2A sequences derived from foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), and porcine tescho virus-1 (P2A).

[0064] As used herein, the term “promoter” is a nucleotide sequence that enables the binding of RNA polymerase and induces the transcription of a gene. Typically, a promoter is located in the 5' non-coding region of a gene, proximal to the gene’s transcription start site. The sequence elements within a promoter that function in the initiation of transcription often feature a consensus nucleotide sequence. Examples of promoters include, but are not limited to, those derived from bacteria, yeast, plants, viruses, and mammals (including humans). Promoters can be inductive, repressive, and / or constitutive. An inductive promoter will start to increase the transcription level from the DNA under its control in response to some change in culture conditions, such as a change in temperature.

[0065] As used herein, the term “enhancer” refers to a type of modulatory element that can enhance transcription efficiency, regardless of its distance from or orientation to the transcription initiation site.

[0066] As used herein, the term “variant” refers to a polynucleotide (or polypeptide) having a sequence substantially similar to that of a reference polynucleotide (or polypeptide). In the case of a polynucleotide, the variant may have deletions, substitutions, or additions of one or more nucleotides at the 5' end, 3' end, and / or one or more internal sites compared to the reference polynucleotide. Sequence similarities and / or differences between a variant and a reference polynucleotide can be detected using conventional techniques known in the art, such as polymerase chain reaction (PCR) and hybridization techniques. Variant polynucleotides also include synthetically obtained polynucleotides, such as those produced by site-directed mutagenesis. In general, variants of polynucleotides, including but not limited to DNA, may have at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity with a reference polynucleotide, when determined by sequence alignment programs known to those skilled in the art. In the case of polypeptides, variants may have deletions, substitutions, or additions of one or more amino acids compared to the reference polypeptide. Sequence similarity and / or differences between a variant and a reference polypeptide can be detected using conventional techniques known in the art, such as Western blotting. Generally, polypeptide variants, when determined by sequence alignment programs known to those skilled in the art, may have at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity with respect to a reference polypeptide.

[0067] As used herein, the term “effective dose” refers to an amount sufficient to achieve a beneficial or desirable biological and / or clinical outcome.

[0068] As used herein, “subject” refers to an animal that is the subject of treatment, observation, or experimentation. “Animals” include cold-blooded and warm-blooded vertebrates and invertebrates, such as fish, crustaceans, reptiles, and especially mammals. “Mammals” as used herein refers to individuals belonging to the class Mammalia, and includes, but is not limited to, humans, domesticated and livestock, zoo animals, and animals for sport and pets. Non-exclusive examples of mammals include mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cattle, horses, primates, such as monkeys, chimpanzees, and apes, and especially humans. In some embodiments, mammals are humans. However, in some embodiments, mammals are not humans.

[0069] As used herein, the term “treatment” refers to an intervention performed in response to a disease, disorder, or physiological condition presenting to a patient. The objectives of a treatment may include, but are not limited to, alleviating or preventing symptoms, slowing or halting the progression or worsening of a disease, disorder, or condition, and achieving remission of the disease, disorder, or condition. The terms “to treat” and “treatment” include, for example, therapeutic treatments, preventive treatments, and applications that reduce the risk of a subject developing a disorder or other risk factor. Treatments do not require a complete cure of the disorder and encompass embodiments that reduce symptoms or underlying risk factors. In some embodiments, “treatment” refers to both therapeutic treatments and preventive or anticipatory measures. Those requiring treatment include individuals already suffering from a disease, disorder, or undesirable physiological condition, and those for whom the disease, disorder, or undesirable physiological condition should be prevented. For example, in some embodiments, a treatment may reduce the level of RAS signaling in a subject, thereby reducing, alleviating, or eradicating the symptoms of a disease. As used herein, the term “prevention” refers to any activity that reduces the burden on individuals who subsequently develop those symptoms. This can be done at primary, secondary, and / or tertiary prevention levels, where a) primary prevention is avoiding the onset of symptoms / disorders / conditions, b) secondary prevention is when the intervention aims to treat the condition / disorder / symptoms at an early stage, thereby increasing the chances of preventing the progression of the condition / disorder / symptoms and the appearance of symptoms, and c) tertiary prevention is when the adverse effects of an already established condition / disorder / symptom are reduced, for example, by restoring function and / or reducing any condition / disorder / symptom or associated complications. The term “prevent” does not require 100% elimination of the possibility of the event. Rather, the term “prevent” indicates that the likelihood of the event occurring has been reduced in the presence of the compound or method.

[0070] A "pharmaceutically acceptable" carrier is nontoxic to cells or mammals to which it is exposed at the dose and concentration used. A "pharmaceutically acceptable" carrier may be an organic or inorganic solid or liquid excipient suitable for a selected mode of application, such as oral administration or injection, but may be administered in the form of conventional pharmaceutical preparations, such as solids such as tablets, granules, powders, or capsules, or liquids such as solutions, emulsions, or suspensions. Often, physiologically acceptable carriers are pH-buffered aqueous solutions, such as phosphate buffer or citrate buffer. Physiologically acceptable carriers may also contain one or more of the following: antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, and immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; carbohydrates, including amino acids, glucose, mannose, or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and nonionic surfactants, such as Tween, polyethylene glycol (PEG), and Pluronic. Furthermore, auxiliary agents, stabilizers, emulsifiers, lubricants, binders, pH adjusters, control agents, isotonic agents, and other conventional additives may be added to the carrier.

[0071] miRNA circuits related to regulated gene expression A critical requirement in diverse fields and applications of biomedical science, including gene therapy and cell therapy, is to express a target protein at a predetermined level, either in terms of the number of RNA and / or protein copies per cell or the RNA and / or protein concentration per cell. This is often difficult due to uncontrollable factors such as the number of copies of the transgene delivered to the cell, stochastic variations ("noise") in transcription and translation, molecular and functional heterogeneity between individual cells affecting expression, and variations in the cellular microenvironment, including variations at the level of exogenous signals.

[0072] Small, easily integrated into expression systems and delivery vectors, and capable of compensating for these uncontrollable factors to more precisely control expression levels, regulatory circuits are valuable in multiple applications. Novel regulatory circuits and design principles for limiting variability in gene expression and reducing its sensitivity to gene copy number (gene quantity) and other factors are provided herein. A wide variety of circuit designs and implementations are provided herein.

[0073] The problem of precise control arises in many situations. In gene therapy, most vectors result in a distribution of gene copy numbers in various cells. However, in many diseases that are candidates for gene therapy, therapeutic benefit depends on the expression of proteins within the therapeutic range. Too little expression does not provide benefit, while too much expression can cause other diseases or be toxic. This can occur, for example, in Rett syndrome, Angelman syndrome, and spinal muscular atrophy. Similarly, engineered cell therapies may require a predetermined expression level of a specific functional transgene in cells. Overexpression of receptors, modulators, or effectors in cell therapy can make the cell therapy less effective or toxic. A third category involves metabolic engineering to create cells that can produce drugs, biologics, materials, or other components. In these systems, enzymes and other proteins need to be expressed at specific levels to maximize the quantity and quality of production. A fourth category is for research purposes. There, the ability to accurately assess the function of wild-type proteins, mutant proteins (mutae), newly designed proteins, or RNA including mRNA, miRNA, lncRNA, or other classes of RNA, whether modified from natural sequences or synthetically designed, may be sensitively dependent on the ability to achieve precise expression levels in cells. Similarly, the ability to accurately determine the spatial pattern of intracellular protein localization by labeling a target protein or RNA with a fluorescent protein or other dye may depend, in some cases, on restricting the expression of the labeled protein to a specific level by fusing it with a particular binder, such as a single-chain antibody or nanobody or other binder. In other situations, excessive expression can cause background fluorescence and obscure the signal. While these examples highlight specific use cases, the problem of restricting the expression of specific RNAs, proteins, and other components to specific expression ranges applies to a wide range of situations. The methods and compositions provided herein may enable precise control in such use cases.

[0074] In some embodiments, a set of miRNA regulatory circuits is provided herein, constructed by expressing a gene of interest in one ("forward") direction from a bidirectional promoter having a miRNA target site at the 3'UTR, while the corresponding miRNA is generated within the intron of the gene, expressed in the reverse direction. The circuit architecture is an example of a broader class of circuit architectures referred to as incoherent feedforward loops. In its precise operational state, the circuit architecture can provide gene dose compensation by increasing miRNA expression to a sufficient level to compensate for increased transcription of the target gene by increasing the number of copies of the gene in the cell, thereby limiting the target expression to a certain maximum level. Previous studies have shown that synthetic IFFL circuits can successfully buffer gene expression against gene dose fluctuations, noise from upstream regulators, competition for cellular resources, or general disturbances. Furthermore, synthetic miRNA incoherent feedforward loop circuits may enable gene therapies and cell therapies to express their components within physiological ranges where unregulated expression would be harmful.

[0075] However, in all previous approaches, perfectly complementary miRNA target sites have been used to execute gene dose compensation circuits. These designs cannot fully compensate for changes in gene dose as shown herein (see Example 1). Rather, circuits designed with a single copy of a perfectly complementary target tend to exhibit output expression that scales as the square root of the gene dose. The methods and compositions provided herein can overcome this problem through novel circuit designs.

[0076] More specifically, in some embodiments of the disclosed methods and compositions, without being bound by any particular theory, this problem is modified by making two significant changes to the design. First, the complementarity of the target site is reduced. Second, the number of tandem binding sites in the target gene is increased. This combined change is not trivial because it depends on two characteristics of miRNA regulation: first, the use of a “scaffold” protein known as TNRC6 or GW182, and second, the fact that varying levels of complementarity result in various types of regulation. More specifically, complete complementarity results in “slicer” activity, which can lead to target-mediated miRNA degradation (TDMD), which triggers effective feedback that disrupts the ability of the conventional circuit to achieve complete gene dose compensation. By combining these two changes, along with further optimizations described in the accompanying manuscript, improved gene dose compensation can be achieved.

[0077] Furthermore, a method is provided herein for quantitatively adjusting the steady-state output of a circuit, enabling the user to specify the expression level through sequence design. Specifically, as detailed in Example 1, sequences having variable target region phase complementarity, an appropriate number of miRNA-generating cassettes within the synthetic intron, and variable intensity of the promoter expressing the miRNA are provided herein. It is demonstrated how these three parameters can be modified together to achieve various gene-quantity-compensated expression levels. In addition, means for generating orthogonal variants of miRNA circuits that can be used to simultaneously and independently regulate multiple genes in the same cell are provided herein. A set of sequences called synmiR, which can enable independent regulation of the expression of multiple target genes, is described. To ensure the generation of these orthogonal variants, a constrained random generation system has been created that reliably generates multiple fully orthogonal miRNAs and target sequences. This makes it possible to independently regulate multiple genes using multiple circuits in the same cell or system and maintain them at various setpoints. To demonstrate the usefulness of these circuits, Example 1 shows how they can be used to increase the signal-to-noise ratio in imaging applications. Furthermore, the system was demonstrated to enhance the control of gene expression from transgenes that are stably integrated at various copy numbers in transiently expressed DNA and genomes. Overall, this technology dramatically improves upon existing miRNA-based gene expression control circuits. It expands the dynamic range of gene dose-independent control and improves the consistency of circuit outputs at both low and high gene doses. This technology makes functionality more consistent and robust, enabling greater flexibility in the design of miRNA circuits.

[0078] In some embodiments of the methods and compositions provided herein, the target gene of interest in this circuit (e.g., payload gene) may be replaced to allow regulation of the expression of any gene, including both protein-coding genes and non-coding (e.g., lncRNA) genes. For example, the target gene may be a therapeutic gene in the context of gene therapy, including but not limited to MeCP2, SMN, UBE3A, or other genes used or expected to be used in gene therapy. In the context of cell therapy, the gene being regulated may consist of a synthetic chimeric antigen receptor (CAR), T cell receptor (TCR), SynNotch receptor, or other synthetic receptor. In some embodiments, the methods and compositions provided herein may allow controlled regulation of cell signaling molecules, including cytokines and chemokines, in cell therapy or other contexts. The expression of these proteins is expected to enhance cell therapy, but overexpression may be harmful.

[0079] In some embodiments, the methods and compositions provided herein may also be used for therapeutic and non-therapeutic gene editing. Overexpression of gene editors may cause off-target editing and nonspecific toxicity. In this context, the target gene being regulated may include any of many CRISPR-related proteins. These include Cas9, Cas12, and Cas13; multiple gene editors such as CRISPR base-editing nickase or dcas9-APOBEC1; CRISPR transcription modulators such as dcas9-KRAB or dcas9-VP64; and epigenetic regulators having various repressive, activating, or other effector domains; and catalytically inactive "dead" variants of Cas9 and Cas13 used for imaging and other applications, such as CRISPR imaging using Cas9 or Cas13 or common protein-fluorescent protein fusions, for example. In some embodiments, the methods and compositions provided herein may also be used to modulate non-CRISPR gene editors, for example, those based on TALE-based CRISPR free base editors, which can also be modulated using these systems.

[0080] In some embodiments, the methods and compositions provided herein may also be used to regulate the expression of proteins used in RNA extratransport systems and the production of virus-like particles (VLPs) in cells to avoid overexpression toxicity. More generally, the regulated gene may be any transcription factor, protease, kinase, phosphatase, or other regulatory protein involved in the construction of synthetic biological circuits. In the context of regenerative medicine, the methods and compositions provided herein may enable the expression of ectopic transcription factors at predetermined levels to optimally reprogram cellular states to achieve desired cell types more efficiently and selectively. In each of the application categories considered above, the circuits may play a crucial role in helping to express proteins at non-toxic physiological levels in the presence of noise, reduce off-target effects from overexpression, and reduce background activity to enhance the signal-to-background ratio.

[0081] The methods and compositions provided herein have been demonstrated in mammalian cells, and in some embodiments, the methods and compositions provided herein may function in cells of other species that contain the core mechanisms necessary for miRNA regulation. The miRNA-based gene regulatory circuits disclosed herein enable a wide range of precise control of gene expression in numerous situations, including both basic research and applications such as gene therapy. The miRNA-based gene regulatory circuits disclosed herein introduce novel designs and mechanisms that improve gene dose independence and other properties compared to currently available technologies.

[0082] Synthetic biology enables the rational design of circuits that confer novel functions to living cells. For example, CHOMP (Hacking Orthogonal Modular Protease Circuits) allows for the design of assembleable protein circuit components. Many innate cellular functions can be performed by protein-level circuits, where proteins specifically modify each other's activity, localization, or stability. Synthetic protein circuits are described in Gao, Xiaojing J., et al. "Programmable protein circuits in living cells." Science 361.6408(2018):1252-1258 and WO2019 / 147478, and the contents of each of these, including any supporting or supplementary information or materials, are incorporated herein by reference in their entirety. In some embodiments, the synthetic protein circuit responds only to inputs above or below certain adjustable threshold concentrations, such as those provided in US2020 / 0277333, and the contents of these are incorporated herein by reference in their entirety. In some embodiments, the synthetic protein circuit includes one or more synthetic protein circuit design components and / or concepts from US2020 / 0071362, the contents of which are incorporated herein by reference in whole. In some embodiments, the synthetic protein circuit includes a reasonably designed circuit including an incoherent feedforward loop circuit at the miRNA level and / or protein level that maintains payload expression at an effective level, for example, one provided in US2021 / 0171582, the contents of which are incorporated herein by reference in whole. The compositions, methods, systems and kits provided herein can be used in conjunction with the international patent application publication PCT / US2021 / 048100, titled "Synthetic Mammalian Signaling Circuits For Robust Cell Population Control," filed on 27 August 2021, the contents of which are incorporated herein by reference in whole.The systems, methods, compositions, and kits provided herein can, in some embodiments, be used in conjunction with the systems, methods, compositions, and kits described in PCT Patent Application Publication WO2022 / 125590, entitled "A synthetic circuit for cellular multistability," the contents of which are incorporated herein by reference in their entirety. The systems, methods, compositions, and kits provided herein can, in some embodiments, be used in conjunction with the systems, methods, compositions, and kits described in U.S. Patent Applications Publications 2018 / 0142307 and 2020 / 0172968, the contents of which are incorporated herein by reference in their entirety. The systems, methods, compositions, and kits provided herein may, in some embodiments, be used in conjunction with the systems, methods, compositions, and kits described in U.S. Patent Application Publication No. 2023 / 0076395, entitled "CELL-TO-CELL DELIVERY OF RNA CIRCUITS," and in U.S. Patent Application Publication No. 2023 / 0071834, entitled "EXPORTED RNA REPORTERS FOR LIVE-CELL MEASUREMENT," the contents of which are incorporated herein by reference in their entirety. The systems, methods, compositions, and kits provided herein may, in some embodiments, be used in conjunction with the systems, methods, compositions, and kits described in PCT Application Publication PCT / US23 / 69663, entitled "A SYNTHETIC PROTEIN-LEVEL NEURAL NETWORK IN MAMMALIAN CELLS," filed on July 5, 2023, the contents of which are incorporated herein by reference in their entirety. The systems, methods, compositions, and kits provided herein may, in some embodiments, be used in conjunction with the systems, methods, compositions, and kits described in PCT publication WO2020117713A1, entitled "In situ readout of dna barcodes," the contents of which are incorporated herein by reference in their entirety.The systems, methods, compositions, and kits provided herein may, in some embodiments, be used in conjunction with the systems, methods, compositions, and kits described in U.S. Patent Publications 17 / 820,232, 17 / 820,235, and 18 / 757,460, the contents of which are incorporated herein by reference in their entirety. The systems, methods, compositions, and kits provided herein may, in some embodiments, be used in conjunction with the systems, methods, compositions, and kits described in PCT Application Publication WO2024081912A1, titled "PROTEIN-BASED SIGNAL AMPLIFICATION," the contents of which are incorporated herein by reference in their entirety. The systems, methods, compositions, and kits provided herein may, in some embodiments, be used in conjunction with the systems, methods, compositions, and kits described in U.S. Patent Application Publication No. 18 / 799,870, entitled "MOLECULAR RECORDING METHODS AND SYSTEMS TO CAPTURE LINEAGE RELATIONSHIPS IN DIFFERENTIATING STEM CELLS," filed August 9, 2024, the contents of which are incorporated herein by reference in their entirety. The systems, methods, compositions, and kits provided herein may, in some embodiments, be used in conjunction with the systems, methods, compositions, and kits described in Du, Rongrong, et al. ("miRNA circuit modules for precise, tunable control of gene expression." BioRxiv (2024)), the contents of which, including any supporting or supplementary information or materials, are incorporated herein by reference in their entirety.

[0083] A composition (e.g., a nucleic acid composition, one or more cells) is disclosed herein. In some embodiments, the nucleic acid composition comprises a first promoter sequence operably ligated to a first polynucleotide comprising one or more miRNA cassettes. In some embodiments, the first promoter sequence is capable of inducing transcription of the first polynucleotide to produce a first transcript. In some embodiments, the first transcript is capable of being processed to produce the miRNA. In some embodiments, the nucleic acid composition comprises a second promoter sequence operably ligated to a second polynucleotide comprising a payload gene. In some embodiments, the payload gene comprises a miRNA target region comprising one or more miRNA target sequences. In some embodiments, the second promoter sequence is capable of inducing transcription of the second polynucleotide to produce a payload transcript. In some embodiments, the payload transcript is capable of being translated to produce a payload protein. In some embodiments, the first promoter sequence and the second promoter sequence are components of a bidirectional promoter. In some embodiments, the first promoter sequence and the second promoter sequence are oriented in a reverse complementary orientation with respect to each other in the bidirectional promoter.

[0084] A composition comprising one or more cells is disclosed herein. In some embodiments, the one or more cells comprises a first promoter sequence operably ligated to a first polynucleotide comprising one or more miRNA cassettes. In some embodiments, the first promoter sequence is capable of inducing transcription of the first polynucleotide to produce a first transcript. In some embodiments, the first transcript is capable of being processed to produce the miRNA. In some embodiments, the one or more cells comprises a second promoter sequence operably ligated to a second polynucleotide comprising a payload gene. In some embodiments, the payload gene comprises a miRNA target region comprising one or more miRNA target sequences. In some embodiments, the second promoter sequence is capable of inducing transcription of a second polynucleotide to produce a payload transcript. In some embodiments, the payload transcript is capable of being translated to produce a payload protein. In some embodiments, the first promoter sequence and the second promoter sequence are components of a bidirectional promoter. In some embodiments, the first promoter sequence and the second promoter sequence are oriented in a reverse complementary orientation with respect to each other in the bidirectional promoter.

[0085] In some embodiments, the miRNA can bind to one or more miRNA target sequences, thereby reducing the stability of the payload transcript and / or reducing the translation of the payload transcript. In some embodiments, the first polynucleotide comprises a gene quantity gene. In some embodiments, the first transcript can be translated to produce a gene quantity indicator protein. In some embodiments, the intron is located at the 3'UTR of the gene quantity gene, at the 5'UTR of the gene quantity gene, or between the exons of the gene quantity gene, and is, as appropriate, a synthetic intron. In some embodiments, the intron comprises one or more miRNA cassettes. In some embodiments, the intron comprises one or more of the following: (i) an intron insertion encoding a miRNA, (ii) a donor splice site, (iii) an acceptor splice site, (iv) a branching domain, and (v) a polypyrimidine tract. In some embodiments, miRNA or its precursor can be released from the intron by an intron excision mechanism selected from the group including intracellular RNA splicing and / or processing mechanisms, nonsense mutation-dependent degradation (NMD) processing, or any combination thereof, and the precursor may optionally include pri-miRNA. In some embodiments, the gene dose indicator protein is detectable, and the gene dose indicator protein may optionally include green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), TagRFP, Dronpa, Padron, mApple, mCherry, mruby3, rsCherry, rsCherryRev, their derivatives, or any combination thereof.

[0086] In some embodiments, one or more cells comprise two or more cells. In some embodiments, two or more cells comprise a first cell and a second cell. In some embodiments, the first cell is a first cell type, and / or the second cell is a second cell type. In some embodiments, (i) a first cell type and a second cell type and / or (ii) one or more cells are antigen-presenting cells, dendritic cells, macrophages, nerve cells, brain cells, astrocytes, microglia cells, and neurons, spleen cells, lymphoid cells, lung cells, lung epithelial cells, skin cells, keratinocytes, endothelial cells, alveolar cells, alveolar macrophages, alveolar lung cells, vascular endothelial cells, mesenchymal cells, epithelial cells, colon epithelial cells, hematopoietic cells, bone marrow cells, Claudius cells, Hensen cells, Merkel cells, Müller cells, Paneth cells, Purkinje cells, Schwann cells, Sertoli cells, eosinophilic cells, acinar cells, lipoblasts, adipocytes, brown or white alpha cells, amacrine cells, beta cells, capsule cells, cementocytes, chief cells, chondrocytes, chondrocytes, chromaffin cells, pigment apathetoid cells, adrenocorticotropic hormone-secreting cells, and Luta cells, Langerhans cells, follicular dendritic cells, enterochromaffin cells, ependymal cells, epithelial cells, basal cells, squamous epithelial cells, endothelial cells, transitional epithelial cells, erythroblasts, erythrocytes, fibroblasts, fibrous cells, follicular cells, germ cells, gametes, eggs, sperm, oocytes, primary oocytes, secondary oocytes, spermatids, spermatocytes, primary spermatocytes, secondary spermatocytes, germ epithelium, giant cells, glial cells, astroblasts, astrocytes, oligodendrocytes Loblast, oligodendrocyte, glioblast, goblet cell, gonadotropin-secreting cell, granulosa cell, hemocytoblast, hair cell, hepatoblast, hepatocyte, vitreous cell, stromal cell, juxtaglomerular cell, keratinocyte, corneal stromal cell, Remmal cell, leukocyte, granulocyte, basophil, eosinophil, neutrophil, lymphoblast, B-lymphoblast, T-lymphoblast, lymphocyte, B-lymphoblast, T-lymphoblast, helper-induced T-lymphoblast, Th1 T-lymphoblast, Th2T lymphocytes, natural killer cells, thymocytes, macrophages, Kupffer cells, alveolar macrophages, foam cells, histiocytes, luteal cells, lymphoid stem cells, lymphoid cells, lymphoid stem cells, macroglia, mamotoroves, mast cells, medulloblasts, megakaryoblasts, megakaryocytes, melanoblasts, melanocytes, mesangial cells, mesothelial cells, metamyelocytes, monoblasts, monocytes, myxone neck cells, myoblasts, muscle cells, muscle cells, cardiomyocytes, skeletal muscle cells, smooth muscle cells, myelocytes, myeloid cells, myeloid stem cells, myoblasts, myoepithelial cells, myofibroblasts, neuroblasts, neuroepithelial cells, neurons, odontoblasts, The group is selected from a group including osteoblasts, osteoclasts, osteocytes, acid-secreting cells, parafollicular cells, paraluteal cells, digestive cells, pericytes, peripheral blood mononuclear cells, pheochromocytes, phalangeal cells, pineal cells, pituitary cells, plasma cells, platelets, podocytes, proerythroblasts, promonocytes, promyeloblasts, promyelocytes, pronortherythroblasts, reticulocytes, retinal pigment epithelial cells, retinoblasts, small cells, growth hormone-secreting cells, stem cells, supporting cells, teloglycytic cells, enzymatic cells, or any combination thereof, and optionally, stem cells include embryonic stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem / progenitor cells (HSPCs), or any combination thereof.

[0087] In some embodiments, the first polynucleotide is transcribed at a rate at least 1.1 times faster in the first cell compared to the second cell (e.g., 1.1 times, 1.3 times, 1.5 times, 1.7 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 1000 times, 10000 times, or any number or range between these values). In some embodiments, the first polynucleotide is translated at a rate at least 1.1 times faster in the first cell compared to the second cell (e.g., 1.1 times, 1.3 times, 1.5 times, 1.7 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 1000 times, 10000 times, or any number or range between these values). In some embodiments, the first polynucleotide is present in the first cell with at least 1.1 times more copies than the second cell (e.g., 1.1 times, 1.3 times, 1.5 times, 1.7 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 1000 times, 10000 times, or any number or range between these values). In some embodiments, the threshold gene amount at which payload protein expression is saturated is at least 1.1 times higher in the first cell compared to the second cell (e.g., 1.1 times, 1.3 times, 1.5 times, 1.7 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 1000 times, 10000 times, or any number or range between these values). In some embodiments, the transcription rate of the second polynucleotide and / or the translation rate of the payload transcript vary between the first and second time points in a single cell, and / or between the first and second cells at the same time point. In some embodiments, in the absence of miRNA, the payload protein reaches an unadjusted steady-state payload protein level in one or more cells.In some embodiments, the unadjusted steady-state payload protein level falls within the range between the lower and upper unadjusted thresholds of the unadjusted expression range. In some embodiments, the steady-state gene quantity indicator protein level reflects the unadjusted steady-state payload protein level. In some embodiments, in the presence of miRNA, the payload protein reaches the adjusted steady-state payload protein level in one or more cells.

[0088] In some embodiments, the adjusted steady-state payload protein level falls within the range between the lower and upper adjusted thresholds of the adjusted expression range, where the upper adjusted threshold is the saturation expression level, as appropriate. In some embodiments, at first and second time points in a single cell, the steady-state level of the payload protein remains within the adjusted expression range. In some embodiments, at the same time points in the first and second cells, the steady-state level of the payload protein remains within the adjusted expression range. In some embodiments, the lower and / or upper adjusted thresholds of the adjusted expression range can be set by adjusting one or more of the following: (i) the number of miRNA cassettes in the first polynucleotide, (ii) the number of miRNA target sequences in the miRNA target region, (iii) the complementarity between the miRNA and one or more miRNA target sequences, and (iv) the intensity of the first and / or second promoter sequences. In some embodiments, the difference between the lower and upper unadjusted thresholds of the unadjusted expression range is greater than about two orders of magnitude. In some embodiments, the difference between the lower and upper adjusted thresholds of the adjusted expression range is less than approximately one order of magnitude. In some embodiments, the payload protein is effective at steady-state payload protein levels within the adjusted expression range. In some embodiments, the payload protein is ineffective and / or toxic at steady-state payload protein levels above and / or below the adjusted expression range. In some embodiments, the payload protein can induce an immunogenic response and / or a cytokine storm at steady-state payload protein levels outside the adjusted expression range. In some embodiments, the adjusted steady-state payload protein level includes therapeutic levels of the payload protein. In some embodiments, the steady-state payload protein level remains within the adjusted expression range across multiple cell types, viral vector titers, and / or viral vector capsid types. In some embodiments, the adjusted steady-state payload protein level is robust to tissue-targeted and stochastic expression.

[0089] In some embodiments, miRNAs are at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, This includes nucleotide sequences, parts thereof, and / or their complementary (e.g., reverse complementary) sequences that are identical in number or range between any two of these values, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any two of these values. In some embodiments, the miRNA cassette contains at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, and 79% of the miRNAs in sequence numbers 1 through 13. This includes nucleotide sequences, parts thereof, and / or their complementary (e.g., reverse complementary) sequences, which are identical in number or range between any two of these values, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any two of these values. In some embodiments, the miRNA target region comprises at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, and 79% of SEQ ID NOs: 14 to 74. This includes nucleotide sequences, parts thereof, and / or their complementary (e.g., reverse complementary) sequences that are identical in number or range between any two of these values, including %, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any two of these values.

[0090] In some embodiments, the miRNA is approximately 20, 21, 22, 23, or 24 nucleotides (nt) long. In some embodiments, the miRNA sequence is orthogonal to one or more cells and, as appropriate, is a miRNA sequence that targets sea urticaria luciferase. In some embodiments, the miRNA target region is located in the 3'UTR, 5'UTR, or coding region of the payload gene. In some embodiments, the miRNA target region contains at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 miRNA target sequences. In some embodiments, the payload transcript can be simultaneously bound by multiple miRNA-loading Argonaut (Ago) complexes, as appropriate, via TNRC6. In some embodiments, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 nucleotides (nt) of the miRNA target sequence are complementary to the miRNA, and, as appropriate, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nt at the 3' end of the miRNA are not complementary. In some embodiments, the complementarity between the miRNA and the miRNA target sequence is at least, or at most, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, or a number or range between any two of these values. In some embodiments, the miRNA contains 5 to 8 GC nucleotides, optionally 1 to 4 in the seed region and 1 to 2 in the broad region. In some embodiments, the composition includes one or more additional payload genes and one or more additional miRNAs, wherein the additional miRNAs are sequence-different with respect to one another. [Table 1-1] [Table 1-2] Table 1-3 Table 1-4 Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 2

[0091] Some embodiments of the methods and compositions provided herein include circuits having the tunable expression of two or more payloads (e.g., a payload and one or more additional payloads, secondary payloads). In some embodiments, the circuit includes two or more distinct payloads regulated by distinct miRNAs (see Example 1). In some embodiments, two or more payloads are regulated by the same miRNA. Two or more payloads may be regulated by the same miRNA in the circuit but possess distinct miRNA target regions. The total number of payloads whose expression is regulated in the circuit may vary depending on the embodiment. In some embodiments, the composition includes, for each distinct additional miRNA, an additional first promoter sequence operably ligated to an additional first polynucleotide containing one or more additional miRNA cassettes, and an additional second promoter sequence operably ligated to an additional second polynucleotide containing an additional payload gene. In some embodiments, an additional first promoter sequence can induce the transcription of an additional first polynucleotide to produce an additional first transcript, which can be processed to produce the additional miRNA. In some embodiments, an additional payload gene includes an additional miRNA target region containing one or more additional miRNA target sequences, where an additional second promoter sequence can induce the transcription of an additional second polynucleotide to produce an additional payload transcript, which can be translated to produce an additional payload protein. The secondary / additional payload protein may contain any of the payloads described herein. The payload protein and one or more secondary proteins may be expressed as a fusion protein (and may be separated by one or more self-cleaving peptides). The 3'UTR of a transgene encoding one or more secondary proteins may contain one or more miRNA binding sequences.The payload protein and one or more secondary proteins may be expressed on separate payload transcripts. The payload protein and one or more secondary proteins may also be encoded on a single transcript, where the translation of the payload protein and one or more secondary proteins may be driven by separate internal ribosome entry sites. The sequences of the internal ribosome entry sites may be identical or different.

[0092] Nucleic acid compositions may comprise DNA, RNA, or a mixture of DNA and RNA. In some embodiments, the nucleic acid composition comprises one or more mRNAs (e.g., comprising a first promoter sequence operably linked to a first polynucleotide comprising one or more miRNA cassettes and a second promoter sequence operably linked to a second polynucleotide comprising a payload gene). In some embodiments, the composition is a vector, a ribonucleoprotein (RNP) complex, a liposome, nanoparticles, an exosome, a microvesicle, or any combination thereof. In some embodiments, the nucleic acid composition is complexed or bound to one or more lipid or lipid-based carriers, thereby forming liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes, which, as appropriate, encapsulate the nucleic acid composition. In some embodiments, the composition is one or more vectors, comprises one or more vectors, or further comprises one or more vectors. In some embodiments, at least one of the one or more vectors is a viral vector, a plasmid, a translocation element, a naked DNA vector, a lipid nanoparticle (LNP), or any combination thereof. In some embodiments, the transposing element is a piggyback transposon or a sleeping beauty transposon. In some embodiments, the first polynucleotide and the second polynucleotide are contained in one or more vectors. In some embodiments, the first polynucleotide and the second polynucleotide are contained in the same vector and / or different vectors. In some embodiments, the first polynucleotide and the second polynucleotide are located on the same nucleic acid and / or different nucleic acids.In some embodiments, the nucleic acid composition includes approximately 1.0 kb, 1.5 kb, 2.0 kb, 2.5 kb, 3.0 kb, 3.5 kb, 4.0 kb, 4.5 kb, 5.0 kb, 5.5 kb, 6.0 kb, 6.5 kb, 7.0 kb, 7.5 kb, 8.0 kb, 8.5 kb, 9.0 kb, 9.5 kb, 10 kb, 12 kb, 15 kb, 20 kb, 25 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, or less than 100 kb. In some embodiments, miRNA expression disrupts the endogenous gene expression of one or more cells by less than approximately 10%. The method may include TNRC6, GW182, one or more miRNA biogenesis components, Exportin-5, Dicer, and / or one or more nucleic acids encoding Argonaut proteins. In some embodiments, the first promoter sequence and the first polynucleotide are not operably ligated in the absence of a recombination event, where the first promoter sequence and the first polynucleotide are operably ligated after a recombination event so that the first promoter sequence can induce transcription of the first polynucleotide to produce a first transcript. In some embodiments, the first polynucleotide and the second polynucleotide are integrated into the genome of one or more cells.

[0093] Research methods are disclosed herein. In some embodiments, the research method includes introducing one or more cells with the nucleic acid composition disclosed herein and obtaining biological information of one or more cells, as appropriate, via sequencing or imaging. In some embodiments, imaging includes CRISPR imaging and / or super-resolution imaging, and as appropriate, DNA-PAINT (Point Accumulation for Imaging in Nanoscale Topography). Production methods are disclosed herein. In some embodiments, the production method includes introducing one or more cells with the nucleic acid composition disclosed herein and isolating a payload protein or a payload conjugated thereto.

[0094] Methods for treating a disease or disorder in a subject are disclosed herein. In some embodiments, the method includes administering an effective amount of one or more cells disclosed herein to a subject to treat or prevent a disease or disorder in the subject. Methods for treating a disease or disorder in a subject are disclosed herein. In some embodiments, the method includes introducing a nucleic acid composition disclosed herein into one or more cells and administering an effective amount of the resulting cells to a subject to treat or prevent a disease or disorder in the subject. The method includes isolating one or more cells from the subject prior to the introduction step. Methods for treating a disease or disorder in a subject are disclosed herein. In some embodiments, the method includes administering an effective amount of a nucleic acid composition disclosed herein to a subject to treat or prevent a disease or disorder in the subject. Methods relating to the controlled gene quantity-independent expression of a payload protein in one or more cells are disclosed herein. In some embodiments, the method includes introducing a nucleic acid composition disclosed herein into one or more cells. In some embodiments, the introduction step is carried out in vivo, in vitro, and / or ex vivo. In some embodiments, the transfection step includes calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, electronuclear transport, chemotransfection, electrotransfection, lipofectamine-mediated transfection, effectene-mediated transfection, lipid nanoparticle (LNP)-mediated transfection, or any combination thereof. In some embodiments, one or more cells include one or more cells of a subject, which may optionally be suffering from a disease or disorder. The method may include introducing an inducible promoter inducer into one or more cells, which may optionally include doxycycline, and the transfection step may further optionally include administering an initial dose of the inducer, followed by one or more lower maintenance doses of the inducer.

[0095] In some embodiments, one or more cells include eukaryotic cells, and as appropriate, eukaryotic cells include antigen-presenting cells, dendritic cells, macrophages, nerve cells, brain cells, astrocytes, microglia cells, and neurons, spleen cells, lymphoid cells, lung cells, lung epithelial cells, skin cells, keratinocytes, endothelial cells, alveolar cells, alveolar macrophages, alveolar lung cells, vascular endothelial cells, mesenchymal cells, epithelial cells, colon epithelial cells, hematopoietic cells, bone marrow cells, Claudius cells, Hensen cells, Merkel cells, Müller cells, Paneth cells, Purkinje cells, Schwann cells, Sertoli cells, eosinophilic cells, acinar cells, lipoblasts, adipocytes, brown or white alpha cells, amacrine cells, beta cells, capsule cells, cementocytes, chief cells, chondrocytes, chondrocytes, chromaffin cells, pigment aphobe cells, adrenocorticotropin-secreting cells, delta cells, and lan Gerhans cells, follicular dendritic cells, enterochromaffin cells, ependymal cells, epithelial cells, basal cells, squamous epithelial cells, endothelial cells, transitional epithelial cells, erythroblasts, erythrocytes, fibroblasts, fibrous cells, follicular cells, germ cells, gametes, eggs, sperm, oocytes, primary oocytes, secondary oocytes, spermatids, spermatocytes, primary spermatocytes, secondary spermatocytes, germ epithelium, giant cells, glial cells, astroblasts, astrocytes, oligodendrocytes Oligodendrocytes, glioblasts, goblet cells, gonadotropin-secreting cells, granulosa cells, hemoblasts, hair cells, hepatoblasts, hepatocytes, vitreous cells, stromal cells, juxtaglomerular cells, keratinocytes, corneal stromal cells, Lemmar cells, leukocytes, granulocytes, basophils, eosinophils, neutrophils, lymphoblasts, B-lymphoblasts, T-lymphoblasts, lymphocytes, B-lymphoblasts, T-lymphoblasts, helper-induced T-lymphoblasts, Th1 T-lymphoblasts, Th2T lymphocytes, natural killer cells, thymocytes, macrophages, Kupffer cells, alveolar macrophages, foam cells, histiocytes, luteal cells, lymphoid stem cells, lymphoid cells, lymphoid stem cells, macroglia, mamotoroves, mast cells, medulloblasts, megakaryoblasts, megakaryocytes, melanoblasts, melanocytes, mesangial cells, mesothelial cells, metamyelocytes, monoblasts, monocytes, myxone neck cells, myoblasts, muscle cells, muscle cells, cardiomyocytes, skeletal muscle cells, smooth muscle cells, myelocytes, myeloid cells, myeloid stem cells, myoblasts, myoepithelial cells, myofibroblasts, neuroblasts, neuroepithelial cells, neurons, ivory This includes blast cells, osteoblasts, osteoclasts, osteocytes, acid-secreting cells, parafollicular cells, paraluteal cells, digestive cells, pericytes, peripheral blood mononuclear cells, pheochromocytes, phalangeal cells, pineal cells, pituitary cells, plasma cells, platelets, podocytes, proerythroblasts, promonocytes, promyeloblasts, promyelocytes, pronortherythroblasts, reticulocytes, retinal pigment epithelial cells, retinoblasts, small cells, growth hormone-secreting cells, stem cells, supporting cells, teloglycytic cells, enzymatic cells, or any combination thereof, further optionally including embryonic stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem / progenitor cells (HSPCs), or any combination thereof.

[0096] A disease or disorder may be an expression-sensitive disease or disorder. An expression-sensitive disease or disorder may be characterized by a decrease in the expression of one or more proteins, where ectopic overexpression of the one or more proteins at steady-state levels above an upper-adjusted threshold causes cytotoxicity and / or disease. The disease or disorder may be one provided in Table 3. Table 3 provides an exemplary list of so-called "Goldilocks" diseases and disorders in which the disease phenotype is due to a decrease in gene expression, but similarly exhibits cytotoxicity or obvious disease when overexpressed. Surprisingly, the methods and compositions provided herein can treat or prevent the above diseases and disorders via the adjustable and robust expression means provided herein. In some embodiments, the payload gene includes one of the genes provided in Table 3. [Table 3-1] [Table 3-2]

[0097] promoter The nucleic acid promoters provided herein (e.g., first promoter sequence, second promoter sequence) may vary depending on the embodiment. Promoter may include ubiquitous promoters. Ubiquitous promoters include: cytomegalovirus (CMV) initial promoter, CMV promoter, viral simian virus 40 (SV40) (e.g., early or late), Moloney's mouse leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR, RSV promoter, herpes simplex virus (HSV) (thymidine kinase) promoter, vaccinia virus-derived H5, P7.5, and P11 promoters, and elongation factor 1. -The following may be selected from the group including the alpha (EF1a) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5 (HSPA5), heat shock protein 90kDa beta, member (HSP90B1), heat shock protein 70kDa (HSP70), β-kinesin (β-KIN), human ROSA 26 locus, ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, 3-phosphoglycerate kinase promoter, cytomegalovirus enhancer, human β-actin (HBA) promoter, chicken β-actin (CBA) promoter, CAG promoter, CBH promoter, or any combination thereof.

[0098] In some embodiments, one or more cells of the subject (e.g., human) contain an endogenous version of the payload gene, and the promoter may contain or be derived from the endogenous version of the promoter. The promoter may be approximately 25% (e.g., 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%) relative to the promoter of the endogenous version of the payload gene. The promoter may include homology (%), 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, or the number or range between any two of these values). The promoter may be a methyl CpG-binding protein 2 (MeCP2) promoter or a derivative thereof (e.g., a MeCP2 promoter cleaved to about 229 bp and / or a MeCP2 promoter cleaved to about 406 bp). The promoter may include an intron sequence. The promoter may include a bidirectional promoter and / or an enhancer (e.g., a CMV enhancer).

[0099] The promoter may be an inductive promoter (e.g., a tetracycline-responsive promoter, a TRE promoter, a Tre3G promoter, an ecdysone-responsive promoter, a cumate-responsive promoter, a glucocorticoid-responsive promoter, and an estrogen-responsive promoter, a PPAR-γ promoter, and / or a RU-486-responsive promoter). The promoter may include tissue-specific promoters and / or lineage-specific promoters. Tissue-specific promoters may include a liver-specific thyroxine-binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, an α-myosin heavy chain (α-MHC) promoter, or a cardiac troponin T (cTnT) promoter. Tissue-specific promoters include neuron-specific promoters (e.g., synapsin-1 (Syn) promoter, CaMKIIa promoter, calcium / calmodulin-dependent protein kinase IIa promoter, tubulin alpha I promoter, neuron-specific enolase promoter, platelet-derived growth factor beta chain promoter, TRPV1 promoter, Na v 1.7 promoter, Na v 1.8 promoter, Na v 1.9 The promoter may be a promoter (or Advillin promoter). A tissue-specific promoter may be a muscle-specific promoter (e.g., a creatine kinase (MCK) promoter).

[0100] In some embodiments, payload expression may be gated by a drug / small molecule. In some embodiments, the method may include an inductive or repressive promoter. The method may include administering one or more doses (e.g., a higher starting dose and a lower maintenance dose) of a drug that acts on the promoter (e.g., an inducer of the inductive promoter). Some such embodiments induce payload expression only at a specific point in time or expression profile, for example, when a higher starting dose versus a longer-term maintenance dose is required. Some embodiments of the compositions, methods, and systems provided herein may include one or more components of a transactivator rtTA (reverse tetracycline-regulated transactivator) system. By using the rrTA system, the expression of the gene of interest (e.g., payload) can be further regulated by an inductive system, thereby expressing the IFFL regulatory construct only when a small molecule doxycycline is added.

[0101] The compositions provided herein may include tetracycline-on (Tet-On) systems. For example, a tetracycline-on (Tet-On) system may use a reverse tetracycline trans-activator (rtTA) to induce gene expression. The reverse tetracycline trans-activator (rtTA) comprises a mutant tetracycline repressor DNA-binding protein (TetR) and a transactivation domain. These transactivators can be activated in the presence of tetracycline (e.g., doxycycline) and subsequently bind to a promoter containing a tetracycline-responsive element (TRE) to induce gene expression. The TRE comprises at least one Tet operator (Tet-O) sequence (e.g., multiple repeats of the Tel-0 sequence) and may be located upstream of a minimal promoter (e.g., a minimal promoter sequence derived from the human cytomegalovirus (hCMV) initial promoter). The "Tet-On" system, as used herein, is a type of inducible system capable of inducing the expression of a specific payload gene in the presence of tetracycline (e.g., doxycycline (DOX)). In certain embodiments, the Tet-On system comprises a tetracycline-responsive promoter and a reverse tetracycline-regulated transactivator (rtTA) operably linked to a payload gene (e.g., a therapeutic sequence, a gene-targeting nucleic acid, and / or a protein-coding nucleic acid). The expression cassette encoding the tetracycline-responsive promoter (e.g., a TRE-containing promoter including TRE3G, P tight, and TRE2) and the reverse tetracycline-regulated transactivator may be encoded on the same vector or on separate vectors. In some embodiments, the promoter includes a Tet response element (TRE). A tetracycline-dependent promoter can be constructed by placing a TRE upstream of a minimal promoter.

[0102] "Reverse tetracycline transactivator" ("rtTA"), as used herein, is interpreted in its usual sense and refers to an inducer capable of binding to a TRE promoter (e.g., TRE3G, P tight, or TRE2 promoter) in the presence of tetracycline (e.g., doxycycline) to drive the expression of a payload gene operably linked to the TRE promoter. rtTA generally comprises a mutant tetracycline repressor DNA-binding protein (TetR) and a transactivation domain. Any suitable transactivation domain may be used. Non-limiting examples include VP64, P65, RTA, and MPH MS2-P65-HSF1. In some embodiments, the rtTA of this disclosure comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100 transactivation domains. Mutant TetR domains are capable of binding to the TRE promoter when bound to tetracycline.

[0103] The methods and compositions provided herein may include tetracycline repressors. The terms “tetracycline repressor” or “TetR” are to be interpreted in their usual sense and refer to proteins that can bind to a Tet-O sequence (e.g., a Tet-O sequence in a TRE) in the absence of tetracycline (e.g., doxycycline) and prevent the binding of rtTA (e.g., rtTA3, rtTA4, or their variants) in the absence of tetracycline (e.g., doxycycline). TetR prevents gene expression from a promoter containing a TRE in the absence of tetracycline (e.g., doxycycline). In the presence of tetracycline, TetR cannot bind to a promoter containing a TRE, and therefore TetR cannot prevent transcription.

[0104] The term “promoter” shall be interpreted in its ordinary sense and shall refer to a regulatory region of a nucleic acid sequence that controls the initiation and rate of transcription of the remainder of the nucleic acid sequence. Promoters may also contain subregions to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. Promoters may be constitutive, inducible, activatable, repressive, tissue-specific, or any combination thereof. Promoters drive the expression of the nucleic acid sequence they regulate or drive its transcription. Herein, a promoter is considered “operably linked” if it is present in a precise functional location and orientation relative to the nucleic acid sequence it regulates in order to control ("drive") the transcription initiation of the nucleic acid sequence, the expression of the sequence, or a combination thereof.

[0105] Promoter may promote ubiquitous or tissue-specific expression of operablely linked nucleic acid sequences of any species, including humans. In some embodiments, the promoter is a eukaryotic promoter. Non-limiting examples of eukaryotic promoters, as known to those skilled in the art, include TDH3, PGK1, PKC1, TDH2, PYK1, TPI1, AT1, CMV, EFla, SV40, PGK1 (human or mouse), Ubc, human beta-actin, CAG, TRE, UAS, Ac5, polyhedrin, CaMKIIa, GAL1, GAL10, TEF1, GDS, ADH1, CaMV35S, Ubi, Hl, and U6.

[0106] Non-limiting examples of ubiquitous promoters include tetracycline-responsive promoters (under appropriate conditions), CMV, EF1 alpha, SV40 promoters, PGK1, Ubc, CAG, human beta-actin gene promoters, and promoters including upstream activating sequences (UAS). In certain embodiments, the promoter is a mammalian promoter. Non-limiting examples of tissue-specific promoters include brain-specific, liver-specific, muscle-specific, nerve-specific, lung-specific, heart-specific, bone-specific, intestine-specific, skin-specific promoters, and eye-specific promoters.

[0107] Non-exclusive examples of constitutive promoters include CP1, CMV, EF1 alpha, SV40, PGK1, Ubc, human beta-actin, beta-tubulin, CAG, Ac5, polyhedrin, TEF1, GDS, CaM3 5S, Ubi, Hl, and U6.

[0108] "Inducible promoter" is to be interpreted in its usual sense and refers to a promoter characterized by initiating or enhancing transcriptional activity in the presence of, being influenced by, or being contacted by an inducer. The inducer may be an endogenous or usually exogenous condition, compound, drug, or protein that is contacted with the engineered nucleic acid so as to become active in inducing transcriptional activity from the inducible promoter. In certain embodiments, the inducer is a tetracycline-sensitive protein (e.g., rtTA).

[0109] Examples of inducible promoters for use as described herein include any inducible promoters described herein or known to those skilled in the art. Examples of inducible promoters include chemically / biochemically regulated promoters and physically regulated promoters, e.g., alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, which include tetracycline repressor protein (tetRTetR), tetracycline operator sequence (tetO), and tetracycline transactivator fusion protein (tTA), and tetracycline operator sequence (tetO) and reverse tetracycline transactivator fusion protein (rtTA)), steroids Examples of regulated promoters include, but are not limited to, promoters based on rat glucocorticoid receptors, human estrogen receptors, moth ecdysone receptors, and steroid / retinoid / thyroid 25 receptor superfamilies; metal-regulated promoters (e.g., promoters derived from metallothionein (metal ion-binding and sequestering protein) genes from yeast, mouse, and human); pathogenicity-regulated promoters (e.g., those induced by salicylic acid, ethylene, or benzothiadiazole (BTH)); temperature / heat-inducible promoters (e.g., heat shock promoters); and photo-regulated promoters.

[0110] payload In some embodiments, the payload protein includes disease-related proteins, where the abnormal expression of the disease-related protein correlates with the onset and / or progression of the disease. The payload protein may include proteins associated with expression-sensitive diseases or disorders, as provided in Table 3. The payload protein may include methyl CpG-binding protein 2 (MeCP2), DRK1A, KAT6A, NIPBL, HDAC4, UBE3A, EHMT1, one or more genes encoded on chromosome 9q34.3, NPHP1, LIMK1, one or more genes encoded on chromosome 7q11.23, P53, TPI1, FGFR1 and related genes, RA1, SHANK3, CLN3, NF-1, TP53, PFK, CD40L, CYP19A1, PGRN, CHRNA7, PMP22, CD40LG, derivatives thereof, or any combination thereof.

[0111] The payload protein may include fluorescent activity, polymerase activity, protease activity, phosphatase activity, kinase activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, or any combination thereof.

[0112] The payload protein may include nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidative activity, pyrimidine dimerization activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosylase activity, acetyltransferase activity, deacetylase activity, adenylation activity, deadenylation activity, or any combination thereof.

[0113] The payload may include one or more components (e.g., programmable nucleases, PegRNAs, sgRNAs) of gene editing systems (e.g., CRISPR-Cas9 editing (clustered, regularly spaced short palindromic repeats (CRISPR)-CRISPR-related protein 9 (Cas9)) systems, single-base editing, or prime editing) configured to remove or edit one or more nucleic acid bases, for example, by substituting, inserting, and / or deleting sequences present in the genome of a subject or patient. CRISPR-Cas9 may be used to inactivate or modify a gene, or a base editor or prime editor may be utilized. CRISPR-Cas9 systems and single-base editors include a guide RNA (gRNA) or single guide RNA (sgRNA) and a CRISPR-related protein 9 (Cas9) nuclease. Methods for identifying a DNA target strand and performing changes in the target DNA (e.g., gene knockout, knock-in of a desired sequence, or base substitution in the target DNA strand) are within the scope of the skills of those skilled in the art. The circuits provided herein can provide tunable gene quantity-independent expression of gene editing system components and, in some embodiments, may result in reduced off-target editing effects compared to currently available approaches for expressing gene editing system components.

[0114] In some embodiments, methods and compositions are provided for autoregulatory gene therapy that can provide gene-quantity-independent regulated expression. In some embodiments, the autoregulatory gene therapy payload may provide gene-quantity-independent regulated expression of the payload (e.g., MeCP2). In some embodiments, the methods and compositions provide gene-quantity independence of the payload. When used herein, gene-quantity independence is to be interpreted in its usual sense and also refers to a lower multiplier change in protein output compared to gene quantity. The methods and compositions provided herein may result in a multiplier increase of at least 1.1 times (e.g., 1.1x, 1.3x, 1.5x, 1.7x, 1.9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 1000x, 10000x, or any number or range between these values) of protein output compared to gene amount. In some embodiments, the methods and compositions provided herein enable payload gene amount independence in vivo (e.g., in mice, in humans).

[0115] In Crispr-Cas9 editing, a double-strand break is induced in the gRNA sequence when the Cas9 nuclease binds to the PAM and the gRNA binds to the target DNA strand. Endogenous repair mechanisms such as non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or homologous recombination repair (HDR) are triggered by the double-strand break, resulting in gene knockout on the target DNA strand, or knock-in of the desired sequence if a DNA template exists. The DNA template contains the desired sequence adjacent to sequences homologous to the upstream and downstream regions of the double-strand break. Examples of gRNAs include CRISPR RNA (crRNA), which is a 17-20 nucleotide sequence complementary to the target DNA strand, and tracrRNA, which functions as a binding scaffold for the Cas9 nuclease. crRNA and tracrRNA can exist as two separate RNA molecules. Alternatively, the sgRNA may contain both a crRNA sequence and a tracrRNA sequence, where the crRNA sequence is fused to a scaffold tracrRNA sequence. The gRNAs for the base editing techniques described below have standard structures specific to each technique. Those skilled in the art will select a gRNA or sgRNA that maximizes the efficiency of on-target DNA cleavage while minimizing the effects of unintended off-target binding and cleavage.

[0116] Base editing is a genome editing technique that uses DNA base editors to directly generate precise point mutations without producing or involving double-strand breaks. DNA base editors may include fusions between catalytically dead Cas9 (dCas9) or nickase Cas9 (nCas9) fused to a single-strand DNA (ssDNA) specific deaminase and a single guide RNA (sgRNA). d / nCas9 recognizes a specific sequence called a protospacer-adjacent motif (PAM), and the DNA unwinds as a result of complementarity between the sgRNA and a DNA sequence typically located upstream of the PAM ("protospacer"). Subsequently, the opposite DNA strand becomes accessible to a deaminase that translates the base located at the specific DNA extension of the protospacer (see, e.g., Antoniou P, et al. Base and Prime Editing Technologies for Blood Disorders. Front Genome Ed. 2021 Jan 28;3:618406). When a DNA base editor binds to a target DNA strand, base pairing between the sgRNA and the target DNA strand results in the movement of a small segment of ssDNA as an "R loop." Thus, the DNA bases within the ssDNA are substrates for deamination and subsequently modified by the deaminase enzyme. DNA base editors are either cytosine base editors (CBEs) that convert C / G base pairs to T / A base pairs, or adenine base editors (ABEs) that convert A / T base pairs to G / C base pairs.

[0117] Another method of gene editing is prime editing, which is disclosed in U.S. Patent No. 11,447,770B1 and related publications (see also International Patent Application Publication WO2020191242 A1 and Anzalone AV, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019 Dec;576(7785):149-157), which are incorporated herein by reference for their technical disclosure. Prime editors (PEs), including a complete description of pegRNA, are provided in these references, as are methods for the in vivo delivery of prime editor materials, such as viral vectors, e.g., AAV particles encoding prime editors, in vivo. Prime editing is a "search and replace" gene editing method in which Moloney mouse leukemia virus reverse transcriptase (M-MLV RT) is fused to the C-terminus of Cas9 H840A nickase. The fusion enzyme uses prime-edit guide RNA (pegRNA) to define the target insertion, deletion, and all possible base conversions. The pegRNA guides the nickase to the target site by homology to the genomic DNA locus. Longer pegRNAs also encode the desired edit on the primer-binding site (PBS) and RT template. Prime editing has gone through numerous versions. In PE1, the pegRNA guides the Cas9 nickase to the target sequence, where PE1 cuts the non-target strand, generating a 3' flap. This 3' flap binds to the primer-binding site (PBS) of the pegRNA, and the desired edit is incorporated into the DNA by reverse transcription. The edited DNA strand moves the unedited 5' flap, and the resulting heteroduplex is resolved by the cell's mismatch repair (MMR) system. Alternatively, the edited 3' flap may be excised, leaving the target sequence unchanged but available as a substrate for another prime-edit.In the PE2 system, mutations are introduced into the RT enzyme to increase its activity, enhance binding between the template and PBS, increase processability, and improve thermal stability. PE3 uses a simpler sgRNA in addition to the PE2 Cas9 nickase-vaginal mutant RT-type fusion enzyme and pegRNA, which induces Cas9 nickase to cleave the unedited strand at a nearby site. Subsequently, the newly edited strand is suitable as a template for repair during heterodouble helix dissolution. However, the double nicking process slightly increases indel formation. Similar to the PE3b system, nicking of the unedited strand is induced only after editing has occurred by designing an sgRNA with a spacer that binds only to the edited strand. PE4 and PE5 are also described (Chen PJ, et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell. 2021 Oct 28;184(22):5635-5652.e29). "Prime editing" includes all variations of prime editing methods, including but not limited to PE1, PE2, PE3, PE3b, PE4, and PE5 versions. pegRNA includes, for example, epegRNA, and its variations for use in many variations of prime editing (Nelson JW, et al. Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol. 2022 Mar;40(3):402-410).

[0118] The payload protein may contain a programmable nuclease. Programmable nucleases may be selected from the group including SpCas9 or its derivatives; VRER, VQR, EQR SpCas9; xCas9-3.7; eSpCas9; Cas9-HF1; HypaCas9; evoCas9; HiFi Cas9; ScCas9; StCas9; NmCas9; SaCas9; CjCas9; CasX; Cas9 H940A nickase; Cas12 and its derivatives; dCas9-APOBEC1 fusion, BE3, and dCas9-deaminase fusion; dCas9-Krab, dCas9-VP64, dCas9-Tet1, and dCas9-transcription regulator fusion; Dcas9-fluorescent protein fusion; Cas13-fluorescent protein fusion; RCas9-fluorescent protein fusion; and Cas13-adenosine deaminase fusion. Programmable nucleases may include zinc finger nucleases (ZFNs) and / or transcription activator-like effector nucleases (TALENs). Programmable nucleases include Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), zinc finger nucleases, TAL effector nucleases, meganucleases, MegaTAL, and Tev-m. TALEN, MegaTev, Homing Endonuclease, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr 6 may include Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, C2c1, C2c3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, their derivatives, or any combination thereof. In some embodiments, the nucleic acid further comprises (i) a targeting molecule and / or (ii) a polynucleotide encoding a donor nucleic acid.The composition may include (i) a targeting molecule or a nucleic acid encoding a targeting molecule and / or (ii) a donor nucleic acid or a nucleic acid encoding a donor nucleic acid. The targeting molecule may be capable of binding to a programmable nuclease. The targeting molecule may include single-stranded DNA or single-stranded RNA. The targeting molecule may include a single guide RNA (sgRNA). The programmable nuclease may include zinc finger nucleases, TAL effector nucleases, meganucleases, MegaTAL, Tev-m TALEN, MegaTev, homing endonucleases, their derivatives, or any combination thereof. The targeting molecule may be capable of binding to a programmable nuclease. The targeting molecule may include single-stranded DNA or single-stranded RNA. The targeting molecule may include a single guide RNA (sgRNA). The targeting molecule may include synthetic nucleic acids.

[0119] In some embodiments, the payload comprises one or more programmable nucleases disclosed in Table 4. In some embodiments, the payload comprises one or more prime editors. In some embodiments, a gene editing method is provided that serves a genetic purpose and / or regulatory purpose as described in Table 4. [Table 4]

[0120] The payload protein may include a chimeric antigen receptor. The methods and compositions provided herein are used in cell therapies (e.g., adoptive therapy). The methods and compositions provided herein may include adoptive transfer of immune system cells, such as T cells, that are specific to a selected antigen, such as a tumor-associated antigen. For example, T cells can be genetically modified by altering the specificity of the T cell receptor (TCR) using various strategies, for example, by introducing a payload containing a novel TCR and b chain having selected peptide specificity. As an alternative to, or in addition to, TCR modification, chimeric antigen receptors (CARs) may be used to generate immune-responsive cells, such as T cells, that are specific to a selected target, such as malignant cells, and a wide variety of receptor chimeric constructs are described. The methods and compositions provided herein may include adoptive immunotherapy comprising (1) knock-in of an exogenous gene encoding a chimeric antigen receptor (CAR) or T cell receptor (TCR), (2) knock-out or knock-down of the expression of an immune checkpoint receptor, (3) knock-out or knock-down of the expression of an endogenous TCR, (4) knock-out or knock-down of the expression of a human leukocyte antigen class I (HLA-I) protein, and / or (5) knock-out or knock-down of the expression of an endogenous gene encoding an antigen targeted by an exogenous CAR or TCR.

[0121] The payload protein may be associated with a desired agricultural trait selected from the group consisting of increased yield, increased abiotic stress tolerance, increased drought tolerance, increased flood tolerance, increased heat tolerance, increased cold and frost tolerance, increased salt tolerance, increased heavy metal tolerance, increased low nitrogen tolerance, increased disease resistance, increased pest resistance, increased herbicide resistance, increased biomass production, male sterility, or any combination thereof.

[0122] The payload protein may be associated with a biological manufacturing process selected from the group including fermentation, distillation, biofuel production, compound production, polypeptide production, or any combination thereof.

[0123] The payload may include non-coding (e.g., lncRNA) genes. The payload may be an RNA gene product. One or more payload genes of nucleic acid may include siRNA, shRNA, antisense RNA oligonucleotide, antisense miRNA, trans-splicing RNA, guide RNA, single guide RNA, crRNA, tracrRNA, trans-splicing RNA, premRNA, mRNA, or any combination thereof.

[0124] Payload proteins can be any protein, including naturally occurring and non-naturally occurring proteins. Examples of payload proteins include, but are not limited to, luciferase; fluorescent proteins (e.g., GFP); growth hormone (GH) and its variants; insulin-like growth factor (IGF) and its variants; granulocyte colony-stimulating factor (G-CSF) and its variants; erythropoietin (EPO) and its variants; insulin such as proinsulin, preproinsulin, insulin, and insulin analogs; antibodies and their variants such as hybrid antibodies, chimeric antibodies, humanized antibodies, and monoclonal antibodies; antigen-binding fragments (Fab fragments) and single-chain variable fragments (scFV fragments) of antibodies; dystrophin and its variants; coagulation factors and their variants; cystic fibrosis transmembrane conductance regulator (CFTR) and its variants; and interferons and their variants.

[0125] In some embodiments, the payload protein is a therapeutic protein or a variant thereof. Non-limiting examples of therapeutic proteins include blood factors such as β-globin, hemoglobin, tissue plasminogen activator, and coagulation factors; colony-stimulating factor (CSF); interleukins such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, and IL-9; keratinocyte growth factor (KGF), stem cell factor (SCF), fibroblast growth factor (FGF, such as basic FGF and acidic FGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), bone morphogenetic protein (BMP), and epithelial cells. Growth factors such as growth factors (EGF), growth differentiation factor-9 (GDF-9), hepatocellular carcinoma-derived growth factor (HDGF), myostatin (GDF-8), nerve growth factor (NGF), neurotrophin, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β); soluble TNF receptors, soluble VEGF receptors, soluble interleukin receptors (e.g., soluble IL-1 receptor and soluble type II IL-1 receptor), and soluble γ / δ Soluble receptors such as T cell receptors and ligand-binding fragments of soluble receptors; enzymes such as as-glucosidase, imiglucarase, β-glucocerebrosidase, and alglucerase; enzyme activators such as tissue plasminogen activator; chemokines such as IP-10, interferon-gamma-induced monokines (Mig), Gro / IL-8, RANTES, MIP-1, MIP-1β, MCP-1, and PF-4; angiogenic agents such as vascular endothelial growth factor (VEGF, e.g., VEGF121, VEGF165, VEGF-C, VEGF-2), transforming growth factor-beta, basic fibroblast growth factor, glioma-derived growth factor, angiogenin, and angiogenin-2; anti-angiogenic agents such as soluble VEGF receptors; protein vaccines;Nerve growth factor (NGF), bradykinin, cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dynorphin, warfarin, neurotensin, motilin, thyroid-stimulating hormone, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyroid-stimulating hormone-releasing hormone, vasoactive intestinal peptide, sleep peptide Examples include neuroactive peptides such as peptides; thrombolytic agents; atrial natriuretic peptide; relaxin; glial fibrillary acidic protein; follicle-stimulating hormone (FSH); human alpha-1 antitrypsin; leukemia inhibitory factor (LIF); transforming growth factor (TGF); tissue factor, luteinizing hormone; macrophage activator; tumor necrosis factor (TNF); neutrophil chemotactic factor (NCF); nerve growth factor; metalloproteinase tissue inhibitors; vasoactive intestinal peptides; angiogenin; angiotropin; fibrin; hirudin; and IL-1 receptor antagonists. Some other non-limiting examples of payload proteins include ciliary neurotrophic factor (CNTF); brain-derived neurotrophic factor (BDNF); neurotrophin 3 and 4 / 5 (NT-3 and 4 / 5); glial cell-derived neurotrophic factor (GDNF); aromatic amino acid decarboxylase (AADC); hemophilia-related coagulation proteins such as factor VIII, factor IX, and factor X; dystrophin or minidystrophin; lysosomal acid lipase; phenylalanine hydroxylase (PAH); glycogen storage disease-related enzymes such as glucose-6-phosphatase, acid maltase, glycogen debranching enzyme, muscle glycogen phosphorylase, hepatic glycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase (e.g., PHKA2), glucose transporter (e.g., GLUT2), aldolase A, β-enolase, and glycogen synthase; lysosomal enzymes (e.g., beta-N-acetylhexosaminidase A); and any variants thereof.

[0126] In some embodiments, the payload protein is an active fragment of a protein, such as one of the proteins described above. In some embodiments, the payload protein is a fusion protein comprising some or all of two or more proteins. In some embodiments, the fusion protein may comprise all or part of any of the proteins described above.

[0127] In some embodiments, the payload protein is a multi-subunit protein. For example, the payload protein may contain two or more subunits or two or more independent polypeptide chains. In some embodiments, the payload protein may be an antibody. Examples of antibodies include, but are not limited to, antibodies of various isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM), provided that the antibody is capable of binding to an antigen; monoclonal antibodies produced by any means known to those skilled in the art, including antigen-binding fragments of monoclonal antibodies; humanized antibodies; chimeric antibodies; single-chain antibodies; and antibody fragments such as Fv, F(ab')2, Fab', Fab, Fabc, and scFv. In some embodiments, the antibody is a full-length antibody.

[0128] In some embodiments, the payload gene encodes a survival-promoting protein (e.g., Bcl-2, Bcl-XL, Mcl-1, and A1). In some embodiments, the payload gene encodes, for example, AIF, Apaf (e.g., Apaf-1, Apaf-2, and Apaf-3), or APO-2(L), APO-3(L), ​​apopine, Bad, Bak, Bax, Bcl-2, Bcl-xL, Bcl-xS, bik, CAD, calpain, caspase (e.g., caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, and caspase-11), ced-3, ced-9, c-Jun, c-Myc, crm A, Cytochrome C, CdR1, DcR1, DD, DED, DISC, DNA-PKcs, DR3, DR4, DR5, FADD / MORT-1, FAK, Fas (Fas-ligand CD95 / fas (receptor)), FLICE / MACH, FLIP, Fodrin, fos, G-actin, Gas-2, Gerzolin, Granzyme A / B, ICAD, ICE, JNK, Lamin A / B, MAP, MCL-1, Mdm-2, MEKK-1, MORT-1, NEDD, NF- kappa It encodes apoptotic factors or apoptosis-related proteins such as B, NuMa, p53, PAK-2, PARP, perforin, PITSLRE, PKC delta, pRb, presenilin, prICE, RAIDD, Ras, RIP, sphingomyelinase, herpes simplex-derived thymidine kinase, TRADD, TRAF2, TRAIL-R1, TRAIL-R2, TRAIL-R3, and / or transglutaminase.

[0129] In some embodiments, the payload gene encodes a cell reprogramming factor capable of converting at least partially differentiated cells into less differentiated cells, such as Oct-3, Oct-4, Sox2, c-Myc, Klf4, Nanog, Lin28, ASCL1, MYT1L, TBX3b, SV40 Large T, hTERT, miR-291, miR-294, miR-295, or any combination thereof. In some embodiments, the payload gene encodes a programming factor capable of differentiating a given cell to a desired differentiation state, such as nerve growth factor (NGF), fibroblast growth factor (FGF), interleukin-6 (IL-6), bone morphogenetic protein (BMP), neurogenin 3 (Ngn3), pancreatic and duodenal homeobox 1 (Pdx1), Mafa, or any combination thereof.

[0130] In some embodiments, the payload genes include, for example, cytokines that induce or enhance the innate immune response, including IL-2, IL-12, IL-15, IL-18, IL-21, CCL21, GM-CSF, and TNF-alpha; cytokines released from macrophages, including IL-1, IL-6, IL-8, IL-12, and TNF-alpha; C1q, MBL, C1r, C1s, C2b, Bb, D, MASP-1, MASP-2, C4b, C3b, C5a, C3a, C4a, C5b, C6, C7, C8, C9, CR1, CR2, CR3, CR4, C1qR, C1INH, C4bp, MC The components of the complement system, including P, DAF, H, I, P, and CD59; and the components of the pattern recognition receptor signaling network, including TLR and IL-1R1, are ligands for pattern recognition receptors, such as IL-1 alpha, IL-1 beta, beta-defensin, heat shock proteins, such as HSP10, HSP60, HSP65, HSP70, HSP75, and HSP90, gp96, fibrinogen, and fibronectin, including the Typl II repeat extradomain A; IL-1 Receptors including RI, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, and TLR11; signaling molecules including components of Small-GTPase signaling (e.g., RhoA, Ras, Rac1, Cdc42), components of PIP signaling (e.g., PI3K, Src kinase), components of MyD88-dependent signaling (e.g., MyD88, IRAK1, IRAK2), and components of MyD88-independent signaling (e.g., TICAM1, TICAM2); activating transcription factors including, for example, NF-κB, c-Fos, c-Jun, and c-Myc; and inducible target genes including, for example, IL-1 alpha, IL-1 beta, beta-defensin, IL-6, IFN gamma, IFN alpha, and IFN beta; costimulatory molecules including CD28 or CD40 ligands or PD1; protein domains including LAMP; and cell surface proteins.Alternatively, it may be a human adjuvant protein containing CD80, CD81, CD86, trif, flt-3 ligand, thymopentin, Gp96, or fibronectin, or any species homolog of any of the above human adjuvant proteins.

[0131] In some embodiments, the payload gene encodes an immunogenic material capable of stimulating an immune response (e.g., an adaptive immune response), such as an antigenic peptide or protein derived from a pathogen. Antigen expression can stimulate the body's adaptive immune system to provide an adaptive immune response. Therefore, in some embodiments, the nucleic acids provided herein are envisioned to be used as vaccines (e.g., as vaccines) for the prevention or treatment of infectious diseases.

[0132] As described herein, the nucleotide sequences encoding payload proteins can be modified to improve the efficiency of protein expression. The methods that may be used to improve gene transcription and / or translation described herein are not particularly limited. For example, nucleotide sequences can be modified to better reflect host codon use frequency and increase gene expression (e.g., protein production) in the host (e.g., mammals).

[0133] The degree of payload gene expression in target cells can vary. For example, in some embodiments, the payload gene encodes a payload protein. The amount of payload protein expressed in the subject (e.g., the subject's serum) can vary. For example, in some embodiments, the protein may be expressed in the subject's serum at amounts of at least about 9 μg / ml, at least about 10 μg / ml, at least about 50 μg / ml, at least about 100 μg / ml, at least about 200 μg / ml, at least about 300 μg / ml, at least about 400 μg / ml, at least about 500 μg / ml, at least about 600 μg / ml, at least about 700 μg / ml, at least about 800 μg / ml, at least about 900 μg / ml, or at least about 1000 μg / ml. In some embodiments, the payload protein is expressed in the serum of the subject at amounts of approximately 9 μg / ml, 10 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, 1000 μg / ml, 1500 μg / ml, 2000 μg / ml, 2500 μg / ml, or in a range between any two of these values. The expression level of the payload protein required for the method to be effective may vary depending on non-limiting factors such as the specific payload protein and the subject being treated, and it will be understood by those skilled in the art that the effective amount of protein can be readily determined by those skilled in the art using conventional methods known in the art without excessive experimentation.

[0134] The payload protein encoded by the payload gene can have a variety of lengths. For example, the payload protein may be at least about 200 amino acids, at least about 250 amino acids, at least about 300 amino acids, at least about 350 amino acids, at least about 400 amino acids, at least about 450 amino acids, at least about 500 amino acids, at least about 550 amino acids, at least about 600 amino acids, at least about 650 amino acids, at least about 700 amino acids, at least about 750 amino acids, at least about 800 amino acids, or longer. In some embodiments, the payload protein is at least about 480 amino acids long. In some embodiments, the payload protein is at least about 500 amino acids long. In some embodiments, the payload protein is about 750 amino acids long.

[0135] Payload genes may have different lengths in different executions. The number of payload genes may vary in different embodiments. In some embodiments, the number of payload genes in the nucleic acid may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or any number or range between any two of these values, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or any number or range between any two of these values. In some embodiments, the number of payload genes in the nucleic acid may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the payload genes are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 50 0, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300,2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or a number or range of nucleotide lengths between any two of these values, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 8, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 5 20, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, The nucleotide length is 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or any number or range between any two of these values. In some embodiments, the payload gene is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150, 160, 170, 180, 190, 200, 210,220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 5 30, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 84 0, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 220 0, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 950 The nucleotide length is 0, or 1000, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 43 0, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740 ,750,760,770,780,790,800,810,820,830,840,850,860,870,880,890,900,910,920,930,940,950,960,970,980,990,1000,1100,1200,1300,1400,The nucleotide lengths are 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 1000.

[0136] The payload may be an inducer of cell death. The payload may induce cell death via a non-endogenous cell death pathway (e.g., bacterial pore-forming toxins). In some embodiments, the payload may be a pro-survival protein. In some embodiments, the payload is a modulator of the immune system. The payload may activate adaptive immune responses and innate immune responses, or both. In some embodiments, the payload gene encodes immunogenic material capable of stimulating an immune response (e.g., an adaptive immune response), such as an antigenic peptide or protein derived from a pathogen. Antigen expression may stimulate the organism's adaptive immune system to provide an adaptive immune response. Therefore, in some embodiments, the compositions provided herein are envisioned to be used as vaccines (e.g., as vaccines) for the prevention or treatment of infectious diseases. The payload protein may include CRE recombinase, GCaMP, cell therapy components, knockdown gene therapy components, epitopes exposed on the cell surface, or any combination thereof.

[0137] Examples of payload genes include sequences related to signaling biochemical pathways, e.g., signaling biochemical pathway-related genes or polynucleotides (e.g., signaling molecules). In some embodiments, the methods and compositions disclosed herein include knockdown of an endogenous signaling molecule with the controlled expression of a payload protein containing an appropriate version of the signaling molecule. Examples of target polynucleotides include disease-related genes or polynucleotides. A “disease-related” gene or polynucleotide refers to any gene or polynucleotide that produces a transcription or translation product at abnormal levels or in abnormal forms in cells derived from diseased tissue compared to non-disease control tissue or cells. A “disease-related” gene or polynucleotide may be a gene that becomes expressed at abnormally high levels, or a gene that becomes expressed at abnormally low levels, where the alteration of expression correlates with the onset and / or progression of the disease. A disease-related gene also refers to a gene that is directly involved in the pathogenesis of a disease or that possesses a mutation or genetic variant that is in linkage disequilibrium with a gene involved in the pathogenesis of a disease. The transcribed or translated product may be known or unknown, and may be at normal or abnormal levels. The signaling molecule may be associated with one or more diseases or disorders. In some embodiments, the disease or disorder is characterized by abnormal signaling of one or more signaling molecules disclosed herein. In some embodiments, the activation level of the signaling molecule correlates with the onset and / or progression of the disease or disorder. The activation level of the signaling molecule may be directly or indirectly involved in the pathogenesis of the disease or disorder. Non-limiting examples of signaling molecules, signaling pathways, and diseases and disorders characterized by abnormal signaling of the signaling molecules are listed in Tables 5 to 7. In some embodiments, the methods and compositions disclosed herein prevent or treat one or more of the diseases and disorders listed in Tables 5 to 7. In some embodiments, the payload includes a replacement version of the signaling molecule.In some embodiments, the method and composition further include knockdown of the corresponding endogenous signaling molecule. The payload may include the product of the genes listed in Tables 5 to 7. In some embodiments, the payload restores a disease or disorder characterized by abnormal signaling of one or more signaling molecules. In some embodiments, the payload reduces the activation level of one or more signaling molecules (e.g., signaling molecules with abnormal hyperactivity signaling, listed in Tables 5 to 7). In some embodiments, the payload increases the activation level of one or more signaling molecules (e.g., signaling molecules with abnormal hypoactivity signaling). In such embodiments, the payload can modulate the abundance, location, stability, and / or activity of the activators or repressors of the signaling molecules. [Table 5] [Table 6-1] [Table 6-2] [Table 6-3] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8] [Table 7-9] [Table 7-10] [Table 7-11]

[0138] vector In some embodiments, a vector (e.g., a viral vector) is provided. The viral vector may be an RNA viral vector. The polynucleotide may be derived from a positive-sense RNA virus, a negative-sense RNA virus, an ambient-sense RNA virus, or any combination thereof. The polynucleotide may be derived from a single-stranded RNA virus. The polynucleotide may be derived from a negative-sense RNA virus. The polynucleotide may be derived from one or more negative-sense RNA viruses of the order Mononegavirales. The nucleoprotein (N), phosphoprotein (P), matrix protein (M), and / or RNA-dependent RNA polymerase (L) may be derived from one or more negative-sense RNA viruses of the order Mononegavirales (e.g., bornaviridae viruses, filoviridae viruses, nyamiviridae viruses, paramyxoviridae viruses, rhabdoviridae viruses, or any combination thereof). Mononegaviral viruses may include rabies virus, Sendai virus, vesicular stomatitis virus, or any combination thereof. Mononegaviral-based viral vectors may include one or more attenuated mutations. In some embodiments, one or more negative-strand RNA viruses of the Mononegaviral order may include attenuated rabies virus strains (e.g., CVS-N2c, CVS-B2c, DRV-4, RRV-27, SRV-16, ERA, CVS-11, SAD B19, SPBN, SN-10, SN10-333, PM, LEP, SAD, or any combination thereof).

[0139] Viral vectors and methods of use are provided in PCT application publication WO2020 / 210655A1 and U.S. Patent application publication 2020 / 0165576, the contents of which are incorporated herein by reference in their entirety. Viral vectors may be modified so that they are targeted to a specific target environment of interest, such as the central nervous system, and to enhance their directivity to the target environment of interest (e.g., CNS directivity). In some embodiments, the viral vector is AAV-CAP.B22. Broad gene expression throughout the mouse and marmoset brain after intravenous delivery of engineered AAV capsids is described in Flytzanis et al. ("Broad gene expression throughout the mouse and marmoset brain after intravenous delivery of engineered AAV capsids," biorxiv, 2020), the contents of which are incorporated herein by reference in their entirety.

[0140] Examples of viral vectors that may be used in the methods, compositions, systems, and kits described herein include those provided in US20200071723A1, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the vector may include an adenovirus vector, an adeno-associated virus (AAV) vector, an Epstein-Barr virus vector, a herpesvirus vector, an attenuated HIV vector, a retrovirus vector, a vaccinia virus vector, or any combination thereof. In some embodiments, the vector may include an RNA virus vector. In some embodiments, the vector may be derived from one or more minus-strand RNA viruses of the Mononegavirales order. In some embodiments, the vector may be a rabies virus vector. Many such vectors are available that are useful for transferring exogenous genes into target mammalian cells. The vector may be, for example, an episomal vector, such as a plasmid, a virus-derived vector such as cytomegalovirus or adenovirus, or it may be incorporated into the genome of the target cell through homologous recombination or random insertion, and may be a retrovirus-derived vector such as MMLV, HIV-1, or ALV. In some embodiments, a combination of a retrovirus and a suitable packaging cell system may also be used, where the capsid protein is functional for infecting target cells. The retroviral vector may be “deficient,” meaning it may be unable to produce the viral proteins required for productive infection. Vector replication may require growth in the packaging cell system. As used herein, the term “vector” refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector may be viral or nonviral. The term “vector” encompasses any genetic element that is replicable when bound to an appropriate regulatory element and that can transfer a gene sequence into a cell.Examples of vectors include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, artificial chromosomes, viruses, and virions.

[0141] In some embodiments, the vector may include regulatory sequences that enable the translation of multiple proteins derived from a single mRNA, for example. Non-limiting examples of such regulatory sequences include internal ribosome entry sites (IRESs) and 2A self-processing sequences. In some embodiments, the 2A sequence is a 2A peptide site (F2A sequence) derived from foot-and-mouth disease virus. In some embodiments, the F2A sequence has a standard furin cleavage site. In some embodiments, the vector may also include regulatory elements known to those skilled in the art to affect the expression of polynucleotide-encoded RNA and / or protein products in the desired cell of interest. In some embodiments, functionally, polynucleotide expression is at least partially controllable by operably linked regulatory elements such that the elements regulate the transcription of the polynucleotide, the transport, processing and stability of the polynucleotide-encoded RNA, and, if necessary, the translation of the transcript. A specific example of an expression regulatory element is a promoter, typically located at 5' of the transcribed sequence. Another example of an expression regulatory element is an enhancer, which may be located at 5' or 3' of the transcribed sequence, or within the transcribed sequence. Another example of a regulatory element is the recognition sequence of microRNA. Another example of a regulatory element is the splice donor and splice acceptor sequences that regulate rations and the splicing of the introns mentioned above. Another example of a regulatory element is the transcription termination signal and / or polyadenylation sequence.

[0142] Examples of regulatory elements and promoters include those that are active in specific tissues or cell types, referred herein as “tissue-specific regulatory elements / promoters.” Tissue-specific regulatory elements are typically active in specific cells or tissues (e.g., in the liver, brain, central nervous system, spinal cord, eye, retina, or lung). Regulatory elements are typically active in these cells, tissues, or organs because they are recognized by transcription-activating proteins or other transcriptional regulators specific to those cells, tissues, or organ types.

[0143] Furthermore, expression regulatory elements include ubiquitous or promiscuous promoters / enhancers capable of driving polynucleotide expression in many different cell types. Such elements include, but are not limited to, the earliest promoter / enhancer sequence of cytomegalovirus (CMV), the promoter / enhancer sequence of Rous sarcoma virus (RSV), and other viral promoters / enhancers active in various mammalian cell types, as well as promoter / enhancer sequences derived from mammalian genes that are expressed ubiquitously or indiscriminately, including but not limited to beta-actin, ubiquitin, or EF1 alpha, or synthetic elements that do not exist naturally.

[0144] Expression regulatory elements can also confine expression in a regulated manner, i.e., a signal or stimulus increases or decreases the expression of an operably linked polynucleotide. A regulated element that increases the expression of an operably linked polynucleotide in response to a signal or stimulus is also called an “inducible element” (i.e., it is induced by a signal). Specific examples include, but are not limited to, hormone (e.g., steroid)-inducible promoters. A regulated element that decreases the expression of an operably linked polynucleotide in response to a signal or stimulus is called an “inhibitory element” (i.e., the signal decreases expression so that expression increases when the signal is removed or absent). Typically, the amount of increase or decrease confine by such an element is proportional to the amount of signal or stimulus present, with greater amounts of signal or stimulus resulting in greater increases or decreases in expression.

[0145] Detection and monitoring methods In some embodiments, the methods and compositions provided herein are useful for detecting a disease or disorder and / or monitoring its progression. As used herein, the term “diagnosis” means determining the presence or absence or nature of a disease or disorder. Such detection methods can be used, for example, for the early diagnosis of a condition, to determine whether a subject is predisposed to a disease or disorder, to monitor the progression of a disease or disorder or the progress of a treatment protocol, to assess the severity of a disease or disorder, to predict the prognosis and / or recovery prospects of a disease or disorder, or to assist in determining appropriate treatment for a subject. Detection can be performed in vitro or in vivo. The payload protein may include, for example, an imaging agent such as a diagnostic agent. The payload protein may include a diagnostic contrast agent. The diagnostic agent may include green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), TagRFP, Dronpa, Padron, mApple, mCherry, mruby3, rsCherry, rsCherryRev, their derivatives, or any combination thereof.

[0146] In some embodiments, the payload protein encodes a diagnostic agent. In some embodiments, the diagnostic agent helps identify specific cell types and / or specific cellular states. Diagnostic agents can be detectable molecules, including but not limited to fluorescent agents, chemiluminescent agents, chromophores, bioluminescent proteins, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, isotope labels, semiconductor nanoparticles, dyes, metal ions, metal sols, and ligands (e.g., biotin, streptavidin, or hapten). The term "fluorescent agent" refers to a substance or part thereof that is capable of exhibiting fluorescence to a detectable extent. For example, in some embodiments, diagnostic agents may include fluorescent proteins, including but not limited to green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), TagRFP, Dronpa, Padron, mApple, mCherry, rsCherry, rsCherryRev, or any combination thereof. In some embodiments, the expression, stability, and / or activity (e.g., fluorescence) of the diagnostic agent are configured to respond to a disease or impairment state.

[0147] In some embodiments, the diagnostic agent helps identify specific cell types and / or specific cellular conditions. These specific cell types and / or specific cellular conditions may include lesions (e.g., tumors, infected cells). Detection and / or imaging of the diagnostic agent may enable clinicians to detect such lesions during surgery, laparoscopy, intravascularly, or endoscopically. In some such embodiments, the distinction between lesions (e.g., tumors) and non-lesions (e.g., non-tumor tissue) is enhanced by detection and / or imaging of the diagnostic agent. In some embodiments, detection and / or imaging of the diagnostic agent may enable clinicians to precisely locate lesions in patients, thereby assisting in resection, irradiation, biopsy, and / or lesion removal. In some embodiments, detection and / or imaging of the diagnostic agent assists in the detection of non-malignant pathological lesions such as infarctions including myocardium, atherosclerotic foci, blood clots including thrombosis, pulmonary embolism, infectious or inflammatory lesions, non-tumorous or non-infectious inflammation, or hyperplasia. Diagnostic detection and / or imaging can also be used to detect various stages or severity of disease progression (e.g., benign, pre-malignant, and malignant thoracic lesions, tumor growth, or metastasis). Diagnostic detection and / or imaging can also be used to detect the disease's response to prophylactic or therapeutic measures or other interventions. Furthermore, diagnostic detection and / or imaging can be used to assist healthcare professionals in determining a patient's prognosis (e.g., deterioration, maintenance, partial recovery, or complete recovery) and appropriate course of action. Diagnostic detection and / or imaging can be performed using, for example, ultrasound scanners, magnetic resonance imaging (MRI) scanners, X-ray sources with film or detectors (e.g., conventional or digital radiography systems), X-ray computed tomography (CT) or computed axial tomography (CAT) scanners, gamma cameras, or positron emission tomography (PET) scanners.

[0148] Pharmaceutical composition and administration method Pharmaceutical compositions comprising one or more nucleic acids, vectors, and / or compositions provided herein, and one or more pharmaceutically acceptable carriers are also disclosed herein. Such compositions may also comprise further components such as diluents, stabilizers, excipients, and adjuvants. When used herein, “pharmaceutically acceptable” carriers, excipients, diluents, adjuvants, or stabilizers are non-toxic (preferably inactive) to cells or subjects to which they are exposed at the doses and concentrations used, or have toxic levels that can be determined by those skilled in the art.

[0149] Examples of carriers, diluents, and adjuvants include buffers such as phosphates, citrates, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides (e.g., less than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween®, Pluronics®, or polyethylene glycol (PEG). In some embodiments, a physiologically acceptable carrier is a pH-buffered aqueous solution.

[0150] The titer of the vector to be administered varies depending on, for example, the specific viral vector, mode of administration, treatment target, individual, and target cell type, and can be determined by standard methods in the art.

[0151] As will be readily apparent to those skilled in the art, useful in vivo dosages of the nucleic acids, vectors, and / or compositions to be administered, as well as specific modes of administration, will vary depending on age, body weight, severity of disease, the animal species to be treated, the particular IFFL employed, and the specific use for which the IFFL is applied. Determination of an effective dosage level, which is the dosage level necessary to achieve the desired result, can be accomplished by those skilled in the art using routine pharmacological methods. Typically, human clinical application of products is initiated at lower dosage levels, and the dosage level is increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful dosages and routes of administration for the administration of the compositions identified by the present methods using established pharmacological methods.

[0152] The exact dosage will be determined on a drug-by-drug basis, and in most cases, some generalizations regarding the dosage can be made. The dosage of the provided nucleic acids, vectors, and / or compositions may depend primarily on factors such as the condition being treated, age, body weight and health status of the patient, and thus may vary among patients. For example, a therapeutically effective human dosage of a viral vector is generally about 1×10 9 to 1×10 16 in the range of about 0.1 ml to about 100 ml of a solution containing a concentration of viral genomes. A preferred human dosage may be about 1×10 13 to 1×10 16 viral vector genomes. The dosage is adjusted to balance therapeutic benefits with any side effects, and such dosage may vary depending on the therapeutic application for which the recombinant vector is used. In some embodiments, the level of payload expression can be monitored to determine the amount of dosage and / or the frequency of administration attributable to the viral vector.

[0153] The nucleic acids, vectors, and / or compositions disclosed herein may be administered to subjects (e.g., humans) that require them. The route of administration is not particularly limited. For example, therapeutically effective amounts of nucleic acids, vectors, and / or compositions may be administered to subjects via routes standard in the art. The route of administration can be readily determined by those skilled in the art, taking into account the infectious and / or disease state being treated and the target cells / tissues that should express the payload protein.

[0154] Administration may include systemic administration (e.g., intravenous, intramuscular, intraperitoneal, or intra-articular). Administration may include intrathecal administration, intracranial injection, aerosol delivery, nasal delivery, vaginal delivery, rectal delivery, oral delivery, intraocular delivery, local delivery, local delivery, intracisional delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous injection, subcutaneous injection, intra-lymph node injection, intratumoral injection, intraperitoneal injection, intradermal injection, or any combination thereof.

[0155] Administration may include injection into brain regions (e.g., direct administration into the brain parenchyma). Brain regions include the lateral parabrachial nucleus, brainstem, medulla oblongata, medullary pyramidal body, olivary body, inferior olivary nucleus, rostral ventrolateral part of the medulla oblongata, respiratory center, dorsal respiratory group, ventral respiratory group, Pre-Betzinger complex, Betzinger complex, paramedian reticular nucleus, cuneate nucleus, gracile nucleus, internuclei, area postparenchyma, medullary cranial nerve nuclei, inferior salivary nucleus, nucleus ambiguus, dorsal vagus nucleus, hypoglossal nucleus, nucleus tractus solitarius, pons, pontine nuclei, and pontine nerves. The trigeminal nerve nucleus, the main nucleus or pontine nucleus of the trigeminal sensory nucleus (V), the motor nucleus of the trigeminal nerve (V), the abducens nerve nucleus (VI), the facial nerve nucleus (VII), the vestibulocochlear nerve nucleus (vestibular and cochlear nerve nuclei) (VIII), the superior salivary nucleus, the peduncleopontine tegmental nucleus, the respiratory center, the respiratory control center, the sustained respiratory center, the pontine micturition center (Barlington nucleus), the locus coeruleus, the pontine peduncle, the dorsolateral tegmental nucleus, the pontine tegmental reticular nucleus Superior olivary complex, paramedian pontine reticular formation, cerebellar peduncle, superior cerebellar peduncle, middle cerebellar peduncle, inferior cerebellar peduncle, cerebellum, cerebellar vermis, cerebellar hemispheres, anterior lobe, posterior lobe, flocculonodular lobe, cerebellar nuclei, fastigial nucleus, insertion nucleus, globose nucleus, embolus nucleus, dentate nucleus, tectum, quadrige, inferior colliculus, superior colliculus, pretectal area, tegmentum, periaqueductal gray matter, paracerebellar peduncle area, medial parabrachial nucleus, connective parabrachial nucleus (Koelliler-Fuse nucleus) ), medial longitudinal fasciculus rostral interstitial nucleus, midbrain reticular formation, dorsal raphe nucleus, red nucleus, ventral tegmental area, substantia nigra, parenchymal compacta, parenchymal reticular formation, interpeduncle nuclei, cerebral peduncle, cerebral peduncle branches, mescratic nerve nuclei, oculomotor nerve nucleus (III), trochlear nerve nucleus (IV), mesencephalic duct (cerebral aqueduct, Sylvian aqueduct), pineal gland, reticular nucleus, stria medullaris, thalamic cord, subcommittal organ, thalamus, anterior thalamic nuclei, anterior ventral nucleus (also known as ventral anterior nucleus) nucleus)), anterodorsal nucleus, anteromedial nucleus, medial nucleus group, dorsomedial nucleus, median nucleus group, thalamic paraphimone nucleus, Leniens nucleus, rhomboid nucleus, intralaminar nucleus group, median central nucleus, parafascicular nucleus, paracentral nucleus, lateral central nucleus, medial central nucleus, lateral nucleus group, dorsolateral nucleus, posterolateral nucleus, pulvinar, ventral nucleus group, anteroventral nucleus, lateral ventral nucleus, posteroventral nucleus, posteroventral lateral nucleus, posteroventral medial nucleus, posterior thalamus, medial geniculate body, lateral geniculate body, thalamic reticular nucleus, optic Hypofloor, limbic system, HPA axis, preoptic area, medial preoptic nucleus, suprachiasmatic nucleus, paraventricular nucleus, supraoptic nucleus, prehypothalamic nucleus, lateral preoptic nucleus, median preoptic nucleus, periventricular preoptic nucleus, eminence, dorsomedial hypothalamic nucleus, ventral Medial nucleus, arcuate nucleus, lateral field, lateral nucleus bulge, lateral nucleus bulge, mammillary nucleus, posterior nucleus, lateral field, optic chiasm, subfornical organ, periventricular nucleus, pituitary stalk, gray ridge, nucleus bulge, tuberomammillary nucleus, bulge region, mammillary body, mammillary nucleus,Hypothalamic region, subthalamic nucleus, zona inconsistency, pituitary gland, neurohypopituitary gland, middle lobe, adenohypopituitary gland, cerebral hemispheres, corona radiata, internal capsule, external capsule, outermost capsule, arcuate fasciculus, uncinate fasciculus, perforating fibers, hippocampus, dentate gyrus, Ammon's horn, Ammon's horn area 1, Ammon's horn area 2, Ammon's horn area 3, Ammon's horn area 4, amygdala, central nucleus, medial nucleus (accessory olfactory system), cortical and basomedial nuclei, lateral and basolateral nuclei, dilated amygdala, stria terminalis, bed nucleus of stria terminalis, claustrum, basal ganglia, striatum, dorsal striatum (also known as neostriatum), putamen, caudate nucleus, ventral striatum Striatum, striatum, nucleus accumbens, olfactory tubercle, globus pallidus, subthalamic nucleus, basal forebrain, anterior perforated nucleus, nucleus nomina, basal ganglia, Broca's diagonal zone, septal nucleus, medial septal nucleus, lamina terminalis, vascular organs of lamina terminalis, olfactory bulb, piriform cortex, anterior olfactory nucleus, olfactory tract, anterior commissure, uncinate, cerebral cortex, frontal lobe, frontal cortex, primary motor cortex, supplementary motor cortex, premotor cortex, prefrontal cortex, frontal pole cortex, orbitofrontal cortex, dorsolateral prefrontal cortex, dorsomedial prefrontal cortex, ventrolateral prefrontal cortex, superior frontal gyrus, middle frontal gyrus, inferior frontal gyrus, Brodmann's area (4, 6, 8, 9, 1) 0, 11, 12, 24, 25, 32, 33, 44, 45, 46, and / or 47), parietal lobe, parietal cortex, primary somatosensory cortex (S1), secondary somatosensory cortex (S2), posterior parietal cortex, postcentral gyrus, precuneus, Brodmann area (1, 2, 3 (primary somatosensory cortex), 5, 7, 23, 26, 29, 31, 39, and / or 40), occipital lobe, primary visual cortex (V1), V2, V3, V4, V5 / MT, extococcal gyrus, cuneiform, Brodmann area (17 (V1, primary visual cortex), 18, and / or 19), temporal lobe, primary auditory cortex (A1), secondary auditory cortex (A2), inferior temporal cortex, posterior inferior temporal cortex, superior temporal gyrus, middle temporal gyrus, inferior temporal gyrus, entorhinal cortex, perirhinal cortex, parahippocampal gyrus, fusiform gyrus, Brodmann's area (9, 20, 21, 22, 27, 34, 35, 36, 37, 38, 41, and / or 42), medial superior temporal lobe (MST), insular cortex, cingulate cortex, anterior cingulate cortex, posterior cingulate cortex, dorsal cingulate cortex, posterior corpus callosum cortex, gray zone, subgenual area (Subgenual) Area 25, Brodmann's area (23, 24, 26, 29, 30 (posterior corpus callosum area), 31, and / or 32), cranial nerves (olfactory nerve (I), optic nerve (II), oculomotor nerve (III), trochlear nerve (IV), trigeminal nerve (V), abducens nerve (VI), facial nerve (VII), vestibular-cochlear nerve (VIII), glossopharyngeal nerve (IX), vagus nerve (X), accessory nerve (XI), hypoglossal nerve (XII)),Or may include any combination thereof. Brain regions may include the neural pathways such as the superior longitudinal fasciculus, arcuate fasciculus, thalamocortical radiation, cerebral peduncle, corpus callosum, posterior commissure, pyramidal or corticospinal tract, medial longitudinal fasciculus, dopamine system, mesocortical tract, mesolimbic tract, nigrostriatal tract, tuberculous infundibular tract, serotonin system, norepinephrine tract, posterior columnar-medial lemniscus tract, spinothalamic tract, lateral spinothalamic tract, anterior spinothalamic tract, or any combination thereof. It may include.

[0156] The nucleic acids, vectors, and / or compositions to be used may be available in liquid or lyophilized form (combined with one or more suitable preservatives and / or protective agents to protect the virus during the lyophilization process). For gene therapy (e.g., for neurological disorders that can be restored by a specific gene product), nucleic acids, vectors, and / or compositions expressing a therapeutically effective dose of a therapeutic protein are administered to a host requiring such treatment. The use of the nucleic acids, vectors, and / or compositions provided herein in the manufacture of agents for inducing immunity in a host or for providing gene therapy to a host is within the scope of this application.

[0157] A therapeutically effective amount of the nucleic acids, vectors, and / or compositions provided herein can be administered to a subject at various points in time. For example, the nucleic acids, vectors, and / or compositions provided herein can be administered to a subject before, during, or after the onset of a disease, disorder, and / or infection. The nucleic acids, vectors, and / or compositions provided herein can also be administered to a subject before, during, or after the onset of a disease, disorder, and / or infection. In some embodiments, the nucleic acids, vectors, and / or compositions provided herein are administered to a subject during remission of a disease or disorder. In some embodiments, the nucleic acids, vectors, and / or compositions provided herein are administered before the onset of a disease or disorder in the subject. In some embodiments, the nucleic acids, vectors, and / or compositions provided herein are administered to a subject at risk of developing a disease or disorder.

[0158] The dosage frequency of the nucleic acids, vectors, and / or compositions provided herein can vary. For example, the nucleic acids, vectors, and / or compositions provided herein can be administered to a subject about once a week, about once every two weeks, about once a month, about once every six months, about once a year, about once every two years, about once every three years, about once every four years, about once every five years, about once every six years, about once every seven years, about once every eight years, about once every nine years, about once every ten years, or about once every fifteen years. In some embodiments, the nucleic acids, vectors, and / or compositions provided herein are administered to a subject at most about once a week, at most about once every two weeks, at most about once a month, at most about once every six months, at most about once a year, at most about once every two years, at most about once every three years, at most about once every four years, at most about once every five years, at most about once every six years, at most about once every seven years, at most about once every eight years, at most about once every nine years, at most about once every ten years, or at most once every fifteen years.

[0159] Some aspects of the embodiments discussed above are disclosed in more detail in the following embodiments, but they are not intended to limit the scope of this disclosure in any way. [Examples]

[0160] [Example 1] miRNA circuit modules for precise and tunable control of gene expression The ability to express transgenes at specified levels is crucial for understanding cell behavior and for applications in gene therapy and cell therapy. Transfection, viral vectors, and other gene delivery methods result in varying protein expression levels and limited quantitative control, while targeted knock-in and stable selection are inefficient and slow. Although accuracy can be improved with active compensation mechanisms, their widespread use has been hindered by the need for further proteins or a lack of tunability. A toolkit of small, synthetic miRNA-based circuit modules that provide precise and tunable control of transgenes across diverse cell types is disclosed herein. These circuits, referred to as DIMMERs (parameter-independent miRNA-mediated expression regulators), in some embodiments achieve nearly uniform protein expression over variations of more than two orders of magnitude in underlying parameters or transcription rates by utilizing multivalent miRNA regulatory interactions within an incoherent feedforward loop architecture, without being constrained by any particular theory. In some embodiments, these circuits also allow for coarse and fine control of expression, are portable, and function across diverse cell types. Furthermore, heuristic miRNA design algorithms enable the creation of orthogonal circuit variants that independently control multiple genes within the same cell. These circuits have enabled dramatic improvements in CRISPR imaging and super-resolution imaging of the EGFR receptor using transient transfection. The toolbox provided herein can enable precise, tuneable, and quantity-independent gene expression for research, gene therapy, and other biotechnology applications.

[0161] introduction Biotechnology and biomedical research heavily rely on the ectopic expression of transgenes in living cells. Typical expression systems produce a wide range of expression levels across individual cells. This applies to non-integrated approaches such as DNA transfection and AAV vectors, as well as integrated systems such as lentiviruses or piggyback transposons. This heterogeneity reflects the inevitable variability in the number of gene copies taken up, integrated, and expressed by each cell, and gene expression noise. While variability or noise may be acceptable, or even useful, in some situations, more often it hinders accurate analysis and precise control of cellular behavior. Selecting stable clones can reduce expression variability, but this is time-consuming and susceptible to stochastic silencing. An ideal gene regulation system compensates for this variability, enabling more precise control of expression levels, reduced toxicity in gene and cell therapies, and lower background when using reporters (Figure 1A).

[0162] The incoherent feedforward loop (IFFL) circuit motif provides an ideal mechanism for providing these capabilities. In an IFFL, a target gene and its negative regulator are simultaneously regulated by the same input (Figure 1B). Gene quantity can be considered such an input and proportionally affects the expression of both the regulator and its target. In some parameter regimes, these two effects cancel each other out, and target expression approaches a fixed level at high gene quantities (Figure 1C, supplementary modeling). An ideal IFFL system should further allow for adjustment of this expression setpoint, creation of multiple orthogonal regulatory systems for the simultaneous control of multiple genes, and the ability to operate in multiple cell types (Figure 1C).

[0163] Previous studies have demonstrated that IFFL circuits can provide gene-powder-independent expression over ranges of 50-fold and 100-fold, respectively, in bacterial and mammalian cells. However, these systems require the expression of additional proteins, complicating their routine use. miRNAs can be ideal regulators for IFFL gene-powder compensation systems because they can be expressed from within introns or from small transcripts. Bleris et al. demonstrated that miRNA-based IFFLs can achieve gene-powder compensation. Strovas et al. improved this by incorporating multiple repeats of a native miRNA and its target sequence into the gene. This reduced expression fluctuations in single-copy incorporation, compensating for potential crosstalk with endogenous genes, and achieved gene-powder compensation over a range of approximately 20-fold. More recently, Yang et al. introduced an "equalizer" architecture combining transcriptional negative feedback of transcription by the TetR protein with miRNA. This expanded the range of effective gene-powder compensation but added complexity and potential immunogenicity, requiring the expression of bacterial TetR protein. Despite numerous studies, it remains unclear which sequence features are sufficient to enable orthogonally adjustable dose-compensated miRNA IFFLs, and consequently, a widely useful toolkit for such circuits does not yet exist.

[0164] To address this need, a set of miRNA-based gene dose compensation systems called DIMMER (Pass-Independent miRNA-Mediated Expression Regulators) was designed. These circuits can utilize specific configurations of miRNA expression cassettes and their target sequences, leveraging the ability to achieve multivalent miRNA regulation through the natural TNRC6 scaffold system. These circuits can enable systematic adjustment of expression levels by modulating the number of miRNA cassettes, the number of target binding sites, and the phase complementarity of the miRNA target sites. Furthermore, they can be used to orthogonally regulate multiple genes in the same cell and operate similarly across different cell types. A toolkit of 10 mutually orthogonal, ready-to-use expression systems that can be incorporated into a variety of systems was designed. Finally, their usefulness was demonstrated for CRISPR imaging and super-resolution protein imaging techniques. The DIMMER provided herein may enable routine research and biotechnology applications to operate with greater precision, control, and predictability.

[0165] result Mathematical modeling identifies parameter schemes for gene dose compensation. First, we aimed to identify the general conditions under which a miRNA-based IFFL circuit can achieve gene dose-compensated target expression (Figure 1B, Supplementary Modeling). We developed a minimal model based on several assumptions. First, we assumed that pri-miRNA and target mRNA are transcribed constitutively in fixed ratios and in direct proportion to gene dose. Second, we assumed that the total rate of RISC complex production per gene copy is constant. This rate incorporates the combined processes of pri-miRNA transcription, post-transcriptional processing, and binding to the Argonaut protein. Third, we assumed that RISC and its target mRNA reversibly bind to form a RISC-mRNA complex. Finally, we assumed that this complex can cause degradation of the bound mRNA.

[0166] This model showed an expression profile independent of gene load (Figure 2A). More specifically, the target protein expression level initially increased linearly at low gene loads, but K / k c At this value, the expression level approached the extreme level of gene dose independence (Figure 2A). Changing the values ​​of mRNA and miRNA binding or dissociation rates, or miRNA inhibition intensity, can be used to adjust the steady-state level of mRNA while maintaining compensation (Figures 7A-7B). It should be noted that hypersensitive or quasi-linear regulation of mRNA by RISC may result in a gene dose-dependent profile (Supplementary Modeling, Figure 7C). Nevertheless, these results suggest that, under general conditions, miRNA-based IFFLs can achieve gene dose-independent protein expression.

[0167] miRNA-based IFFLs enable gene-quantity independent expression. Based on these modeling results, we designed and constructed a miRNA-based IFFL set. After exploring multiple circuit configurations, we focused on a design in which miRNAs are expressed on separate adjacent transcripts derived from the same DNA region (Figure 2B) because this configuration offers some flexibility in relative miRNA and target gene expression levels (Figure 8).

[0168] To execute the circuit, a platform including a branched promoter pair was constructed (Figure 2B). One promoter (facing left in Figure 2B) constitutively expressed mRuby3, a fluorescent protein gene quantity indicator, along with a synthetic miRNA. To create a miRNA expression cassette, a miR-E backbone for pri-miRNA expression was inserted into a synthetic intron. The second promoter (facing right in Figure 2B) constitutively expressed the target gene, EGFP. To enable regulation by synthetic miRNA, one or more complementary target miRNA sites were incorporated into the 3'UTR of the target gene. This small two-transcript construct enabled systematic analysis of the composition of various miRNAs and target sites.

[0169] To perform miRNA regulation orthogonal to natural miRNAs, we first used a previously described miRNA sequence targeting sea urticaria luciferase (miR-L) along with a single copy of its perfectly complementary 21 bp target site. U2OS cells were transiently transfected with the resulting construct, and cells were analyzed by flow cytometry after 48 hours, plotting targeted EGFP expression against gene load as shown by mRuby3 (Figure 2C). Compared to an unregulated control lacking the miRNA target site, IFFL strongly reduced targeted EGFP expression, as expected (Figure 2C). However, the circuit failed to achieve the gene load compensation behavior predicted by mathematical modeling (Figure 2A).

[0170] Next, we investigated whether the lack of gene dose compensation could be related to the strongly repressive regime resulting from complete miRNA-target complementarity. We designed a set of IFFL mutants in which the complementarity of a single target site was gradually reduced from 21 bp to 17 bp (Table 1). Designs with reduced 3' complementarity, particularly below 19 bp, showed weak or no repression of target expression (Figure 2D), while those that efficiently repressed maintained strong repression comparable to that of the full-length 21 bp construct. Nevertheless, within this set, the constructs did not achieve complete gene dose independence. Therefore, regulation of complementarity alone was not sufficient to provide gene dose compensation.

[0171] The loss of regulation with reduced complementarity contrasted with the well-known regulatory capacity of miRNAs with much shorter complementary regions of only about 8 bp. One mechanism enabling specific regulation by short sequences involves multivalent recognition of multiple target binding sites on the same mRNA. TNRC6 is a scaffold protein that enables multivalent recognition by simultaneously binding to multiple miRNA-loaded Argonaut (Ago) complexes (Figure 2G). Consistent with its role in multivalent regulation, tandem repeats of 2-4 copies of the 17 bp target site gradually increased regulation and strongly reduced gene dose sensitivity at higher expression levels (Figure 2E). For example, with four tandem binding sites, target expression increased only fourfold over gene doses ranging from 200-fold to 200-fold (Figure 2F). The “tail” of increased expression at the highest gene doses may reflect saturation of the miRNA binding mechanism, as observed in other studies. In summary, these results demonstrate that miRNA-based IFFLs, based on 17 bp of miRNA-target complementarity and four tandem binding sites, can achieve near-perfect compensation over gene volume variations of more than two orders of magnitude.

[0172] To elucidate whether this compensatory behavior depends on TNRC6, a previously identified fragment of the native TNRC6B protein, we utilized a T6B peptide that competitively inhibits TNRC6 activity (Figure 2G). When co-transfected with a 4×17bp IFFL, the T6B inhibitory peptide caused a loss of regulation, resulting in dose-dependent expression nearly identical to that produced by the unregulated construct (Figure 2H). In contrast, the T6B inhibitory peptide had little effect on a single, fully complementary 21bp construct, suggesting that it regulates in a TNRC6-independent manner (Figure 2I). Finally, a negative control mutant of T6B lacking the Ago2-binding domain failed to abolish regulation, as expected (Figure 9A). Taken together, these results suggest that the 4×17bp and 1×21bp designs operate via TNRC6-dependent and TNRC6-independent mechanisms, respectively. Due to their ability to restrict expression, these circuits are referred to herein as DIMMERs. More generally, these results suggest that multivalent regulation by multiple individually weak miRNA binding sites can achieve robust regulation and gene dose compensation within the context of the IFFL circuit.

[0173] The DIMMER circuit enables independent adjustment of gene expression levels. An ideal regulatory system would allow for control not only of gene dose independence but also of saturation expression levels. In principle, this can be achieved by adjusting the complementarity of repeated target sites, the number of miRNA cassettes within synthetic introns, or the strength of promoters controlling miRNA cassette expression. We systematically varied each of these parameters and analyzed their effects on expression.

[0174] First, the complementarity of the miR-L target site was varied from 8 bp to 21 bp, and in each case, four tandem target site copies were incorporated (Figure 3A, Figure 10). At each length, the expression of the IFFL regulatory target was quantified at high gene loads (Figure 3A, right panel, shaded area) (Figure 3A, left panel). Repression was mild at 8 bp, decreased with increasing complementarity in the central region, and then strengthened again after the central region as more complementarity was added (Figure 3A, left panel). These results are generally consistent with previous observations that miRNA inhibition does not increase monotonically with complementarity. For the miR-L target site, repression was most sensitive at 16-20 base pair complementarity (Figure 3A). In fact, the three designs of 4×17, 4×18, and 4×19 achieved gene load-independent expression at more than an order of magnitude more than an order of magnitude at saturated expression levels (Figure 3B).

[0175] Next, we varied the copy number of the miRNA expression cassette in the synthetic intron to effectively regulate the miRNA-to-mRNA stoichiometric ratio (a second design feature of these circuits is the separation of miRNA and target, 3C). Compared to a single copy, two or three copies of miRNA provided a means of fine-tuning expression regulation, doubling and tripling expression, respectively, while preserving gene dose compensation.

[0176] Finally, we altered the promoter controlling miRNA cassette expression. Compared to EF1α, the weaker PGK promoter enabled approximately 3.5 times higher target gene expression at a given gene dose level (Figure 3D). Nevertheless, gene dose compensation was conserved here as well. Furthermore, we addressed whether our circuit functions in inducible system settings. Using a 4-epi-tetracycline-modulated promoter, we expressed the target and verified the circuit's performance in TRex cells (Figure 3E). By adjusting the concentration of 4-epi-tetracycline, it was possible to change the circuit's setting point by approximately two orders of magnitude while preserving gene dose compensation (Figure 3E, Figure 11). In summary, these results demonstrate that expression levels can be tuned in a coarse or fine manner using four distinct mechanisms.

[0177] Orthogonal gene dose compensation circuits enable independent control of target genes. Manipulated gene systems increasingly require multiple genes and transcripts, many or all of which may benefit from gene-independent control. Therefore, we aimed to construct an expanded set of orthogonal synthetic miRNA-target site pairs, referred to as synmiR. Designing synmiR began by generating five random miRNA sequences, each similar in structure to miR-L, each containing the initial A, a congeneral U at the 3' end of a single target site, and 25% GC content. Next, dose-response curves were measured for each candidate miRNA. To do this, we designed an "open-loop" system that allows for the measurement of miRNA expression-independent control and its effect on the target miRNA reporter gene (Figure 4A). For a single, perfectly complementary miR-L site, consistent with the previous closed-loop results (Figure 2C), inhibition was found to increase with the gene dose of the miRNA (Figure 4B). Of the five sequences initially examined, synmiR1, synmiR4, and synmiR5 repressed miRNA expression by at least an order of magnitude compared to a control lacking miRNA (Table 1, Figure 4D). In contrast, synmiR2 and synmiR3 achieved little substantial repression (a second design feature of these circuits is the separation of miRNA and target, 12A). Scrutiny of these designs revealed subsequences with two or more A / T pairs over a wide area. We hypothesized that these A / T dinucleotides could potentially induce miRNA degradation mechanisms to recognize miRNA tailing and trimming signals, leading to miRNA destabilization. Consistent with this hypothesis, A-to-G substitutions at position 20 in synmiR-2 or at position 19 in synmiR-3 restored miRNA inhibition of target gene expression (Figure 4D).

[0178] Based on these results, an empirical two-step sequence design algorithm was developed (Figure 4C). In the first step, random 21 bp miRNA candidate sequences were generated, constrained to have a 5'U in the mature miRNA. In the second step, based on known requirements for miRNA loading, the sequences were constrained to a total of 5-8 GC nucleotides, 1-4 in the seed region and 1-2 in the broad region (Figure 4C). Five candidate sequences (synmiR6-synmiR10) were generated and validated using this simplified algorithm, and their open-loop behavior was analyzed (Figure 4D, Figure 12B). When used as a single, perfectly complementary site, all five sequences produced potent repression comparable to that of miR-L. However, one sequence, synmiR-6, was discarded due to its similarity to the human endogenous miRNA hsa-mir-5697 (Methods). In summary, these results yielded a set of 10 miRNA sequences capable of potent suppression in a completely complementary manner (Figure 4D). Pairing each of these 10 miRNAs with all 10 target sequences in the open-loop system revealed the desired strong orthogonality in regulation (Figure 4E).

[0179] Using these synmiR sequences, a set of 10 orthogonal dose compensation systems was developed using the framework shown in Figure 2B. Based on our previous analysis of miR-L (Figures 2-3), the complementarity and number of tandem target sites were varied for each sequence design. Various sequences required alternative configurations to yield dose compensation. For example, with synmiR-4 and synmiR-5, a 4×17bp configuration produced a potent inhibitory profile more similar to that of fully complementary targets (Figure 13). This was inferred to reflect a higher GC content in the seed and supplement regions of these two miRNAs compared to miR-L. This increase in GC content allowed for shorter 8bp or 9bp target sites present during 4-8 repeats, successfully resulting in dose compensation designs for these miRNAs (Figure 4F). A similar process was repeated for other sequences, ultimately identifying nine further closed-loop systems that exhibited substantial levels of dose compensation (Figure 4F). Generally, the regulatory behavior was highly sensitive to both the number and complementarity of target sites (Figure 14). However, decisively, each of these nine synmiR sequences, and the original sequence, was capable of resulting in at least partial gene dose compensation in one or more configurations (Figure 4F), and many of them could be tuned to various expression setpoints through these features (Figure 14). These results provide a toolkit for gene dose compensation systems and, more generally, suggest that it should be possible to manipulate further systems with various expression setpoints.

[0180] By using multiple dose compensation systems in combination, it should be possible to simultaneously and independently specify the expression of multiple target genes (Figure 1C, Panel 2). To verify this possibility, a second set of dose compensation expression systems was constructed using separate fluorescent reporters (Figure 4G). Cells were transfected with pairs of systems having various regulatory setpoints, and the resulting expression profiles of two regulated target genes were analyzed (Figure 4H). Four pairs of systems were analyzed. Each produced a separate two-dimensional expression distribution based on the setpoints for the two reporters. This demonstrates that manipulated dose compensation systems can specify two-dimensional expression distributions, and suggests that higher-dimensional control of the distributions of more genes should also be attainable.

[0181] The gene dose compensation system is portable and minimally disruptive. An ideal gene dose compensation system is portable, capable of operating similarly across different cell types, functional in both transient transfection and genomic integration, and minimizing disruption to host cells. To examine these characteristics, several circuit variants, including a 4×17 miR-L system (Figure 2E), were transiently transfected into four mammalian cell lines: U2OS, CHO-K1, HEK293, and N2A. Strong and qualitatively similar gene dose compensation was observed in each cell line (Figure 5A). The cell lines were diverse in the threshold gene dose at which expression saturates (Figure 5A, gray vertical line) and in the saturation expression level (Figure 5A, gray horizontal line), as measured by arbitrary fluorescence units. However, the ratios of these values ​​were conserved (Figure 5B). Similar results were obtained for other circuits, including synmiR-4 with eight repeats of a 9 bp target site, as well as synmiR-L and synmiR-5, each with eight repeats of an 8 bp target site (Figure 15A). Furthermore, the ratio of saturated expression level to threshold gene amount was similar across cell lines for each construct (Figure 15B). This suggests a model in which miRNA circuits function equally well across different cell types, but protein expression intensities diverge, sometimes due to differences in translational capacity or basal proteolytic rates. Taken together, these results indicate that gene dose compensation circuits can function across different cellular conditions.

[0182] Stable cell lines are crucial for research and applications such as cell therapy. To elucidate whether the gene dose compensation circuit can function even under stable integration conditions, we used piggyback transposition in combination with the iON system, which enables expression only from constructs that have been successfully integrated and undergone site-directed recombination in the genome. Subsequently, we selected monoclones and analyzed reporter expression by flow cytometry (Figure 5C). The integrated copy number varied by more than two orders of magnitude between clones, as indicated by the fluorescence intensity of mRuby3 (Figure 5D, x-axis). Nevertheless, cargo EGFP expression remained almost constant (Figure 5D, y-axis). Therefore, the gene dose compensation circuit functions under stable integration conditions.

[0183] The expression of synthetic miRNAs can, in principle, disrupt endogenous gene expression. To identify such effects, bulk RNA sequencing was performed on cells transfected with miR-L and nine orthogonal synmiRs, and these were compared to negative control transfections with a BFP expression vector. Only a few genes were significantly up- or down-regulated by the miRNAs (Figure 5E). These were rich in heat shock proteins such as HSPA6. Crucially, the gene sets up-regulated by different miRNAs showed strong duplication (Figure 5F, Table 8, Figure 16). Therefore, for the synmiRs described herein, off-target regulation appears to reflect only the non-specific effects of miRNA expression, rather than sequence-specific disruption. [Table 8]

[0184] Gene dose compensation systems can suppress background noise and enhance biological imaging. Gene dose compensation has diverse applications across biology. In quantitative imaging, restricting the expression of a fluorescent protein fused to another protein of interest can reduce background fluorescence, enable more precise spatial localization of fusion protein distribution, and facilitate single-molecule imaging by creating exogenous protein expression that matches endogenous expression, even with transient transfection. To validate this, expression constructs of EGFR-mEGFP membrane marker fusion proteins were transfected either unregulated or controlled by 4×17 or 4×18 circuits (Figure 6A). Both circuits restricted expression, with 4×18 providing the lowest expression levels (Figure 17A). Furthermore, the unregulated control showed heterogeneous expression, with approximately 20% of cells in the region overexpressing EGFR, making it difficult to recognize its membrane localization (Figure 6B, left). In contrast, in the 4×18bp DIMMER system, most cells showed a more clearly defined pattern with strong plasma membrane localization (Figure 6B, right).

[0185] The advantage of gene-compensated expression is that it can be directly and quantitatively observed using super-resolution imaging with DNA-PAINT (Point Accumulation for Imaging in Nanoscale Topography), which enables the analysis of single protein molecules on the cell surface. CHO-K1 cells were transfected with unregulated and regulated EGFR-mEGFP plasmids. After 48 hours of transient transfection, cells were fixed and prepared for DNA-PAINT imaging using DNA-conjugate anti-GFP nanobodies targeting the intracellular mEGFP tag of the EGFR receptor (Figure 6C). Raw DNA-PAINT localization (Figure 6D) was subjected to a recently developed clustering procedure to obtain the location of single EGF receptor proteins on the cell membrane (Figure 6E). From these datasets, the overall receptor density for various plasmid constructs was determined.

[0186] As expected, the receptor density from the unregulated construct was significantly higher than that from the regulated plasmid (a second design feature of these circuits is the separation of miRNA and target, 6F). Specifically, the receptor density for the unregulated plasmid was 29 ± 40 μm². -2 While the average density was (mean ± sd), the density of receptors in cells transfected with the modified plasmid was approximately 15 times lower (4 × 17 bp: 1.9 ± 1.2 μm). -2 4×18bp:1.0±0.8μm -2 , 4×19bp:0.6±0.4μm -2 Interestingly, not only did the average density follow the expected trend, but the distribution was considerably narrower for the regulated plasmids compared to the unregulated ones (Figure 6F).

[0187] The gene dose compensation circuit also improved CRISPR imaging. For example, telomeres could be imaged by expressing dCas9-EGFP with or without gene dose compensation, along with gRNAs targeting repeating telomere sequences (Figure 6G). Flow cytometry showed that the circuit reduced dCas9-EGFP expression and its gene dose sensitivity, but not to the same extent as seen with other genes (Figure 17B). In the unregulated system, dCas9-EGFP hardly labeled telomeres; instead, consistent with previous observations, it formed sharp aggregates in the nucleolus (Figure 6H, left panel). In contrast, the 4×17bp circuit reduced nucleolar labeling, with most fluorescence limited to dots, consistent with telomere labeling. Furthermore, the more potent 4×19bp circuit removed almost all nucleolar labeling while maintaining clear telomere labeling. Therefore, these circuits improved the signal-to-background ratio, as is evident in the line scan of the images (Figure 6H, center and right panels) and the contrast at each dot. In summary, these results demonstrate how the gene dose compensation circuits developed in this invention improve the imaging of proteins and cellular structures.

[0188] Consideration Ectopic gene expression is fundamental to modern biology, as well as gene and cell therapies; however, most approaches result in variable, dose-sensitive expression distributions. This invention designs a set of miRNA-based circuits that enable more precise and dose-independent control of protein expression. These circuits also allow for regulating expression levels through sequence (Figures 2-3), orthogonal control of multiple target genes (Figure 4), and portability across cell types and delivery modes (Figure 5). Therefore, the circuits provided herein can serve as a standard system for controlling gene expression in diverse fields of biomedical science and biotechnology, including imaging (Figure 6).

[0189] The process of designing robust, dose-independent circuits required identifying design principles for miRNA regulation. A single, perfectly complementary target site resulted in a setpoint of extremely low expression (Figure 2C). Reducing complementarity was thought to modulate the binding affinity between miRNA and the target, as well as the inhibitory intensity, thus increasing the circuit's expression level. However, systematic variations in the complementarity of single target sites demonstrated that reducing complementarity could completely eliminate regulation (Figure 2D). Surprisingly, however, TNRC6-dependent co-existence between multiple target sites restored regulation to individually weak sites, enabling dose-independent action (Figure 2E). Thus, while co-existence in other regulatory systems is associated with oversensitivity, in this invention, it allows for a linear dependence of target expression on miRNA levels, which is necessary for dose-independent action (see supplementary modeling below). These results suggest that weak, polyvalent miRNA regulation may provide the linear response required for effective dose compensation, sometimes by avoiding direct cleavage. It should also be noted that the behavior observed in this invention is generally consistent with the evidence regarding the important role of weak synergistic interactions in natural miRNA regulatory systems.

[0190] It has been found that altering miRNA regulation strength through complementarity is straightforward for one instance of miR-L, but more complex for other generated synmiRs, indicating that additional factors beyond base pairing are important for determining the activity of a miRNA towards its target. In some embodiments, without being bound by any particular theory, the molecular regulatory mechanism in a gene dosage-compensated regime is catalytic, and in some cases slicer-independent.

[0191] A second design feature of these circuits is the separation of the miRNA and the target gene into separate, divergently transcribed genes. Initial designs that incorporated the miRNA and target into a single transcript failed to achieve complete gene dosage independence (Figure 8). The divergent design allows for strong expression of the miRNA, avoiding effective inhibition of the miRNA's own production. Further engineering may enable even more compact designs.

[0192] Empirical rules effective for designing orthogonal miRNA sequences are provided herein. In some embodiments, the circuit achieves gene dosage compensation across multiple cell lines (Figure 5A, Figure 15), while total protein levels vary. Through modulation of translation or proteolysis, cell-type independent control of protein concentration can be achieved in some embodiments. In some embodiments, the use of these systems includes regulating expression via an inducing agent.

[0193] The DIMMER circuits provided herein constitute a powerful toolbox for researchers and biomedical engineers. These circuits can reduce background noise in imaging (Figure 6), enable detailed investigation of concentration-dependent effects in numerous studies, ensure fixed expression levels for receptors or other components in cell therapy, and help refine ectopic transcription factor expression in regenerative medicine. One of the primary application categories is gene therapy. Many diseases that can be targeted by gene therapy exhibit toxicity at high levels of therapeutic genes, making it crucial to suppress overexpression. Therefore, these systems can be useful components in a wide range of manipulated research and therapeutic settings.

[0194] method Supplemental Modeling Plasmid construction

[0195] Table 1 lists the constructs used in this study. Some constructs were generated using standard cloning procedures. Inserts were generated using PCR or gBlock synthesis (IDT) and ligated to a linearized skeleton using restriction enzyme digestion by either T4 ligase (NEB #M0202M) or in-fusion (Takara #102518) assembly. The remaining constructs were designed by the authors and synthesized by GenScript. Selected constructs have been deposited in Addgene, and maps are available.

[0196] miRNA alignment to databases

[0197] Synthetic miRNA sequences (mature miRNA, 22nt) were aligned to known miRNA sequence databases (mirbase.org) to determine whether any similarities existed between the synthetic and natural sequences.

[0198] tissue culture

[0199] U2OS cells, T-Rex cells, CHO cells, and N2A cells were cultured at 37°C in a humidity-controlled chamber with 5% CO2. The growth medium consisted of DMEM (Dulbecc's modified Eagle medium, ThermoFisher #11960-069) supplemented with 10% FBS, 1 U / ml penicillin, 1 μg / ml streptomycin, 1 mM sodium pyruvate, 1 × NEAA (ThermoFisher #11140-050), 1 mM L-glutamine, and 0.1 mg / mL Normocin (InvivoGen #ant-nr).

[0200] Transient transfection

[0201] Cells were seeded at a density of 50,000 cells per well in a standard 24-well glass-bottom plate for flow cytometry or imaging experiments, and cultured overnight under standard conditions. The following day, the cells were transiently transfected using Fugene HD (Promega #E2311) according to the manufacturer's protocol.

[0202] Flow cytometry

[0203] Cells were incubated 2 days after transient transfection, and the culture medium was changed 24 hours after transfection. Cells were trypsinized with 75 μL of 0.25% trypsin at 37°C for 5 minutes. After digestion, cells were resuspended in 125 μL of HBSS containing 2.5 mg / ml BSA and 1 mM EDTA. Subsequently, cells were filtered through a 40 μm cell strainer and analyzed using a CytoFLEX S instrument (Beckman Coulter). Flow data was analyzed using a custom-built Python code.

[0204] Cell sorting

[0205] To prepare monoclones expressing the DIMMER circuit integrated into the genome, cells were harvested and resuspended as monoclones in sorting buffer (BD FACS Pre-Sort Buffer) supplemented with 1 U / ml of DNAseI using a cell sorter (Sony MA900). The cells were sorted into 96-well plates in standard U2OS culture medium. After growing the cells in 24-well plates, they were measured using a flow cytometer.

[0206] In vitro image analysis

[0207] Transiently transfected U2OS cells were imaged at 60x magnification using a Nikon confocal microscope, with each image spaced 0.5 microns apart in the z-direction. The images were processed with Fiji software.

[0208] To analyze relative signal intensities in dCas9 imaging experiments, a maximum intensity projection was applied to 11 slices of a Z-stack. Freehand lines were drawn to select the dot region and determine the signal intensity of the dots. Straight lines approximately 5 microns long were drawn centered on the dots to determine the signal intensity in the background. Signal intensities were generated using the Fiji ROI average intensity function. Relative signal intensities were calculated by normalizing the background intensity to 1.

[0209] To analyze the signal-to-noise ratio (SNR), dot and nuclear regions were selected by drawing freehand lines. Noise intensity was calculated using the intensity within the nuclear region, excluding the dot area. SNR was calculated by dividing the average intensity of each dot by the noise.

[0210] Bulk RNA sequencing Sample preparation and sequencing

[0211] To verify the effects of all off-targets of synmiR, U2OS cells were seeded on 6-well plates at 300,000 cells per well. The next day, cells were transfected with 1,000 ng of either control plasmid or BFP-miRNA plasmid using Fugene HD (Promega #E2311) according to the manufacturer's instructions. The medium was replaced with 2 mL of fresh medium 24 hours after transfection. Cells were harvested 48 hours after transfection by digestion with 0.25% trypsin-EDTA, centrifugation at 300g for 5 minutes, and removal of the supernatant by aspiration. The cell pellet was stored at -80°C prior to purification.

[0212] RNA was extracted using the RNeasy Kit (Qiagen #74106) according to the manufacturer's instructions. RNA was treated with Turbo DNase (ThermoFisher #AM2238) and purified using the RNA cleanup protocol of the RNeasy Kit. mRNA sequencing libraries were prepared by Novogene.

[0213] Preprocessing of sequencing data

[0214] Reads from RNA sequencing were aligned to a custom reference genome using kallisto(0.48.0). This reference consisted of the human genome GRCh38 cDNA and mTagBFP2 coding sequence. Weakly expressed genes were excluded if they showed fewer than three samples expressing at least 10 transcript permillions (TPM), or if the maximum TPM across all samples was less than 105. The filtered counts were then entered into DEseq to eliminate the influence of size factors. Since off-target effects of miRNAs were evaluated using BFP-only cells as a reference, genes showing expression variations across three biological replicas of BFP-only cells should be excluded from the analysis. To achieve this, log(1+x) (where x represents the normalized TPM across three biological replicas of BFP-only cells) was calculated. The logarithmic Fano factors of these were determined and ranked. Transcripts ranked as the top 2.5% in the logarithmic Fano factors were excluded from further analysis. Finally, log(1+x) (where x represents the normalized TPM across all samples) was calculated. Differential functions were defined to calculate the absolute value of the difference in log(1+x) between each sample and the untransfected sample. The median differential functions for the BFP-only group and the experimental group were calculated and used for comparison. The difference between these two differential functions was ranked, and similarly, transcripts ranked in the top 3% were excluded from further analysis.

[0215] The Fano factor is defined as follows:

number

[0216] The equation for a differential function is defined as follows: Δ(BFP, untransfected) = |log(1+x BFP )-log(1+x トランスフェクトされていない )| Δ(experimental, untransfected) = |log(1+x) 実験 )-log(1+x トランスフェクトされていない )| Δ ランク付けされた = Median(Δ(BFP, untransfected)) - Median(Δ(experiment, untransfected))

[0217] Differential gene expression analysis To characterize the disruption of the endogenous transcriptome by synthetic miRNAs, differential expression analysis was performed using DESeq2(1.40.1) in R(4.3.1.), and transcript counts were compared between cells transfected with miRNAs and cells with BFP alone.

[0218] DNA-PAINT buffer solution

[0219] Blocking buffer: 1×PBS, 1mM EDTA (Thermo Fisher, No. AM9260G), 0.02% Tween-20 (Life Science, No. P7949), 2% BSA (Sigma-Aldrich, No. A9647-100G)

[0220] Wash buffer: 1x PBS, 1mM EDTA, 0.01% Tween-20

[0221] Imaging buffer: 1×PBS, 1mM EDTA, 500mM NaCl (Thermo Fisher, No. AM9760G)

[0222] cloning

[0223] The mEGFP Gblock (obtained from Integrated DNA Technologies) was inserted into the pcDNA3.1(+) backbone (ThermoFisher, no. V79020) using Gibson assembly. Two codon-optimized fragments of human EGFR (obtained from Integrated DNA Technologies) were fused to the mEGFP-pcDNA3.1(+) backbone using Gibson assembly.

[0224] cell culture

[0225] CHO-K1 cells (ATCC:CCL-61) were cultured at 37°C and 4% CO2 in Gibco's Ham F-12K (Kaighn) medium (Gibco, No. 11573397) supplemented with 10% FBS. The cells were subcultured every 2-3 days by trypsin treatment with trypsin-EDTA (Gibco, No. 25300096).

[0226] Nanobody-DNA conjugation

[0227] First, GFP nanobodies (clone 1H1, Nanotag Biotechnologies) were conjugated to DBCO-PEG4-maleimide linker (number CLK-A108P, Jena Bioscience). After removing unreacted linker using an Amicon centrifuge filter (10,000 MWCO), the DBCO-nanobodies were conjugated to the docking strand 5'-azide-CTC TCT CTC TCT CTC TCT C-3' (Metabion) via DBCO-azide click chemistry. A detailed description of the conjugation can be found in the previous protocol provided in Strauss, S. & Jungmann, R. (Up to 100-fold speed-up and multiplexing in optimized DNA-PAINT. Nat. Methods 17, 789-791 (2020)).

[0228] DNA-PAINT imaging

[0229] 5,000 cm⁻² CHO-K1 cells were seeded on an ibidi 8-well high-glass-bottom chamber (No. 80807) the day before transfection. Cells were transfected with EGFR-mEGFP plasmid using ThermoFisher's Lipofectamine 3,000 reagent (No. L3000008) with 250 ng of plasmid per well (200 μL of solution and 25 μL of transfection solution per well) at a lower lipofectamine concentration than indicated by the manufacturer. 48 hours after transfection, cells were fixed for 15 minutes in 250 μL of preheated methanol-free 4% PFA (ThermoFisher, No. 043368.9M) in 1× PBS. After washing cells three times with 1× PBS, they were permeabilized for 2 minutes with 0.125% Triton X-100 (Sigma Aldrich, No. 93443) in 1× PBS. After washing three times with 1×PBS, the cells were blocked overnight at 4°C with blocking buffer.

[0230] Next, the cells were washed three times with washing buffer. 25 nM anti-GFP nanobodies were incubated in blocking buffer at RT for 1 hour. After washing three times with washing buffer, the samples were incubated with imaging buffer for 5 minutes. Subsequently, the nanobodies were post-fixed with 4% PFA in 1×PBS for 5 minutes. Then, the cells were washed three times with washing buffer and once with 1×PBS. 90 nm gold nanoparticles (Absource, number G-90-100) in a 1:1 ratio in 1×PBS were incubated at RT for 5 minutes. After washing three times with 1×PBS, the cells were washed once with imaging buffer. The samples were imaged for 40k frames in imaging buffer with a 100 pM imager chain (5'-GAG AGA G-Cy3B 3', obtained from Metabion) at an exposure time of 100 ms per frame and a readout rate of 200 MHz.

[0231] Microscope rotation The samples were measured using an inverted total internal reflection fluorescence (TIRF) microscope (Nikon Instruments, Eclipse Ti2) equipped with an oil immersion objective lens (Nikon Instruments, Apo SR TIRF×100 / numerical aperture 1.49, oil) and a perfect focusing system. The mRuby3 signal was bleached with a 560nm laser (MPB Communications, 1W) using a thin-layer oblique illumination optical sheet (HILO). Subsequently, the TIRF mode was established. A Cy3B imager was excited with the 560nm laser. The laser beam was cleaned with a filter (Chroma Technology, no. ZET561 / 10) and coupled to a microscope equipped with a beam splitter (Chroma Technology, no. ZT561rdc). The fluorescence signal was filtered using emission filters (Chroma Technology, numbers ET600 / 50m and ET575lp) and projected onto an sCMOS camera (Hamamatsu, ORCA-Fusion BT) without further magnification.

[0232] The target acquisition area has a size of 576 × 576 pixels. The obtained effective pixel size is 130 nm. Raw microscope data was acquired via μManager.

[0233] DNA-PAINT analysis The obtained fluorescence data was reconstructed using Picasso software. The data was first corrected for drift using redundant cross-correlation, and then for drift correction using gold particles selected as a reference. To determine receptor density, a homogeneous region of cells was selected, and the DNA-PAINT data was clustered using Picasso's SMLMS clustering algorithm. Using the determined cluster centers, 1 μm 2 The receptor density per unit area (number of cluster centers per unit area) was calculated.

[0234] Supplemental Modeling 1. Modeling of miRNA-based gene dose compensation circuits In this invention, we developed a simplified mathematical model of a miRNA-based incoherent feedforward loop (IFFL) circuit and used it to explore how various parameters control the scaling of target gene expression based on gene dose. This model is based on several reactions:

[0235] 1. RISC Production and Removal. Assume that miRNAs are expressed, processed, and assemble with Argonaut (Ago) proteins to form active (mature) RNA-induced silencing complexes (RISCs). The concentration of mature RISCs (containing miRNAs) is denoted as r. RISCs are released at a total rate of Dβ. r (In the formula, D represents the gene copy number (gene amount), and β r It is assumed that production occurs at a rate of RISC production per gene copy (where γ represents the rate of RISC production per gene copy). This equation implicitly assumes that the miRNA expression level does not saturate the available miRNA processing mechanism, Ago, or other components. Furthermore, the RISC complex is produced at a total rate of γ r r (where γ r It is assumed that it is removed by (the composite rate constant for dilution, decomposition, and other removal processes).

[0236] 2. mRNA of the target gene, indicated by a concentration of m, is similarly produced and removed. mRNA is produced at a rate proportional to the gene copy number Dβ. m (In the formula, β m It is assumed that mRNA is produced at a rate of γ (where γ is the mRNA production rate per gene copy). mRNA is also produced at a rate of γ due to dilution and degradation. m It can be removed by m. Even though mRNA and miRNA are produced from the same manipulated locus, miRNA and mRNA are produced from different promoters, and the production of mRNA and the RISC-miRNA complex involves different biochemical processes, so the production rate constant β m and β r Note that these may differ.

[0237] 3. Formation and dissociation of the RISC-mRNA complex. RISC and target mRNA assemble according to the kinetics of mass action, with a rate constant k on Using velocity k on Assume that a complex is formed at rm, with a concentration of C. Once this complex is formed, the rate k off Dissociates at C, velocity k c The mRNA may undergo catalytic degradation at C. Figure 18 shows a non-restrictive, illustrative schematic diagram of the reaction in the model.

[0238] These chemical reactions can be summarized as follows, using G, which represents the gene present at copy number (gene quantity) D:

number

[0239] Using these definitions and assumptions, we can write a set of ordinary differential equations in terms of three variables r, m, and C.

number

[0240] These equations are similar to previous models of regulation by small RNAs, except for the obvious uptake of gene quantities in the regulation of both mRNA and miRNA.

[0241] To gain insight into the possible behavior of this system, we can first non-dimension it. By rescaling time in units of RISC lifetime, we obtain dimensionless time.

number

number

number

number

[0242] Furthermore, a convenient set of dimensionless parameter ratios,

number

[0243] In a dimensionless system, the differential equation becomes:

number

[0244] By definition, in a steady state, all time derivatives are equal to zero. If we denote the steady state with the subscript 's', then:

number

[0245] Solving the above equation gives us the equation relating to the steady-state mRNA concentration:

number

[0246] From this point forward, for the sake of notation, the tilde will be omitted, and the variables and parameters will be converted to dimensionless forms.

[0247] At the extreme limit of a large gene volume,

number

number

[0248] Figure 2A plots this equation, and Figures 7A and 7B show the results with different parameters, namely k. off and k c The equation is plotted at [the given point].

[0249] Finally, to estimate the biological parameter values, we assumed that miRNA and mRNA are produced at similar rates and selected values ​​based on [Nyayanit, D. & Gadgil, CJ Mathematical modeling of combinatorial regulation suggests that apparent positive regulation of targets by miRNA could be an artifact resulting from competition for mRNA. Rna 21, 307-319(2015)], as listed in Table 9 below. [Table 9]

[0250] 1.1 Incorporation of hypersensitivity Alternatively, a more general phenomenological model was considered, in which the inhibition of miRNA against the target was assumed to follow a Hill function, with the Hill coefficient being n. This procedure omits the intermediate steps, namely the formation and dissociation of the RISC-mRNA complex. Under this assumption, various sets of ordinary differential equations can be described in terms of two variables, r and m.

number

[0251]

number

number

number

number

[0252] Figure 7C shows

number

number

number

number

[0253] In at least some of the embodiments described herein, one or more elements used in one embodiment may be interchangeably used in another embodiment, provided that such substitution is not technically impractical. Those skilled in the art will understand that various other omissions, additions, and modifications can be made to the above-described methods and structures without departing from the subject matter of the claims. All such modifications and changes are intended to fall within the subject matter as defined by the accompanying claims.

[0254] With regard to the use of substantially any plural and / or singular terms herein, those skilled in the art can convert from plural to singular and / or singular to plural as appropriate for the context and / or use. Various singular / plural reorders may be expressly described herein for clarity. Where used herein and in the accompanying claims, the singular forms “a,” “an,” and “the” encompass the plural unless the context clearly indicates otherwise. Any reference to “or” herein is intended to encompass “and / or” unless otherwise noted.

[0255] In general, it will be understood by those skilled in the art that the terms used herein, particularly in concurrent claims (e.g., the body of concurrent claims), are generally intended to be “open” terms (for example, the term “includes” should be interpreted as “includes but not limited to,” the term “has” should be interpreted as “has at least,” and the term “includes” should be interpreted as “includes but not limited to,” etc.). Furthermore, where a specific number of introduced claims are intended, such intent will be explicitly enumerated in the claims, and in the absence of such enumeration, such intent will be understood by those skilled in the art. For example, for the sake of understanding, the following concurrent claims may include the use of the introductory phrases “at least one” and “one or more” to introduce the description of the claims. However, even when the same claim includes the introductory phrase "one or more" or "at least one" and the indefinite article "a" or "an" (for example, "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"), the use of such phrases should not be interpreted as implying that the introduction of a claim enumeration by the indefinite article "a" or "an" limits any particular claim containing such introduced claim enumeration to only the embodiment containing such enumeration, and the same applies to the use of the definite article used to introduce a claim enumeration. Furthermore, even when a specific number of introduced claim enumerations are explicitly enumerated, a person skilled in the art will recognize that such enumeration should be interpreted as meaning at least the number enumerated (for example, the literal enumeration "two enumerations" without other modifying phrases means at least two enumerations, or two or more enumerations). Furthermore, when expressions similar to "at least one of A, B, and C, etc." are used, such constructions are generally intended to be understood by those skilled in the art (for example, "a system having at least one of A, B, and C" includes, but is not limited to, systems having only A, only B, only C, a combination of A and B, a combination of A and C, a combination of B and C, and / or a combination of A, B, and C, etc.).In examples where expressions similar to "at least one of A, B, or C" are used, such configurations are generally intended to be understood by those skilled in the art (for example, "a system having at least one of A, B, or C" includes, but is not limited to, systems having only A, only B, only C, a combination of A and B, a combination of A and C, a combination of B and C, and / or a combination of A, B, and C, etc.). Furthermore, it will be understood by those skilled in the art that virtually any separate word and / or phrase presenting two or more alternative terms, whether in a description, claims, or drawings, should be understood to assume the possibility of including one of those terms, one of them, or both.

[0256] Furthermore, if any feature or aspect of this disclosure is described in relation to the Markush group, it will be recognized by those skilled in the art that this disclosure also describes any individual member or subgroup of members of the Markush group.

[0257] As will be understood by those skilled in the art, for any purpose relating to the provision of written information, for example, all scopes disclosed herein also encompass all conceivable subscopes and combinations thereof. Any enumerated scope is readily apparent as being sufficiently descriptive of its scope and allowing for its decomposition into at least two, three, four, five, ten, etc. As a non-limiting example, each scope considered herein can readily be decomposed into a lower third, a middle third, an upper third, etc. Also, as will be understood by those skilled in the art, all terms such as “maximum,” “at least,” “greater than,” and “less than” include the enumerated number, and subsequently refer to a scope that can be decomposed into subscopes as considered above. Finally, as will be understood by those skilled in the art, a scope includes each individual member. Thus, for example, a group having 1 to 3 articles refers to a group having 1, 2, or 3 articles. Similarly, a group having 1 to 5 articles refers to a group having 1, 2, 3, 4, or 5 articles, and so on.

[0258] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes only and are not intended to limit, and the true scope and spirit are indicated by the accompanying claims.

Claims

1. A first promoter sequence operably ligated to a first polynucleotide containing one or more miRNA cassettes, The first promoter sequence can induce the transcription of the first polynucleotide to produce the first transcript. A first promoter sequence that allows the first transcript to be processed to generate the miRNA; and A second promoter sequence operably ligated to a second polynucleotide containing the payload gene, The payload gene includes a miRNA target region containing one or more miRNA target sequences, The second promoter sequence can induce the transcription of the second polynucleotide to generate a payload transcript. A second promoter sequence that enables the payload transcript to be translated and generate the payload protein. A composition comprising a copolymer composition containing, The first promoter sequence and the second promoter sequence are components of a bidirectional promoter as appropriate. A composition wherein, as appropriate, the first promoter sequence and the second promoter sequence are in an inversely complementary orientation with respect to each other in the bidirectional promoter.

2. A first promoter sequence operably ligated to a first polynucleotide containing one or more miRNA cassettes, The first promoter sequence can induce the transcription of the first polynucleotide to produce the first transcript. A first promoter sequence that allows the first transcript to be processed to generate the miRNA; and A second promoter sequence operably ligated to a second polynucleotide containing the payload gene, The payload gene includes a miRNA target region containing one or more miRNA target sequences, The second promoter sequence can induce the transcription of the second polynucleotide to generate a payload transcript. A second promoter sequence that enables the payload transcript to be translated and generate the payload protein. A composition comprising one or more cells including, The first promoter sequence and the second promoter sequence are components of a bidirectional promoter as appropriate. A composition wherein, as appropriate, the first promoter sequence and the second promoter sequence are in an inversely complementary orientation with respect to each other in the bidirectional promoter.

3. The composition according to any one of claims 1 to 2, wherein the miRNA is capable of binding to one or more miRNA target sequences, thereby reducing the stability of the payload transcript and / or reducing the translation of the payload transcript.

4. The first polynucleotide contains a gene quantity gene, The first transcript can be translated to produce a gene quantity indicator protein. The intron is located in the 3'UTR of the gene quantity gene, in the 5'UTR of the gene quantity gene, or between the exons of the gene quantity gene, and is optionally a synthetic intron. The intron includes one or more miRNA cassettes, The intron includes one or more of the following: (i) an intron insertion encoding miRNA, (ii) a donor splice site, (iii) an acceptor splice site, (iv) a branching point domain, and (v) a polypyrimidine tract, and / or The composition according to any one of claims 1 to 3, wherein the miRNA or its precursor can be released from the intron by an intron excision mechanism selected from the group including intracellular RNA splicing and / or processing mechanisms, nonsense mutation-dependent degradation (NMD) processing, or any combination thereof, and the precursor optionally comprises prim-miRNA.

5. The composition according to any one of claims 1 to 4, wherein the gene quantity indicator protein is detectable, and optionally the gene quantity indicator protein comprises green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), TagRFP, Dronpa, Padron, mApple, mCherry, mruby3, rsCherry, rsCherryRev, derivatives thereof, or any combination thereof.

6. The composition according to any one of claims 1 to 5, wherein the one or more cells comprise two or more cells.

7. The two or more cells mentioned above include the first cell and the second cell, and as appropriate, The first cell is a first cell type, The second cell is a second cell type, The first and second cell types include antigen-presenting cells, dendritic cells, macrophages, nerve cells, brain cells, astrocytes, microglia cells, and neurons, spleen cells, lymphoid cells, lung cells, lung epithelial cells, skin cells, keratinocytes, endothelial cells, alveolar cells, alveolar macrophages, alveolar lung cells, vascular endothelial cells, mesenchymal cells, epithelial cells, colon epithelial cells, hematopoietic cells, bone marrow cells, Claudius cells, Hensen cells, Merkel cells, Müller cells, Paneth cells, Purkinje cells, Schwann cells, Sertoli cells, eosinophilic cells, acinar cells, lipoblasts, adipocytes, brown or white alpha cells, amacrine cells, beta cells, capsule cells, cementocytes, chief cells, chondrocytes, chondrocytes, chromaffin cells, pigment-averse cells, adrenocorticotropic hormone-secreting cells, delta cells, Langerhans cells, and follicular dendritic cells. Enterochromaffin cells, ependymal cells, epithelial cells, basal cells, squamous epithelial cells, endothelial cells, transitional epithelial cells, erythroblasts, erythrocytes, fibroblasts, fibrous cells, follicular cells, germ cells, gametes, eggs, sperm, oocytes, primary oocytes, secondary oocytes, spermatids, spermatocytes, primary spermatocytes, secondary spermatocytes, germ epithelium, giant cells, glial cells, astroblasts, astrocytes, oligodendrocytes, oligodendrocytes Glioblasts, goblet cells, gonadotropin-secreting cells, granulosa cells, hemocytoblasts, hair cells, hepatoblasts, hepatocytes, vitreous cells, stromal cells, juxtaglomerular cells, keratinocytes, corneal stromal cells, Lemmal cells, leukocytes, granulocytes, basophils, eosinophils, neutrophils, lymphoblasts, B-lymphoblasts, T-lymphoblasts, lymphocytes, B-lymphoblasts, T-lymphoblasts, helper-induced T-lymphoblasts, Th1 T-lymphoblasts, Th2T lymphocytes, natural killer cells, thymocytes, macrophages, Kupffer cells, alveolar macrophages, foam cells, histiocytes, luteal cells, lymphoid stem cells, lymphoid cells, lymphoid stem cells, macroglia, mammotropes, mast cells, medulloblasts, megakaryoblasts, megakaryocytes, melanoblasts, melanocytes, mesangial cells, mesothelial cells, metamyelocytes, monoblasts, monocytes, myxone neck cells, myoblasts, muscle cells, muscle cells, cardiomyocytes, skeletal muscle cells, smooth muscle cells, myelocytes, myeloid cells, myeloid stem cells, myoblasts, myoepithelial cells, myofibroblasts, neuroblasts, neuroepithelial cells, neurons, odontoblasts, The method according to claim 6, wherein the group is selected from the group including osteoblasts, osteoclasts, osteocytes, acid-secreting cells, parafollicular cells, paraluteal cells, digestive cells, pericytes, peripheral blood mononuclear cells, pheochromocytes, phalangeal cells, pineal cells, pituitary cells, plasma cells, platelets, podocytes, proerythroblasts, promonocytes, promyeloblasts, promyelocytes, pronortherythroblasts, reticulocytes, retinal pigment epithelial cells, retinoblasts, small cells, growth hormone-secreting cells, stem cells, supporting cells, teloglycytic cells, enzymatic cells, or any combination thereof, and optionally the stem cells further include embryonic stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem / progenitor cells (HSPCs), or any combination thereof.

8. The composition according to any one of claims 1 to 7, wherein the first polynucleotide is transcribed in the first cell at a rate at least 1.1 times faster than in the second cell.

9. The composition according to any one of claims 1 to 8, wherein the first polynucleotide is translated at a rate at least 1.1 times faster in the first cell compared to the second cell.

10. The composition according to any one of claims 1 to 9, wherein the first polynucleotide is present in the first cell in at least 1.1 times more copies than in the second cell.

11. The composition according to any one of claims 1 to 10, wherein the threshold gene amount at which payload protein expression is saturated is at least 1.1 times higher in the first cell compared to the second cell.

12. The composition according to any one of claims 1 to 11, wherein the rate of transcription of the second polynucleotide and / or the rate of translation of the payload transcript varies between a first time point and a second time point in a single cell, and / or varies between the first cell and the second cell at the same time point.

13. The composition according to any one of claims 1 to 12, wherein, in the absence of the miRNA, the payload protein reaches an unadjusted steady-state payload protein level in one or more cells.

14. The composition according to any one of claims 1 to 13, wherein the unadjusted steady-state payload protein level extends to a range between the lower unadjusted threshold and the upper unadjusted threshold of the unadjusted expression range.

15. The composition according to any one of claims 1 to 14, wherein the steady-state gene quantity indicator protein level reflects the unadjusted steady-state payload protein level.

16. The composition according to any one of claims 1 to 15, wherein, in the presence of the miRNA, the payload protein reaches a controlled steady-state payload protein level in one or more cells.

17. The composition according to any one of claims 1 to 16, wherein the adjusted steady-state payload protein level fluctuates between a lower adjusted threshold and an upper adjusted threshold of the adjusted expression range, and the upper adjusted threshold of the adjusted expression range is, as appropriate, the saturation expression level.

18. The composition according to any one of claims 1 to 17, wherein at the first and second time points in a single cell, the steady-state level of the payload protein remains within the adjusted expression range.

19. The composition according to any one of claims 1 to 18, wherein the steady-state level of the payload protein remains within the adjusted expression range at the same time in the first cell and the second cell.

20. The composition according to any one of claims 1 to 19, wherein the lower adjusted threshold and / or upper adjusted threshold of the adjusted expression range can be set by adjusting one or more of the following: (i) the number of miRNA cassettes in the first polynucleotide, (ii) the number of miRNA target sequences in the miRNA target region, (iii) the complementarity between the miRNA and the one or more miRNA target sequences, and (iv) the intensity of the first promoter sequence and / or the second promoter sequence.

21. The composition according to any one of claims 1 to 20, wherein the difference between the lower unadjusted threshold and the upper unadjusted threshold of the unadjusted expression range is greater than approximately two orders of magnitude.

22. The composition according to any one of claims 1 to 21, wherein the difference between the lower adjusted threshold and the upper adjusted threshold of the adjusted expression range is less than approximately one order of magnitude.

23. The composition according to any one of claims 1 to 22, wherein the payload protein is effective at steady-state payload protein levels within the adjusted expression range.

24. The composition according to any one of claims 1 to 23, wherein the payload protein is inactive and / or toxic at steady-state payload protein levels above and / or below the adjusted expression range.

25. The composition according to any one of claims 1 to 24, wherein the payload protein is capable of inducing an immunogenic response and / or a cytokine storm at steady-state payload protein levels outside the adjusted expression range.

26. The composition according to any one of claims 1 to 25, wherein the adjusted steady-state payload protein level includes a therapeutic level of the payload protein.

27. The composition according to any one of claims 1 to 26, wherein the steady-state payload protein level remains within the adjusted expression range across multiple cell types, viral vector titers, and / or viral vector capsid types.

28. The composition according to any one of claims 1 to 27, wherein the adjusted steady-state payload protein level is robust to tissue-targeting and stochastic expression.

29. The composition according to any one of claims 1 to 28, wherein the miRNA comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NOs. 75 to SEQ ID NOs.

87.

30. The composition according to any one of claims 1 to 29, wherein the miRNA cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NOs: 1 to SEQ ID NOs:

13.

31. The composition according to any one of claims 1 to 30, wherein the miRNA is approximately 20, 21, 22, 23, or 24 nucleotides (nt) long.

32. The composition according to any one of claims 1 to 31, wherein the sequence of the miRNA is orthogonal to one or more cells and is optionally a miRNA sequence that targets sea urchin luciferase.

33. The composition according to any one of claims 1 to 32, wherein the miRNA target region is located in the 3'UTR, 5'UTR, or coding region of the payload gene.

34. The composition according to any one of claims 1 to 33, wherein the miRNA target region comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NOs: 14 to 74.

35. The composition according to any one of claims 1 to 34, wherein the miRNA target region comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 miRNA target sequences.

36. The composition according to any one of claims 1 to 35, wherein the payload transcript can be simultaneously bound by a plurality of miRNA-loaded Argonaut (Ago) complexes, optionally via TNRC6.

37. The composition according to any one of claims 1 to 36, wherein 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 nucleotides (nts) of the miRNA target sequence are complementary to the miRNA, and optionally, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nts at the 3' end of the miRNA are not complementary.

38. The composition according to any one of claims 1 to 37, wherein the complementarity between the miRNA and the miRNA target sequence is at least, or at most, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

39. The composition according to any one of claims 1 to 38, wherein the miRNA optionally contains 5 to 8 GC nucleotides, with 1 to 4 in the seed region and 1 to 2 in the broad region.

40. The composition comprises one or more additional payload genes and one or more additional miRNAs, wherein the additional miRNAs have sequences that differ from each other, and the composition may optionally provide for each of the separate additional miRNAs. An additional first promoter sequence operably ligated to an additional first polynucleotide containing one or more additional miRNA cassettes, The additional first promoter sequence can induce the transcription of the additional first polynucleotide to generate an additional first transcript. An additional first promoter sequence, which allows the additional first transcript to be processed to generate the additional miRNA, and An additional second promoter sequence operably ligated to an additional second polynucleotide containing an additional payload gene, The additional payload gene comprises an additional miRNA target region containing one or more additional miRNA target sequences, The additional second promoter sequence can induce the transcription of the additional second polynucleotide to generate an additional payload transcript. An additional second promoter sequence, which allows the additional payload transcript to be translated to generate an additional payload protein. A composition according to any one of claims 1 to 39, comprising:

41. The first promoter sequence and / or the second promoter sequence comprises a ubiquitous promoter, which may optionally be a cytomegalovirus (CMV) initial promoter, a CMV promoter, a viral monkey virus 40 (SV40) (e.g., early or late), a Moloney's mouse leukemia virus (MoMLV) LTR promoter, a Roussarcoma virus (RSV) LTR, an RSV promoter, a herpes simplex virus (HSV) (thymidine kinase) promoter, or a vaccinia virus-derived H 5, P7.5 and P11 promoters, elongation factor 1-alpha (EF1a) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5 (HSPA5), heat shock protein 90kDa beta, member 1 (HSP90B1), heat shock protein 70kDa (HSP70), β-kinesin (β-KIN), human ROSA Selected from a group including 26 gene loci, ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, 3-phosphoglycerate kinase promoter, cytomegalovirus enhancer, human β-actin (HBA) promoter, chicken β-actin (CBA) promoter, CAG promoter, CBH promoter, or any combination thereof, The first promoter sequence and / or the second promoter sequence is an inducible promoter, and the inducible promoter is, as appropriate, a tetracycline-responsive promoter, a TRE promoter, a Tre3G promoter, an ecdysone-responsive promoter, a cumate-responsive promoter, a glucocorticoid-responsive promoter, and an estrogen-responsive promoter, a PPAR-γ promoter, or a RU-486-responsive promoter. The first promoter sequence and / or the second promoter sequence includes a tissue-specific promoter and / or a lineage-specific promoter, The tissue-specific promoter is the liver-specific thyroxine-binding globulin (TBG) promoter, insulin promoter, glucagon promoter, somatostatin promoter, pancreatic polypeptide (PPY) promoter, synapsin-1 (Syn) promoter, creatine kinase (MCK) promoter, mammalian desmin (DES) promoter, α-myosin heavy chain (α-MHC) promoter, or cardiac troponin T (cTnT) promoter. The tissue-specific promoter is a neuron-specific promoter, and optionally, the neuron-specific promoter is a synapsin-1 (Syn) promoter, a CaMKIIa promoter, a calcium / calmodulin-dependent protein kinase IIa promoter, a tubulin alpha I promoter, a neuron-specific enolase promoter, a platelet-derived growth factor beta chain promoter, a TRPV1 promoter, Na ｖ 1.7 promoter, Na ｖ 1.8 promoter, Na ｖ 1.9 Promoter, or Advillin promoter, The tissue-specific promoter is a muscle-specific promoter, and optionally the muscle-specific promoter includes a creatine kinase (MCK) promoter. The first promoter sequence and / or the second promoter sequence is a methyl CpG-binding protein 2 (MeCP2) promoter or a derivative thereof, wherein the MeCP2 promoter or derivative thereof optionally includes a MeCP2 promoter cleaved into about 229 base pairs or a MeCP2 promoter cleaved into about 406 base pairs, and / or The composition according to any one of claims 1 to 40, wherein one or more cells contain an endogenous version of the payload gene, and the promoter contains or is derived from the promoter of the endogenous version.

42. The payload protein Disease-related proteins, wherein the abnormal expression of said disease-related proteins correlates with the onset and / or progression of said disease. Proteins associated with expression-sensitive diseases or disorders, Methyl CpG-binding protein 2 (MeCP2), SMN, DRK1A, KAT6A, NIPBL, HDAC4, UBE3A, EHMT1, one or more genes encoded on chromosome 9q34.3, NPHP1, LIMK1, one or more genes encoded on chromosome 7q11.23, P53, TPI1, FGFR1 and related genes, RA1, SHANK3, CLN3, NF-1, TP53, PFK, CD40L, CYP19A1, PGRN, CHRNA7, PMP22, CD40LG, derivatives thereof, or any combination thereof. An imaging agent, wherein the imaging agent optionally comprises green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), TagRFP, Dronpa, Padron, maple, mCherry, mruby3, rsCherry, rsCherryRev, derivatives thereof, or any combination thereof, wherein the imaging agent optionally conjugates to an endogenous protein or an exogenous protein. A binding domain, an endogenous or exogenous protein bound to a binding domain configured for isolation or imaging, and / or As appropriate, SpCas9 or its derivatives; VRER, VQR, EQR SpCas9; xCas9-3.7; eSpCas9; Cas9-HF1; HypaCas9; evoCas9; HiFi Cas9; ScCas9; StCas9; NmCas9; SaCas9; CjCas9; CasX; Cas9 A programmable nuclease selected from the group including H940A nickase; Cas12 and its derivatives; dCas9-APOBEC1 fusion, BE3, and dCas9-deaminase fusion; dCas9-Krab, dCas9-VP64, dCas9-Tet1, and dCas9-transcription regulator fusion; DCas9-fluorescent protein fusion; Cas13-fluorescent protein fusion; RCas9-fluorescent protein fusion; and Cas13-adenosine deaminase fusion. A composition according to any one of claims 1 to 41, comprising:

43. The composition according to any one of claims 1 to 42, wherein the programmable nuclease comprises a zinc finger nuclease (ZFN) and / or a transcription activator-like effector nuclease (TALEN).

44. The programmable nucleases include Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), zinc finger nuclease, TAL effector nuclease, meganuclease, MegaTAL, and Tev-m. TALEN, MegaTev, Homing Endonuclease, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, C A composition according to any one of claims 1 to 43, comprising sx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, C2c1, C2c3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, derivatives thereof, prime edited versions thereof, or any combination thereof.

45. The composition according to any one of claims 1 to 44, further comprising (i) a targeting molecule and / or (ii) a polynucleotide encoding a donor nucleic acid.

46. The composition according to any one of claims 1 to 45, wherein the targeting molecule is capable of binding to a programmable nuclease, the targeting molecule optionally comprises single-stranded DNA or single-stranded RNA, and the targeting molecule optionally comprises single guide RNA (sgRNA).

47. The composition according to any one of claims 1 to 46, wherein the payload protein is capable of regulating the expression, concentration, localization, stability, and / or activity of one or more endogenous proteins in a cell.

48. The composition according to any one of claims 1 to 47, wherein the payload protein is a therapeutic protein or a variant thereof, optionally a therapeutic protein configured to prevent or treat a disease or disorder of interest, and optionally the subject is deficient in the therapeutic protein.

49. The payload protein includes, and / or comprises fluorescent activity, polymerase activity, protease activity, phosphatase activity, kinase activity, SUMO activity, deSUMO activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, or any combination thereof. The composition according to any one of claims 1 to 48, wherein the payload protein comprises nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimerization activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosylase activity, acetyltransferase activity, deacetylase activity, adenylation activity, deadenylation activity, or any combination thereof.

50. The payload protein CRE recombinase, GCaMP, cell therapy components, knockdown gene therapy components, epitopes exposed on the cell surface, or any combination thereof. Bispecific T cell engagers (BiTEs), As appropriate, interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL- 12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, Interleukin- 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL- 14. Cytokines selected from the group consisting of IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, granulocyte-macrophage colony-stimulating factor (GM-CSF), M-CSF, SCF, TSLP, oncostatin M, leukemia suppressor (LIF), CNTF, cardiotropin-1, NNT-1 / BSF-3, growth hormone, prolactin, erythropoietin, thrombopoietin, leptin, G-CSF, or their receptors or ligands. A member of the TGF-beta / BMP family selected from the group consisting of TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-3a, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-9, BMP-10, BMP-11, BMP-15, BMP-16, endometrial hemorrhage-related factor (EBAF), growth and differentiation factor-1 (GDF-1), GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-12, GDF-14, Müllerian duct inhibitor (MIS), activin-1, activin-2, activin-3, activin-4, and activin-5. A cytokine member of the TNF family selected from the group consisting of TNF-alpha, TNF-beta, LT-beta, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, and 4-1 BBL. A cytokine member of the immunoglobulin superfamily selected from the group consisting of B7.1 (CD80) and B7.2 (B70), Interferon selected as appropriate from interferon alpha, interferon beta, or interferon gamma. As appropriate, a chemokine selected from CCL1, CCL2, CCL3, CCR4, CCL5, CCL7, CCL8 / MCP-2, CCL11, CCL13 / MCP-4, HCC-1 / CCL14, CTAC / CCL17, CCL19, CCL22, CCL23, CCL24, CCL26, CCL27, VEGF, PDGF, Lymphotactin (XCL1), Eotaxin, FGF, EGF, IP-10, TRAIL, GCP-2 / CXCL6, NAP-2 / CXCL7, CXCL8, CXCL10, ITAC / CXCL11, CXCL12, CXCL13, or CXCL15, Interleukins selected as appropriate from IL-10, IL-12, IL-1, IL-6, IL-7, IL-15, IL-2, IL-18, or IL-21, Tumor necrosis factor (TNF) selected as appropriate from TNF-alpha, TNF-beta, TNF-gamma, CD252, CD154, CD178, CD70, CD153, or 4-1BBL, and / or Factors that locally downregulate the activity of endogenous immune cells A composition according to any one of claims 1 to 49, comprising:

51. The composition according to any one of claims 1 to 50, wherein the payload protein is capable of remodeling the tumor microenvironment at a target site and / or reducing immunosuppression.

52. The composition according to any one of claims 1 to 51, wherein the payload protein comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

53. The payload protein is an activity regulator, and this activity regulator can appropriately reduce T cell activity. The activity regulator comprises a ubiquitin ligase involved in TCR / CAR signaling, selected from the group including c-CBL, CBL-B, ITCH, RF125, RF128, WWP2, or any combination thereof. The activity regulator comprises a negative regulatory enzyme selected from the group including SHP1, SHP2, SHTP1, SHTP2, CD45, CSK, CD148, PTPN22, DGK alpha, DGK zeta, DRAK2, HPK1, HPK1, STS1, STS2, SLAT, or any combination thereof. The regulatory factor is a negative regulatory scaffold / adapter protein selected from the group including PAG, LIME, NTAL, LAX31, SIT, GAB2, GRAP, ALX, SLAP, SLAP2, DOK1, DOK2, or any combination thereof. The activity regulator is a dominant-negative version of an activated TCR signaling component selected from the group including ZAP70, LCK, FYN, NCK, VAV1, SLP76, ITK, ADAP, GADS, PLC Gamma 1, LAT, p85, SOS, GRB2, NFAT, p50, p65, API, RAP1, CRKII, C3G, WAVE2, ARP2 / 3, ABL, ADAP, RIAM, SKAP55, or any combination thereof. The regulatory factor comprises the cytoplasmic tail of a negative co-regulating receptor selected from the group including CD5, PD1, CTLA4, BTLA, LAG3, B7-H1, B7-1, CD160, TFM3, 2B4, TIGIT, or any combination thereof. The regulatory factor is targeted to the cell membrane by a targeting sequence derived from LAT, PAG, LCK, FYN, LAX, CD2, CD3, CD4, CD5, CD7, CD8a, PD1, SRC, LYN, or any combination thereof, and / or The composition according to any one of claims 1 to 52, wherein the activity regulator reduces or inhibits the function of a pathway and / or function selected from the group including Ras signaling, PKC signaling, calcium-dependent signaling, NF-kappa B signaling, NFAT signaling, cytokine secretion, T cell survival, T cell proliferation, CTL activity, degranulation, tumor cell death, differentiation, or any combination thereof.

54. The composition according to any one of claims 1 to 53, wherein the payload protein is a cell reprogramming factor capable of converting at least partially differentiated cells into less differentiated cells, and is optionally Oct-3, Oct-4, Sox2, c-Myc, Klf4, Nanog, Lin28, ASCL1, MYT1L, TBX3b, SV40 Large T, hTERT, miR-291, miR-294, miR-295, or any combination thereof.

55. The composition according to any one of claims 1 to 54, wherein the payload protein is a cell reprogramming factor capable of differentiating a given cell into a desired differentiated state, and is optionally nerve growth factor (NGF), fibroblast growth factor (FGF), interleukin-6 (IL-6), bone morphogenetic protein (BMP), neurogenin 3 (Ngn3), pancreatic and duodenal homeobox 1 (Pdx1), Mafa, or any combination thereof.

56. The payload protein A checkpoint inhibitor or checkpoint stimulant molecule-specific agonist or antagonist antibody, or its antigen-binding fragment, optionally PD1, PD-L1, PD-L2, CD27, CD28, CD40, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, PD-1, and / or TIM-3. Constitutive signal peptides for proteolysis, PEST as appropriate. Nuclear localization signals (NLS) or nuclear export signals (NES), Depending on the circumstances, a transcription factor, protease, kinase, phosphatase, synthetic Notch (SynNotch) receptor, modular extracellular sensor architecture (MESA) receptor, Tango, or synthetic protein circuit components including dCas9-synR, A cleavage site of a protease capable of cleaving degron and degron, wherein the exposed degron of the payload protein destabilizes the payload protein, and the degron may include, as appropriate, N-degron, dihydrofolate reductase (DHFR) degron, FKB protein (FKBP) degron, derivatives thereof, or any combination thereof, and / or A protease or split protease wherein the activation level of the protease is, optionally, associated with one or more input signals, and further optionally, the protease includes tobacco etch virus (TEV) protease, tobacco vein spot virus (TVMV) protease, hepatitis C virus protease (HCVP), derivatives thereof, or any combination thereof. A composition according to any one of claims 1 to 55, comprising:

57. The composition according to any one of claims 1 to 56, wherein the payload protein is associated with a target agricultural trait selected from the group consisting of increased yield, increased abiotic stress tolerance, increased drought tolerance, increased flood tolerance, increased heat tolerance, increased cold and frost tolerance, increased salt tolerance, increased heavy metal tolerance, increased low nitrogen tolerance, increased disease resistance, increased pest resistance, increased herbicide resistance, increased biomass production, male sterility, or any combination thereof.

58. The composition according to any one of claims 1 to 57, wherein the payload protein is associated with a biological manufacturing process selected from the group including fermentation, distillation, biofuel production, compound production, polypeptide production, or any combination thereof.

59. The composition according to any one of claims 1 to 58, wherein the payload protein comprises a synthetic receptor, optionally a synthetic Notch (SynNotch) receptor, a modular extracellular sensor architecture (MESA) receptor, Tango, dCas9-synR, or any combination thereof.

60. The composition according to any one of claims 1 to 59, wherein the payload protein comprises components of an RNA transport system, a lipid envelope nanoparticle (LN) production system, or a virus-like particle (VLP) production system, and optionally an RNA transporter protein or fusogen.

61. The composition according to any one of claims 1 to 60, wherein the nucleic acid composition comprises about 1.0 kb, 1.5 kb, 2.0 kb, 2.5 kb, 3.0 kb, 3.5 kb, 4.0 kb, 4.5 kb, 5.0 kb, 5.5 kb, 6.0 kb, 6.5 kb, 7.0 kb, 7.5 kb, 8.0 kb, 8.5 kb, 9.0 kb, 9.5 kb, 10 kb, 12 kb, 15 kb, 20 kb, 25 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, or less than 100 kb.

62. The composition according to any one of claims 1 to 61, wherein the expression of the miRNA disrupts the endogenous gene expression of one or more cells by less than about 10%.

63. The composition according to any one of claims 1 to 62, further comprising TNRC6, GW182, one or more miRNA biogenesis components, Exportin-5, Dicer, and / or one or more nucleic acids encoding one or more Argonaut proteins.

64. The composition according to any one of claims 1 to 63, wherein the first promoter sequence and the first polynucleotide are not operably linked in the absence of a recombination event, and the first promoter sequence and the first polynucleotide are operably linked after a recombination event so that the first promoter sequence can induce transcription of the first polynucleotide to produce a first transcript.

65. The composition according to any one of claims 1 to 64, wherein the first polynucleotide and the second polynucleotide are incorporated into the genome of one or more cells.

66. A composition according to any one of claims 1 to 65, which is a vector, a ribonucleoprotein (RNP) complex, a liposome, nanoparticles, an exosome, a microvesicle, or any combination thereof.

67. The composition according to any one of claims 1 to 66, wherein the nucleic acid composition is complexed with or bound to one or more lipids or lipid-based carriers, thereby forming liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes, which optionally encapsulate the nucleic acid composition.

68. It is one or more vectors, or contains one or more vectors, or further contains one or more vectors, Depending on the circumstances, at least one of the one or more vectors may be a viral vector, plasmid, translocation element, naked DNA vector, lipid nanoparticle (LNP), or any combination thereof. The viral vector may be, as appropriate, an AAV vector, lentivirus vector, retrovirus vector, adenovirus vector, herpesvirus vector, herpes simplex virus vector, cytomegalovirus vector, vaccinia virus vector, MVA vector, baculovirus vector, varicella stomatitis virus vector, human papillomavirus vector, avipox virus vector, Sindbisvirus vector, VEE vector, measles virus vector, influenza virus vector, hepatitis B virus vector, integration-deficient lentivirus (IDLV) vector, or any combination thereof. The composition according to any one of claims 1 to 67, wherein the dislocation element is, as appropriate, a piggyback transposon or a sleeping beauty transposon.

69. The first polynucleotide and the second polynucleotide are included in the one or more vectors, The first polynucleotide and the second polynucleotide may be included in the same vector and / or different vectors as appropriate. The composition according to any one of claims 1 to 68, wherein the first polynucleotide and the second polynucleotide are appropriately positioned on the same nucleic acid and / or different nucleic acids.

70. The composition according to any one of claims 1 to 69, wherein the viral vector is an AAV vector, a lentiviral vector, a retroviral vector, or an integration-deficient lentivirus (IDLV) vector.

71. The AAV vector is Single-stranded AAV (ssAAV) vector or self-complementary AAV (scAAV) vector, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, their derivatives, or any combination thereof, and / or AAV9 variants modified for systemic administration, including AAV-PHP.B, AAV-PHP.eB, or AAV-PHP.S, as appropriate. A composition according to any one of claims 1 to 70, comprising:

72. The composition according to any one of claims 1 to 71, wherein the vector is a neurotropic viral vector, and optionally the neurotropic viral vector includes or is derived from the Herpesviridae family, varicella-zoster virus, pseudorabies virus, cytomegalovirus, Epstein-Barr virus, encephalitis virus, poliovirus, coxsackievirus, echovirus, mumps virus, measles virus, rabies virus, or any combination thereof.

73. A method for treating a disease or disorder in a subject, To administer to the subject an effective amount of one or more cells described in any one of claims 1 to 72, thereby treating or preventing the disease or disorder in the subject. A method that includes this.

74. A method for treating a disease or disorder in a subject, Introducing the nucleic acid composition according to any one of claims 1 to 72 into one or more cells, The subject is administered an effective amount of the obtained cells to thereby treat or prevent the disease or disorder in the subject. A method that includes this.

75. The method according to claim 74, comprising isolating one or more cells from the subject before the introduction step.

76. A method for treating a disease or disorder in a subject, A method comprising administering to a subject an effective amount of the nucleic acid composition according to any one of claims 1 to 72, thereby treating or preventing a disease or disorder in the subject.

77. A method relating to the controlled gene amount-independent expression of a payload protein in one or more cells, Introducing the nucleic acid composition according to any one of claims 1 to 72 into one or more cells. A method that includes this.

78. The method according to any one of claims 74 to 77, wherein the introduction step is carried out in vivo, in vitro, and / or ex vivo.

79. The method according to any one of claims 74 to 78, wherein the introduction step includes calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, electrical nuclear transport, chemical transfection, electrotransfection, lipofectamine-mediated transfection, effectene-mediated transfection, lipid nanoparticle (LNP)-mediated transfection, or any combination thereof.

80. The method according to any one of claims 73 to 79, wherein the one or more cells include one or more cells of a subject, and optionally the subject is suffering from a disease or disorder.

81. The method according to any one of claims 73 to 80, wherein the one or more cells include neurons, and the neurons are optionally associated with a neurological disease or disorder.

82. The method according to any one of claims 73 to 81, wherein the administration includes systemic administration, and the systemic administration is, as appropriate, intravenous, intramuscular, intraperitoneal, or intraarticular.

83. The method according to any one of claims 73 to 82, wherein the administration includes intrathecal administration, intracranial injection, aerosol delivery, nasal delivery, transvaginal delivery, rectal delivery, oral delivery, intraocular delivery, local delivery, local delivery, intracisional delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous injection, subcutaneous injection, intralymph node injection, intratumor injection, intraperitoneal injection, intradermal injection, or any combination thereof.

84. The method according to any one of claims 73 to 83, wherein administration comprises injection into a brain region, and, as may be, administration comprises direct administration into the brain parenchyma.

85. The aforementioned brain regions include the lateral parabrachial nucleus, brainstem, medulla oblongata, medullary pyramidal body, olivary body, inferior olivary nucleus, rostral ventrolateral part of the medulla oblongata, respiratory center, dorsal respiratory group, ventral respiratory group, Pre-Bettzinger complex, Betzinger complex, paramedian reticular nucleus, cuneate nucleus, gracile nucleus, internuclei, area postoderm, medullary cranial nerve nuclei, inferior salivary nucleus, nucleus ambiguus, dorsal vagus nerve nucleus, hypoglossal nerve nucleus, nucleus tractus solitarius, pons, pontine nuclei, pontine nerve nuclei, main nucleus or pontine nucleus of the trigeminal sensory nucleus (V), motor nucleus of the trigeminal nerve nucleus (V), abducens nerve nucleus (VI), facial nerve nucleus (VII), vestibulocochlear nerve nucleus (vestibular and cochlear nerve nuclei) (VIII), superior salivary nucleus, pedunculopontine tegmental nucleus, respiratory center, respiratory control center, sustained respiratory center, pontine micturition center (Barlington nucleus), locus coeruleus, pontine pedunculi nucleus, dorsolateral tegmental nucleus, and pontine tegmental reticular formation. Nuclei, superior olivary complex, paramedian pontine reticular formation, cerebellar peduncle, superior cerebellar peduncle, middle cerebellar peduncle, inferior cerebellar peduncle, cerebellum, cerebellar vermis, cerebellar hemispheres, anterior lobe, posterior lobe, flocculonodular lobe, cerebellar nuclei, fastigial nucleus, insertion nucleus, globose nucleus, embolus nucleus, dentate nucleus, tectum, quadrige, inferior colliculus, superior colliculus, pretectal area, tegmentum, periaqueductal gray matter, paracerebellar peduncle area, medial parabrachial nucleus, connective parabrachial nucleus (Koellliker-Fuse) Nuclei), medial longitudinal fasciculus rostral interstitial nucleus, midbrain reticular formation, dorsal raphe nucleus, red nucleus, ventral tegmental area, substantia nigra, parenchymal compacta, parenchymal reticular formation, interpeduncle nuclei, cerebral peduncle, cerebral peduncle branches, midbrain nerve nuclei, oculomotor nerve nucleus (III), trochlear nerve nucleus (IV), mesencephalic duct (cerebral aqueduct, Sylvian aqueduct), pineal gland, reticular nucleus, stria medullaris, thalamic cord, subcomisticular organ, thalamus, anterior thalamic nuclei, anterior ventral nucleus (also known as ventral anteria) nucleus), anterior dorsal nucleus, anteromedial nucleus, medial nuclei group, dorsomedial nucleus, median nuclei group, parastring nucleus of the thalamus, nucleus Renience, rhomboid nucleus, intralaminar nuclei group, median central nucleus, parafascicular nucleus, paracentral nucleus, lateral central nucleus, medial central nucleus, lateral nuclei group, dorsolateral nucleus, posterolateral nucleus, pulvinar, ventral nuclei group, anterior ventral nucleus, lateral ventral nucleus, posterior ventral nucleus, posteroventromedial nucleus, posteroventromedial nucleus, posterior thalamus, medial geniculate body, lateral geniculate body, reticular nucleus, hypothalamus, limbic system, HPA axis, preoptic tract area, medial preoptic nucleus, suprachiasmatic nucleus, paraventricular nucleus, supraoptic nucleus, prehypothalamic nucleus, lateral preoptic nucleus, median preoptic nucleus, periventricular preoptic nucleus, eminence, dorsomedial hypothalamic nucleus, ventromedial nucleus, arcuate nucleus, lateral field, lateral nucleus eminence, lateral eminence nucleus , mammillary nucleus, posterior nucleus, lateral field, optic chiasm, subfornical organ, periventricular nucleus, pituitary stalk, gray protuberance, nucleus protuberans, tuberomammillary nucleus, protuberant area, mammillary body, mammillary nucleus, subthalamic area, subthalamic nucleus, zona incerta, pituitary gland, neurohypophysis, middle lobe, adenohypophysis,Cerebral hemispheres, corona radiata, internal capsule, external capsule, outermost capsule, arcuate fasciculus, uncinate fasciculus, perforating fibers, hippocampus, dentate gyrus, Ammon's horn, Ammon's horn area 1, Ammon's horn area 2, Ammon's horn area 3, Ammon's horn area 4, amygdala, central nucleus, medial nucleus (accessory olfactory system), cortical and basomedial nuclei, lateral and basolateral nuclei, dilated amygdala, stria terminalis, bed nucleus of stria terminalis, claustrum, basal ganglia, striatum, dorsal striatum (also known as neostriatum), putamen, caudate nucleus, ventral striatum, striatum, nucleus accumbens, olfactory tubercle, globus pallidus, subthalamic nucleus, basal forebrain, anterior foramina, innominate, nucleus basalis, Broca's diagonal zone, septal nucleus, medial septal nucleus, end plate, end plate vascular organ, olfactory bulb, piriform cortex, anterior olfactory nucleus, olfactory tract, anterior commissure, uncinate, cerebral cortex, frontal lobe, frontal cortex, primary motor cortex, supplementary motor cortex, anterior Motor cortex, prefrontal cortex, frontopolar cortex, orbitofrontal cortex, dorsolateral prefrontal cortex, dorsomedial prefrontal cortex, ventrolateral prefrontal cortex, superior frontal gyrus, middle frontal gyrus, inferior frontal gyrus, Brodmann's area (4, 6, 8, 9, 10, 11, 12, 24, 25, 32 , 33, 44, 45, 46, and / or 47), parietal lobe, parietal cortex, primary somatosensory cortex (S1), secondary somatosensory cortex (S2), posterior parietal cortex, postcentral gyrus, precuneus, Brodmann area (1, 2, 3 (primary somatosensory cortex), 5, 7, 23, 26, 29, 31, 39, and / or 40), occipital lobe, primary visual cortex (V1), V2, V3, V4, V5 / MT, extococcal gyrus, cuneiform, Brodmann area (17 (V1, primary visual cortex), 18, and / or 40), parietal lobe, parietal cortex, primary somatosensory cortex, secondary somatosensory cortex (S2), posterior parietal cortex, postcentral gyrus, precuneus, Brodmann area (17 (V1, primary visual cortex), 18, and / or 47), parietal lobe, parietal cortex, primary somatosensory cortex (S1), precuneus, Brodmann area (1, 2, 3 (primary somatosensory cortex), 5, 7, 23, 26, 29, 31, 39, and / or 40), occipital lobe, primary visual cortex (V1), (19) Temporal lobe, primary auditory cortex (A1), secondary auditory cortex (A2), inferior temporal cortex, posterior inferior temporal cortex, superior temporal gyrus, middle temporal gyrus, inferior temporal gyrus, entorhinal cortex, perirhinal cortex, parahippocampal gyrus, fusiform gyrus, Brodmann's area (9, 20, 21, 22, 27, 34, 35, 36, 37, 38, 41, and / or 42), medial superior temporal area (MST), insular cortex, cingulate cortex, anterior cingulate cortex, posterior cingulate cortex, dorsal cingulate cortex, posterior corpus callosum cortex, gray zone, subgenuclear area (Subgeneral area) 25, Brodmann area (23, 24, 26, 29, 30 (posterior splenic area of ​​the corpus callosum), 31, and / or 32), cranial nerves (olfactory nerve (I), optic nerve (II), oculomotor nerve (III), trochlear nerve (IV), trigeminal nerve (V), abducens nerve (VI), facial nerve (VII), vestibular-cochlear nerve (VIII), glossopharyngeal nerve (IX), vagus nerve (X), accessory nerve (XI), hypoglossal nerve (XII)), or any combination thereof, wherein the brain region includes, as appropriate, the neural pathways superior longitudinal fasciculus, arcuate fasciculus, thalamocortical radiation,The method according to claim 84, comprising the cerebral peduncle, corpus callosum, posterior commissure, pyramidal tract or corticospinal tract, medial longitudinal fasciculus, dopamine system, mesocortical tract, mesolimbic tract, nigrostriatal tract, tuberculous infundibular tract, serotonin system, norepinephrine tract, posterior column-medial lemniscus tract, spinothalamic tract, lateral spinothalamic tract, anterior spinothalamic tract, or any combination thereof.

86. The method according to any one of claims 73 to 85, further comprising introducing an inductive factor of the inductive promoter into one or more cells, wherein the inductive factor optionally comprises doxycycline, and further optionally the introduction step comprises administering an initial dose of the inductive factor, followed by one or more lower maintenance doses of the inductive factor.

87. The one or more cells mentioned above include eukaryotic cells, and the eukaryotic cells may include antigen-presenting cells, dendritic cells, macrophages, nerve cells, brain cells, astrocytes, microglia cells, and neurons, spleen cells, lymphoid cells, lung cells, lung epithelial cells, skin cells, keratinocytes, endothelial cells, alveolar cells, alveolar macrophages, alveolar lung cells, vascular endothelial cells, mesenchymal cells, epithelial cells, colon epithelial cells, hematopoietic cells, bone marrow cells, Claudius cells, Hensen cells, Merkel cells, Müller cells, Paneth cells, Purkinje cells, Schwann cells, Sertoli cells, eosinophilic cells, acinar cells, lipoblasts, adipocytes, brown or white alpha cells, amacrine cells, beta cells, capsule cells, cementocytes, chief cells, chondrocytes, chondrocytes, chromaffin cells, pigment-averse cells, adrenocorticotropic hormone-secreting cells, delta cells, Langerhan cells Cells, follicular dendritic cells, enterochromaffin cells, ependymal cells, epithelial cells, basal cells, squamous epithelial cells, endothelial cells, transitional epithelial cells, erythroblasts, red blood cells, fibroblasts, fibrous cells, follicular cells, germ cells, gametes, eggs, sperm, oocytes, primary oocytes, secondary oocytes, spermatids, spermatocytes, primary spermatocytes, secondary spermatocytes, germ epithelium, giant cells, glial cells, astroblasts, astrocytes, oligodendrocytes, Oligodendrocytes, glioblasts, goblet cells, gonadotropin-secreting cells, granulosa cells, hematoblasts, hair cells, hepatoblasts, hepatocytes, vitreous cells, stromal cells, juxtaglomerular cells, keratinocytes, corneal stromal cells, Lemmar cells, leukocytes, granulocytes, basophils, eosinophils, neutrophils, lymphoblasts, B-lymphoblasts, T-lymphoblasts, lymphocytes, B-lymphoblasts, T-lymphoblasts, helper-induced T-lymphoblasts, Th1 T-lymphoblasts, Th2T lymphocytes, natural killer cells, thymocytes, macrophages, Kupffer cells, alveolar macrophages, foam cells, histiocytes, luteal cells, lymphoid stem cells, lymphoid cells, lymphoid stem cells, macroglia, mamotoroves, mast cells, medulloblasts, megakaryoblasts, megakaryocytes, melanoblasts, melanocytes, mesangial cells, mesothelial cells, metamyelocytes, monoblasts, monocytes, myxocervus cells, myoblasts, muscle cells, muscle cells, cardiomyocytes, skeletal muscle cells, smooth muscle cells, myelocytes, myeloid cells, myeloid stem cells, myoblasts, myoepithelial cells, myofibroblasts, neuroblasts, neuroepithelial cells, neurons, odontoblasts, osteoblasts, osteoclasts The method according to any one of claims 73 to 86, further comprising cells, osteocytes, acid-secreting cells, parafollicular cells, paracorpus luteum cells, digestive cells, pericytes, peripheral blood mononuclear cells, pheochromocytes, phalangeal cells, pineal cells, pituitary cells, plasma cells, platelets, podocytes, proerythroblasts, promonocytes, promyeloblasts, promyelocytes, pronorthoblasts, reticulocytes, retinal pigment epithelial cells, retinoblasts, small cells, growth hormone-secreting cells, stem cells, supporting cells, teloglycytic cells, enzymatic cells, or any combination thereof, further optionally comprising embryonic stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem / progenitor cells (HSPCs), or any combination thereof.

88. The method according to any one of claims 73 to 87, wherein the disease or disorder comprises a MECP2-related disorder selected from the group comprising typical Rett syndrome, MECP2-associated severe neonatal encephalopathy, PPM-X syndrome, or any combination thereof.

89. The method according to any one of claims 73 to 88, wherein the disease or disorder is a blood disorder, an immune disorder, cancer, an infection, a genetic disorder, a disorder caused by abnormal mtDNA, a metabolic disorder, a disorder caused by an abnormal cell cycle, a disorder caused by abnormal angiogenesis, a disorder caused by abnormal DNA damage repair, or any combination thereof.

90. The aforementioned disease or disorder includes neurological diseases or disorders, and as appropriate, said neurological disease or disorder is Alzheimer's disease, Creutzfeldt-Jakob syndrome / disease, bovine spongiform encephalopathy (BSE), prion-related infections, diseases with mitochondrial dysfunction, diseases with β-amyloid and / or tauopathy, Down syndrome, hepatic encephalopathy, Huntington's disease, motor neuron disease, amyotrophic lateral sclerosis (ALS), olivopontocerebellar atrophy, postoperative cognitive impairment (POCD), systemic lupus erythematosus, systemic sclerosisClerosis, Sjögren's syndrome, neurogenic ceroid lipofuscinosis, neurodegenerative cerebellar ataxia, Parkinson's disease, Parkinson's dementia, mild cognitive impairment, cognitive deficits in various forms of mild cognitive impairment, cognitive deficits in various forms of dementia, Boxer dementia, vascular and frontal lobe dementia, cognitive impairment, learning disability, eye injury, eye disease, eye disorder, glaucoma, retinopathy, macular degeneration, head or brain or spinal cord injury, head or brain or spinal cord trauma, convulsions, epileptic seizures, epilepsy, Temporal lobe epilepsy, myoclonus epilepsy, tinnitus, dyskinesia, chorea, Huntington's chorea, athetosis, dystonia, stereotypic syndrome, ballism, tardive dyskinesia, tic disorder, spasmodic torticollis, blepharospasm, focal and generalized dystonia, nystagmus, hereditary cerebellar ataxia, corticobasal degeneration, tremor, essential tremor, addiction, anxiety disorder, panic disorder, social anxiety disorder (SAD), attention deficit hyperactivity disorder (ADHD), attention deficit syndrome (ADS), restless legs syndrome ( RLS), hyperactivity in children, autism, dementia, Alzheimer's disease dementia, Korsakoff syndrome dementia, Korsakoff syndrome, vascular dementia, dementia associated with HIV infection, HIV-1 encephalopathy, AIDS encephalopathy, AIDS-dementia complex, AIDS-associated dementia, major depressive disorder, major depression, depression, memory loss, stress, bipolar disorder, drug tolerance, opioid tolerance, motor disorders, fragile X syndrome, irritable bowel syndrome (IBS), migraine, multiple sclerosis (MS) The method according to any one of claims 73 to 89, comprising, ), muscle spasms, pain, chronic pain, acute pain, inflammatory pain, neuropathic pain, post-traumatic stress disorder (PTSD), schizophrenia, spasticity, Tourette syndrome, eating disorders, food addiction, bulimia, agoraphobia, generalized anxiety disorder, obsessive-compulsive disorder, panic disorder, social phobia, phobias, substance-induced anxiety disorder, delusional disorder, schizoaffective disorder, schizotypal disorder, substance-induced mental disorders, hypertension, or any combination thereof.

91. The aforementioned disease or disorder is a susceptible disorder or disorder, and as appropriate, the susceptible disorder or disorder is Rett syndrome, Angelman syndrome, spinal muscular atrophy, Smith-Magenis syndrome, Phelan-McDermid syndrome, Cornelia de Lange syndrome and other NIPBL-related disorders, DRK1A, KAT6A and disorders associated with severe intellectual disability, chromosome 2Q37 deletion syndrome and other HDAC4-related disorders, Angelman syndrome, Cleefstra syndrome, Joubert syndrome and other NPHP1-related disorders, Williams syndrome, neurofibromatosis 1 The method according to any one of claims 73 to 90, selected from the group including type 1, Leafraumeni syndrome and similar p53-associated cancer syndromes, phosphofructokinase deficiency, X-linked hyper-IgM syndrome and similar primary immunodeficiency disorders, triose phosphate isomerase deficiency, Kallmann syndrome, aromatase deficiency, Batten disease, frontotemporal dementia and other neurodegenerative disorders associated with progranulin loss, cholinergic receptor nicotinic alpha 7 subunit-associated disorders, and hereditary neuropathy prone to compression palsy.

92. The method according to claim 91, wherein the expression-sensitive disorder or impairment is characterized by a decrease in the expression of one or more proteins, and ectopic overexpression of the one or more proteins at a steady-state level above an upper-adjusted threshold causes cytotoxicity and / or disease.

93. The aforementioned expression sensitivity disorder is Neurodevelopmental syndromes, which are appropriately selected from the group including Rett syndrome, Smith-Magenis syndrome (RA1), Phelan-McDermid syndrome (SHANK3), Cornelia de Lange syndrome (NIPBL) and other NIPBL-related disorders, DRK1A, KAT6A and related disorders of severe intellectual disability, chromosome 2Q37 deletion syndrome and other HDAC4-related disorders, Angelman syndrome, Cleefstra syndrome, Joubert syndrome and other NPHP1-related disorders, and Williams syndrome, Proliferative disorders and / or cancers, as appropriate, selected from the group including neurofibromatosis type 1, Leafraumeni syndrome, and similar p53-related cancer syndromes. Glycogen storage disease, and appropriately phosphofructokinase deficiency, glycogen storage disease, Hematological disorders and / or immunological disorders, which are appropriately selected from the group including X-linked hyper-IgM syndrome and associated primary immunodeficiency disorders, and triose phosphate isomerase deficiency, Endocrine disorders, which are, as appropriate, selected from the group including Kallmann syndrome and aromatase deficiency, and / or Neuropsychiatric disorders, which are appropriately selected from the group including Batten disease, frontotemporal dementia and other neurodegenerative disorders associated with progranulin loss, cholinergic receptor nicotinic alpha 7 subunit-related disorders, and hereditary neuropathy prone to compression palsy. The method according to any one of claims 91 to 92.

94. The payload protein comprises RA1, and the disease or disorder comprises Smith-Magenis syndrome. The payload protein comprises SHANK3, and the disease or disorder comprises Phelan McDermid syndrome. The payload protein comprises CLN3, and the disease or disorder comprises Batten disease. The payload protein comprises NF-1, and the disease or disorder comprises neurofibromatosis type 1. The payload protein comprises TP53, and the disease or disorder comprises Leafraumeni syndrome. The payload protein comprises PFK, the disease or disorder comprises phosphofructokinase deficiency, and / or The method according to any one of claims 73 to 93, wherein the payload protein comprises CD40LG, and the disease or disorder comprises X-linked hyper-IgM syndrome.

95. Introducing the nucleic acid composition according to any one of claims 1 to 72 into one or more cells, The biological information of one or more cells is obtained, as appropriate, through sequencing or imaging. Research methods including those mentioned.

96. The method according to claim 95, wherein the imaging includes CRISPR imaging.

97. The method according to any one of claims 95 to 96, wherein the imaging includes super-resolution imaging and optionally includes DNA-PAINT (Point Accumulation for Imaging in Nanoscale Topography).

98. Introducing the nucleic acid composition according to any one of claims 1 to 72 into one or more cells, Isolating the aforementioned payload protein or the payload bound thereto A production method that includes this.

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