Genetic elements for adjusting expression

Abstract

Most therapeutic treatments require dose adjustment after initial administration. Dosage adjustment is crucial to ensuring optimal safety and efficacy characteristics of the treatment. Gene therapy is inherently limited by the inability to adjust the dose after initial administration. This invention describes a genetic element capable of dose adjustment after the initial administration of gene therapy. The realization of dose adjustment allows for the replacement of multiple subcutaneous protein injection regimens with single-injection gene therapy, thereby improving pharmacokinetics, safety / efficacy characteristics, medication adherence, and reducing healthcare costs.

Description

[0001] Cross-references to related applications

[0002] This application is based on The disclosure of U.S. Provisional Application No. 63 / 536,530, entitled “GENETIC ELEMENTSFOR ADJUSTMENT OF EXPRESSION”, filed on September 5, 2023, is hereby incorporated in its entirety by reference.

[0003] Reference to electronic sequence listing

[0004] The contents of the electronic sequence list (R087270004WO00-SEQ-KVC.xml; size: 246,308 bytes; and creation date: September 4, 2024) are incorporated herein by reference in their entirety. Technical Field

[0005] This invention relates generally to human and veterinary therapeutics, and more specifically to gene therapy treatments for conditions that may require dose adjustments after initial administration. Summary of the Invention

[0006] Due to the diversity of human biology, the effects of drug therapy are neither uniform nor consistent. Therefore, adjusting the dosing regimen after initial treatment, based on the physician's assessment of the treatment's effectiveness, can ensure that most treatments benefit. Dosage adjustment is important to ensure the optimal safety and efficacy characteristics of the treatment. If the initial treatment proves insufficiently effective in managing the pathology or symptoms, and side effects are at an optimally tolerable level, the treatment dose can be increased. Conversely, if the treatment dose causes excessive side effects or is intolerable to the patient, the treatment dose should be decreased. Similarly, if the patient's condition is cured, the dose should be reduced to a maintenance level or completely eliminated, depending on the pathology. However, gene therapy, as a treatment modality, is inherently limited by the fact that its dose cannot be adjusted after initial administration.

[0007] When genetic vectors are delivered by viral or nonviral vectors, they either integrate into the host cell genome and permanently reside within the cell, passing on to offspring, or they enter the nucleus and form episomes that are expressed for many years at levels defined by promoters, chromatin, epigenetics, or the general natural intracellular environment and extracellular signals. As an example, non-integrating gene therapy delivered by AAVs has demonstrated stable transgene expression over multi-year timeframes in preclinical and clinical studies. Similarly, therapeutic transgenes delivered by lentiviruses permanently integrate into the host cell genome, providing stable expression of the therapeutic transgene for many years. Currently, gene expression treated by gene therapy cannot be upregulated or downregulated in a manner capable of achieving therapeutic treatment of a condition requiring dose adjustment with gene therapy. While transcriptional control using inducible promoters is possible, such control is transient and does not provide stable upregulation or downregulation titers. Similarly, suicide genes can be used to completely shut down gene expression in cells (e.g., CAR-T cells) by inducing apoptosis to completely eliminate the cell. However, the latter approach is incompatible with protein replacement therapy because, in cases where previous gene therapies targeted all secretory cell types, creating a protein replacement factory in vivo and eliminating the target cells would result in significant toxicity. In addition to unacceptable toxicity to the currently targeted cell types, this approach also does not allow for dosage adjustments because the suicide gene is universally eliminated in a way that makes it impossible to control the final level of therapeutic transgene expression.

[0008] This invention provides, at least in part, a combination of target cell types, genetic elements, activators, and effectors that enables safe and effective permanent dose adjustment after initial administration of gene therapy. This invention also provides, at least in part, dose adjustment for gene therapy that allows a single-injection gene therapy to replace multiple subcutaneous protein regimens, while improving pharmacokinetics, safety / efficacy characteristics, medication adherence, and reducing healthcare costs.

[0009] Therefore, some aspects of this disclosure relate to a system for permanently reducing gene expression from one or more intracellular therapeutic constructs or multiple constructs, the system comprising at least: (a) a nucleic acid construct containing a therapeutic transgene, delivered in a formulation capable of entering cells and stably expressing the therapeutic transgene for at least 4 months; (b) an actuator composed of one or more chemical or biomolecules; (c) an actuator-responsive element and / or an actuator-responsive sequence enabling the expression, activation, or stabilization of an effector element or effector sequence; (d) an effector sequence delivered in cis with the therapeutic transgene; and (e) an effector element expressed by the effector sequence, capable of permanently reducing the expression of the therapeutic transgene in a manner that causes minimal bystander effect, and thus safe for surrounding tissues, organs, organisms, including the organism as a whole.

[0010] In some embodiments, the system is intended for delivery to subcutaneous tissue. In some embodiments, the system is intended for intraperitoneal delivery. In some embodiments, the system is intended for delivery to adipocytes. In some embodiments, the system is intended for intramuscular delivery. In some embodiments, the system is intended for delivery to the liver. In some embodiments, the system is intended for delivery to the central nervous system. In some embodiments, the system is intended for delivery to the fascia. In some embodiments, the system is intended for delivery to the subarachnoid space. In some embodiments, the system is intended for delivery to the pancreas. In some embodiments, the system is intended for delivery to the epidermis. In some embodiments, the system is intended for transocular delivery. In some embodiments, the system is intended for delivery to the kidney. In some embodiments, the system is intended for delivery to the bladder. In some embodiments, the system is intended for delivery to the mucosa. In some embodiments, the system is intended for delivery to the lung. In some embodiments, the system is intended for delivery to the spleen. In some embodiments, the system is intended for intravascular delivery. In some embodiments, the system is intended for delivery to the heart. In some implementations, the system is designed to be used in conjunction with therapeutic gene therapy or delivered in conjunction with therapeutic transgenes for the treatment of human, plant, or veterinary diseases or for the enhancement of human, veterinary, or plant functions.

[0011] In some embodiments, the therapeutic transgene encodes at least a portion of a therapeutic antibody. In some embodiments, the therapeutic transgene encodes a protein or enzyme required for the replacement therapy. In some embodiments, the therapeutic transgene encodes a therapeutic peptide. In some embodiments, the therapeutic transgene encodes a protein or fusion protein. In some embodiments, the therapeutic transgene encodes non-coding RNA. In some embodiments, the therapeutic transgene encodes an aptamer, aptamer, ribozyme, or a genetic structure capable of exerting enzymatic activity alone or in combination with a protein.

[0012] In some embodiments, the nucleic acid construct is composed of double-stranded DNA. In some embodiments, the nucleic acid construct is composed of single-stranded DNA. In some embodiments, the nucleic acid construct is composed of single-stranded RNA. In some embodiments, the nucleic acid construct is composed of double-stranded RNA. In some embodiments, the nucleic acid construct is composed of single-stranded or double-stranded linear DNA. In some embodiments, the nucleic acid construct is composed of single-stranded or double-stranded linear RNA. In some embodiments, the nucleic acid construct is composed of single-stranded or double-stranded circular DNA. In some embodiments, the nucleic acid construct is composed of single-stranded or double-stranded circular RNA.

[0013] In some embodiments, the system includes a protein delivered along with the nucleic acid. In some embodiments, the system is delivered in vivo or ex vivo as part of a viral or non-viral preparation. In some embodiments, the system is delivered by physical methods, such as electroporation, nanoparticle bombardment, ultrasound-mediated transfection, or chemically mediated transfection.

[0014] In some embodiments, the actuator is at least partially composed of tetracycline or a tetracycline derivative. In some embodiments, the actuator is at least partially composed of doxycycline or a doxycycline derivative. In some embodiments, the actuator is at least partially composed of an antibiotic or an antimicrobial peptide. In some embodiments, the actuator is at least partially composed of rapamycin or a derivative thereof. In some embodiments, the actuator is at least partially composed of ganciclovir or a derivative thereof. In some embodiments, the actuator is at least partially composed of ganciclovir, valganciclovir, valaciclovir, or a derivative thereof. In some embodiments, the actuator is at least partially composed of tamoxifen or a derivative thereof. In some embodiments, the actuator is at least partially composed of estrogen or a derivative thereof. In some embodiments, the actuator is at least partially composed of acetaldehyde or a derivative thereof. In some embodiments, the actuator is at least partially composed of L-arginine or a derivative thereof. In some embodiments, the actuator is at least partially composed of biotinylated AMP or a derivative thereof. In some embodiments, the actuator is at least partially composed of 2-phenylethyl butyrate or a derivative thereof. In some embodiments, the actuator is at least partially composed of 6-hydroxynicotinic acid or a derivative thereof. In some embodiments, the actuator is at least partially composed of erythromycin or a derivative thereof. In some embodiments, the actuator is at least partially composed of macrolides or derivatives thereof. In some embodiments, the actuator is at least partially composed of streptocin or a derivative thereof. In some embodiments, the actuator is at least partially composed of NADH or a derivative thereof. In some embodiments, the actuator is at least partially composed of an element that causes the formation of increased energy or heat. In some embodiments, the actuator is at least partially composed of SCB1 or a derivative thereof. In some embodiments, the actuator is at least partially composed of 3-oxo-C8-HSL or a derivative thereof. In some embodiments, the actuator is at least partially composed of phloretin or a derivative thereof. In some embodiments, the actuator is a small molecule, peptide, protein, or nucleic acid capable of increasing, decreasing, or modulating local or systemic inflammation or a partial inflammatory response. In some embodiments, the actuator is at least partially composed of theophylline or a derivative thereof. In some embodiments, the actuator is at least partially composed of IPTG or a derivative thereof. In some embodiments, the actuator is at least partially composed of 5-fluorocytosine or a derivative thereof. In some embodiments, the actuator is at least partially composed of propionate or a derivative thereof. In some embodiments, the actuator is at least partially composed of acetate or a derivative thereof. In some embodiments, the actuator is at least partially composed of cumate, cumate ester, or cumic acid or a derivative thereof. In some embodiments, the actuator is at least partially composed of gluconate or a derivative thereof.In some embodiments, the actuator is at least partially composed of maltose or a derivative thereof. In some embodiments, the actuator is at least partially composed of arabinose or a derivative thereof. In some embodiments, the actuator is at least partially composed of a monosaccharide or disaccharide. In some embodiments, the actuator is at least partially composed of trimethoprim or a derivative thereof. In some embodiments, the actuator is at least partially composed of an exogenously administered small molecule, peptide, protein, RNA, DNA, or pharmacological agent. In some embodiments, at least a portion of the actuator may serve as at least a part of an effector element. In some embodiments, the actuator is at least partially composed of an endogenously expressed or presented peptide, hormone, protein, RNA, DNA, enzyme, mineral, or chemical or biochemical substance, and is capable of interacting with an actuator response element or actuator response sequence.

[0015] In some embodiments, the effector element is at least partially composed of inducible caspase 9. In some embodiments, the effector element is at least partially composed of native caspase 9. In some embodiments, the effector element is at least partially composed of a zinc finger nuclease. In some embodiments, the effector element is at least partially composed of a transcription activator-like effector nuclease (TALEN). In some embodiments, the effector element is at least partially composed of a meganuclease. In some embodiments, the effector element is at least partially composed of a deoxycytidine deamination-derived editor. In some embodiments, the effector element is at least partially composed of a deoxyadenosine deamination-derived base editor. In some embodiments, the effector element is at least partially composed of a DNA-binding peptide. In some embodiments, the effector element is at least partially composed of a viral or bacterial DNA-binding protein. In some embodiments, the effector element is at least partially composed of native caspase 8. In some embodiments, the effector element is at least partially composed of native caspase or a caspase pathway protein. In some embodiments, the effector element is FK506-binding protein 12 (e.g., with an F36V mutation) fused to a human caspase 8 protein lacking 1-215 AA and linked to an N-myristylation signaling pathway of Src kinase. In some embodiments, the effector element is partially composed of FK506-binding protein 12 (e.g., with an F36V mutation) fused to a human caspase-9 protein lacking 1-134 AA. In some embodiments, the effector element is at least partially composed of at least a portion of viral thymidine kinase. In some embodiments, the effector element is at least partially composed of at least a portion of Escherichia coli cytosine deaminase. In some embodiments, the effector element is a protein having catalytic activity capable of chemically modifying nucleotides, nucleosides, or nucleic acids. In some embodiments, the effector element is at least partially composed of at least a portion of diphtheria toxin subunit A. In some embodiments, the effector element is at least partially composed of at least a portion of diphtheria toxin subunit B. In some embodiments, the effector element is a dimerizable or trimerizable toxin, protein, or enzyme. In some embodiments, the effector element is at least partially composed of at least a portion of a human or simian diphtheria toxin receptor. In some embodiments, the effector element is at least partially composed of at least a portion of a bacterial, viral, or eukaryotic toxin. In some embodiments, the effector element is at least partially composed of at least a portion of a protein capable of inhibiting transcription. In some embodiments, the effector element is at least partially composed of at least a portion of a protein capable of inhibiting translation. In some embodiments, the effector element is at least partially composed of at least a portion of a protein capable of inducing apoptosis.In some embodiments, the effector element is at least partially composed of at least a portion of a protein capable of inducing aging. In some embodiments, the effector element is at least partially composed of at least a portion of a protein capable of reducing general gene expression. In some embodiments, the effector element is at least partially composed of at least a portion of a nuclease. In some embodiments, the effector element is at least partially composed of at least a portion of a nuclease capable of site-specific mutagenesis. In some embodiments, the effector element is at least partially composed of at least a portion of a nuclease and at least a portion of a targeting element. In some embodiments, the effector element is at least partially composed of at least a portion of: a Cas nuclease and a guide RNA that targets the nuclease to a sequence. In some embodiments, the effector element is at least partially composed of at least a portion of a nuclease of a target sequence. In some embodiments, the effector element is at least partially composed of at least a portion of a nuclease of a promoter region of a target sequence. In some embodiments, the effector element is at least partially composed of at least a portion of a nuclease of a coding region of a target sequence. In some embodiments, the effector element is at least partially composed of at least a portion of a nuclease of a post-translational regulatory element region of a target sequence. In some embodiments, the effector element is at least partially composed of at least a portion of a recombinase. In some embodiments, the effector element is at least partially composed of at least a portion of a Cre recombinase. In some embodiments, the effector element is at least partially composed of at least a portion of the Krüppel-associated box domain of the human gene ZNF10. In some embodiments, the effector element is at least partially composed of at least a portion of a zinc finger protein. In some embodiments, the effector element is at least partially composed of at least a portion of a cyclized recombinase having an SV40 nuclear localization signal. In some embodiments, the effector element is at least partially composed of at least a portion of a Cre recombinase having an SV40 T antigen intron inserted into the ORF. In some embodiments, the effector element is at least partially composed of at least a portion of a Flpe recombinase. In some embodiments, the effector element is at least partially composed of at least a portion of a Flpo recombinase. In some embodiments, the effector element is at least partially composed of at least a portion of a Dre recombinase. In some embodiments, the effector element is at least partially composed of at least a portion of the SV40 T antigen sequence. In some embodiments, the effector element is at least partially composed of at least a portion of a piggyBac transposase. In some embodiments, the effector element is at least partially composed of at least a portion of a transposase. In some embodiments, the effector element is at least partially composed of at least a portion of a sleeping beauty transposase. In some embodiments, the effector element is at least partially composed of at least a portion of an agonaut protein.In some embodiments, the effector element is at least partially composed of at least a portion of a DNA methyltransferase. In some embodiments, the effector element is at least partially composed of at least a portion of a protein constituting or interacting with a chromatin remodeling complex. In some embodiments, the effector element is at least partially composed of at least a portion of a protein constituting or interacting with a DNA methylation pathway. In some embodiments, the effector element is at least partially composed of at least a portion of a base editor. In some embodiments, the effector element is at least partially composed of at least a portion of a leader editor. In some embodiments, the effector element is at least partially composed of at least a portion of a nicking enzyme. In some embodiments, the effector element is at least partially composed of at least a portion of a viral, bacterial, or eukaryotic gene silencing protein. In some embodiments, the effector element is at least partially composed of at least a portion of siRNA. In some embodiments, the effector element is at least partially composed of at least a portion of miRNA. In some embodiments, the effector element is at least partially composed of at least a portion of FRT. In some embodiments, the effector element is at least partially composed of at least a portion of an integrase. In some embodiments, the effector element is at least partially composed of at least a portion of RNA capable of reducing gene expression. In some embodiments, the effector element is at least partially composed of at least a portion of a protein capable of degrading the effector in the absence of an inducer. In some embodiments, the effector element is at least partially composed of at least a portion of dihydrofolate reductase. In some embodiments, the effector element is at least partially composed of at least a portion of RNA, said at least a portion of which is at least partially complementary to the genetic construct. In some embodiments, the effector element is at least partially composed of at least a portion of a transposon or retrotransposon. In some embodiments, the effector element is at least partially composed of at least a portion of Streptococcus pyogenes Cas9. In some embodiments, the effector element is at least partially composed of at least a portion of catalytically inactive Cas9. In some embodiments, the effector element is at least partially composed of at least a portion of codon-optimized Cas9. In some embodiments, the effector element is at least partially composed of at least a portion of Cas9 with a D10A mutation. In some embodiments, the effector element is at least partially composed of at least a portion of dCas9 fused with KRAB. In some embodiments, the effector element is at least partially composed of at least a portion of dCas9 fused to a carboxyl-terminal dichotomous repressor domain KRAB-MeCP2 or a derivative thereof. In some embodiments, the effector element is at least partially composed of at least a portion of an ortholog of Campylobacter jujeni Cas9.In some embodiments, the effector element is at least partially composed of at least a portion of: a CRISPR-associated protein or an ortholog. In some embodiments, the effector element is at least partially composed of at least a portion of: a Cas12a or Cas12b protein. In some embodiments, the effector element is at least partially composed of at least a portion of: Cas13. In some embodiments, the effector element is at least partially composed of at least a portion of: Cas12f or Cas13a. In some embodiments, the effector element is at least partially composed of at least a portion of: DNA, RNA-expressing DNA, RNA, protein, or combinations thereof capable of affecting DNA sequences in a sequence-specific manner to induce double-strand breaks, single-strand breaks, methylation, chromatin modification, or general epigenetic silencing. In some embodiments, the effector element is at least partially composed of at least a portion of: a toxin, a toxin derivative, a modified toxin or its homolog, or an ortholog or paralog. In some embodiments, the effector element is at least partially composed of at least a portion of: bacterial, fungal, plant, or animal toxins. In some embodiments, the effector element comprises at least a portion of the following: Vibrio cholerae toxin, Shiga toxin, Shigella toxin, ricin, saponin, Staphylococcus aureus enterotoxin H, Clostridium perfringens enterotoxin, streptococcal hemolysin O, ribosome inactivating protein (RIP) enzyme, immunotoxin, Pseudomonas exotoxin A, diphtheria toxin (DT), or a DT fragment. In some embodiments, the effector element comprises at least a portion of the following: viral thymidine kinase or a derivative thereof. In some embodiments, the effector element comprises at least a portion of the following: bacterial cytosine deaminase or a derivative thereof. In some embodiments, the effector element comprises at least a portion of the following: D-amino acid oxidase or a derivative thereof. In some embodiments, the effector element is at least partially composed of bacterial carboxypeptidase G2 or a derivative thereof. In some embodiments, the effector element is at least partially composed of purine nucleoside phosphorylase or a derivative thereof. In some embodiments, the effector element is at least partially composed of thymidine phosphorylase or a derivative thereof. In some embodiments, the effector element is at least partially composed of xanthine-guanine phosphoribosyltransferase or a derivative thereof.In some embodiments, the effector element is at least partially composed of at least a portion of: nitroreductase or a derivative thereof. In some embodiments, the effector element is at least partially composed of at least a portion of: penicillin-G amidase or a derivative thereof. In some embodiments, the effector element is at least partially composed of at least a portion of: multidrug activating enzyme or a derivative thereof. In some embodiments, the effector element is at least partially composed of at least a portion of: horseradish peroxidase or a derivative thereof. In some embodiments, the effector element is at least partially composed of at least a portion of: β-galactosidase or a derivative thereof. In some embodiments, the effector element is at least partially composed of at least a portion of: deoxyribonucleotide kinase or a derivative thereof. In some embodiments, the effector element is at least partially composed of at least a portion of: β-glucuronide kinase or a derivative thereof. In some embodiments, the effector element is at least partially composed of at least a portion of: carboxypeptidase A or a derivative thereof. In some embodiments, the effector element is at least partially composed of at least a portion of: cytochrome P450 or a derivative thereof. In some embodiments, the effector element is at least partially composed of at least a portion of: deoxycytidine kinase or a derivative thereof.

[0016] In some embodiments, the effector sequence at least encodes an effector element. In some embodiments, the effector sequence encodes at least a portion of: a shared Lox sequence or a sequence capable of mediating the action of a Lox sequence. In some embodiments, the effector sequence encodes at least a portion of: a shared FRT sequence or a sequence capable of mediating the action of an FRT sequence. In some embodiments, the effector sequence encodes at least a portion of: a shared Rox sequence or a sequence capable of mediating the action of a Rox sequence. In some embodiments, the effector sequence is at least partially composed of: (SEQ ID NO: 1) constitutes the sequence. In some embodiments, the effector sequence is at least partially composed of... (SEQ ID NO: 2) constitutes the effector sequence. In some embodiments, the effector sequence is at least partially composed of (SEQ ID NO: 3). In some embodiments, the effector sequence is at least partially composed of (SEQ ID NO: 4) constitutes the effector sequence. In some embodiments, the effector sequence is at least partially composed of... (SEQ ID NO: 5). In some embodiments, the effector sequence is at least partially composed of (SEQ ID NO: 6) constitutes the effector sequence. In some embodiments, the effector sequence is at least partially composed of... (SEQ ID NO: 7) constitutes the effector sequence. In some embodiments, the effector sequence encodes at least a portion of the following: attB, attR, attL, or attP sites. In some embodiments, the effector sequence is at least partially composed of a genetic sequence that can be targeted by an integrase, recombinase, nuclease, nickase, chromatin complex, or methyltransferase. In some embodiments, the effector sequence is at least partially composed of a genetic sequence that can be targeted for silencing RNA or protein binding, or that can be silenced itself. In some embodiments, the effector sequence is a DNA sequence that can be used as a target of a guide RNA for a CRISPR-related protein. In some embodiments, the effector sequence is a DNA sequence derived from a viral or bacterial genome. In some embodiments, the effector sequence forms a secondary structure in the absence of an inducing molecule. In some embodiments, the effector sequence is an inflammatory response promoter. In some embodiments, the effector sequence is at least partially derived from a mammalian promoter or enhancer. In some embodiments, the effector sequence forms a secondary structure in the presence of an inducing molecule.

[0017] In some embodiments, the actuator-responsive element is a nucleic acid sequence or protein that interacts with the actuator to enable activation of an effector element or effector sequence. In some embodiments, the actuator-responsive element is at least partially composed of a tetracycline-controlled transcriptional silencer (tTS). In some embodiments, the actuator-responsive element is at least partially composed of the Kruppel associated box (KRAB) AB domain of a kid-1 transcriptional repressor. In some embodiments, the actuator-responsive element is at least partially composed of a trans-activator controlled by reverse tetracycline. In some embodiments, the actuator-responsive element is at least partially composed of a viral protein, such as VP16, fused with a Tet repressor (TetR) or a reverse Tet repressor (rTetR). In some embodiments, the actuator-responsive element is composed of tTS and rtTA driven by the same promoter. In some embodiments, the actuator-responsive element is composed of tTS and rtTA driven by different promoters. In some embodiments, the actuator response element is at least partially composed of a transcriptional repressor. In some embodiments, the actuator response element is at least partially composed of a transcriptional silencer. In some embodiments, the actuator response element is at least partially composed of a DNA-binding protein. In some embodiments, the actuator response element is at least partially composed of a promoter-binding protein. In some embodiments, the actuator response element is at least partially composed of a transcription initiation complex-binding protein. In some embodiments, the actuator response element is at least partially composed of a protein responsible for DNA methylation. In some embodiments, the actuator response element is at least partially composed of a protein responsible for histone modification. In some embodiments, the actuator response element is at least partially composed of at least a portion of a Tet-ON system. In some embodiments, the actuator response element is at least partially composed of at least a portion of a Tet-OFF system.

[0018] In some embodiments, the actuator response element is at least partially composed of at least a portion of: AlcR, ArgR, BirA, EthR, HdnoR, HucR, MphR(A), PIP, Rex, RheA, ScbR, TraR, TtgR, or their analogues, homologs, or orthologs, or functionally similar protein fragments, proteins, or fusion proteins. In some embodiments, the actuator response element is at least partially composed of at least a portion of: lacUV5, lac, tac, trc, prpB, aceA, aceB, gntP, gntK, CJ10X2, tacM, malE1, git1, BAD, SPL, P4-N14, cspB, aprE, sod, dapA, porB, ilvC, L10, L26, L16, L51, H30, H36, or natural, synthetic, or semi-synthetic analogs, homologs, or orthologs, or functionally similar protein fragments, proteins, or fusion proteins. In some embodiments, the actuator response element is at least partially composed of at least a portion of: ABI1-Gal4DBD, ABA, PVL1-VP16, PHR-p65, CIB1-TetR, or analogs, homologs, or orthologs, or functionally similar protein fragments, proteins, or fusion proteins. In some implementations, the actuator response element comprises at least a portion of the following: a transcriptional activator, an enhancer, a derepressor, or a protein, RNA, or DNA capable of directly or indirectly enhancing gene expression in a targeted or generally cis-acting manner.

[0019] Some aspects of this disclosure relate at least in part to methods using any of the systems described herein. In some embodiments, any of the systems described herein are used in methods for reducing gene expression in cells. In some embodiments, the cells are hepatocytes, muscle cells, central nervous system cells, pancreatic cells, skin cells, kidney cells, bladder cells, lung cells, heart cells, adipocytes, or spleen cells. In some embodiments, any of the systems described herein are used in methods for reducing gene expression in organs. In some embodiments, the organs are liver, pancreas, skin, brain, eye, spleen, kidney, heart, or bladder.

[0020] Some aspects of this disclosure relate, at least in part, to methods of delivering any of the systems described herein to the cells, tissues, or organs of a subject. Some aspects of this disclosure relate, at least in part, to methods of treating a subject in need of treatment. In some embodiments, the subject is a mammalian subject. In some embodiments, the mammalian subject is a human. In some embodiments, the mammalian subject is a rodent. In some embodiments, the rodent is a mouse. In some embodiments, the rodent is a rat. In some embodiments, the mammalian subject is a non-human primate. In some embodiments, the non-human primate is a rhesus monkey. In some embodiments, the non-human primate is a chimpanzee. In some embodiments, the non-human primate is a monkey. In some embodiments, the mammalian subject is a dog. In some embodiments, the mammalian subject is a cat. Attached Figure Description

[0021] The following figures form part of this specification and are included to further illustrate certain aspects of this disclosure, which can be better understood by referring to one or more of these figures in conjunction with a detailed description of some specific embodiments given in this disclosure. The figures are not intended to be drawn to scale. They are illustrative only. For clarity, not every component may be labeled in every figure. In the figures:

[0022] Figure 1 The map of the npRB2.29v1 plasmid is shown.

[0023] Figure 2 The map of the npRB2.28v1 plasmid is shown.

[0024] Figures 3A to 3B It is a bar graph showing the effective expression of reporter genes. Figure 3A Several systems demonstrating efficient expression of reporting genes are shown. Figure 3B The results show a relative decrease in expression after an equal dose of inducer.

[0025] Figure 4 The map of the npRB2.31v1 plasmid is shown.

[0026] Figure 5 The map of the npRB2.33v1 plasmid is shown.

[0027] Figure 6 This is a bar graph showing the relative bioluminescence of primary human adipocytes treated with liquid nanoparticles (LNPs).

[0028] Figure 7 The map of the npRB2.32v1 plasmid is shown.

[0029] Figure 8 The map of the npRB2.30v1 plasmid is shown.

[0030] Figure 9 This is a bar graph showing the relative bioluminescence of primary human adipocytes treated with liquid nanoparticles (LNP).

[0031] Figure 10 The map of the npRB2.35v1 plasmid is shown.

[0032] Figure 11 The map of the npRB2.34v1T plasmid is shown.

[0033] Figure 12 This is a bar graph showing the relative expression in primary human adipocytes treated with liquid nanoparticles (LNP).

[0034] Figure 13 The plasmid map of npRB2.22v1 (CBh-IGF2-P2A-Tet3G+TRE3G-GSDMD-NT) is shown.

[0035] Figure 14 The plasmid map of npRB2.23v1 (CMV-GBA1-P2A-Tet3G+TRE3G-IE2-P2A-tTA) is shown.

[0036] Figure 15 The plasmid map of npRB2.24v1 (attP-CBh-attB-LMNA-P2A-Tet3G+TRE3G-Bxb1) is shown.

[0037] Figure 16 The plasmid map of npRB2.25v1 (CBh-CFTR-P2A-iCasp9) is shown.

[0038] Figure 17 The plasmid map of npRB2.26v1 (CBh-HEXA-P2A-iCasp8) is shown.

[0039] Figure 18 The plasmid map of npRB2.27v2 (TRE3G-FGF18-P2A-LacI-NLS-KRAB+7xLacO promoter-tTS) is shown. Detailed Implementation

[0040] This invention generally describes a combination of target cell types and anatomical regions, genetic elements, actuators, effector elements, effector sequences, and actuator response elements that enables safe and effective permanent dose adjustment after initial gene therapy administration. This invention is the first to enable permanent, durable, safe, and effective dose adjustment of gene therapy. Therefore, this invention, for the first time, allows for the use of gene therapy as a single-injection adjustable gene therapy method, replacing protein replacement, enzyme replacement, or general protein injection therapy.

[0041] Generally, this invention describes genetic constructs for delivery to a wide range of cell types, but most preferably adipocytes or other non-life-sustaining or organ-sustaining cell types, said genetic constructs comprising an actuator, an effector element, an effector sequence, and an actuator-response element. For the purposes of this invention, the actuator may be a small molecule, a macromolecule, a peptide, a protein, a nucleic acid, a physical or chemical stimuli, capable of achieving, enhancing, or activating the expression or activity of the effector element by acting directly on the effector element or the actuator-response element, or both. Similarly, for the purposes of this invention, the effector element is a protein, nucleic acid, fusion protein, peptide, enzyme, ribozyme, ribonucleoprotein, sugar, small molecule, or a combination of one or more of the above elements, capable of targeting a therapeutic genetic sequence or more specifically, an effector sequence, to achieve an overall dose reduction of the therapeutic genetic load or specifically a reduction in the transcriptional, expression, translational, or functional levels of the therapeutic genetic load. Consistent with this, the effector sequence is a target, a downstream medium of an active or recognition site, an active site of an effector element, or generally associated with an effector element to enable downregulation titration or dose reduction in gene therapy. Finally, the actuator response element is a protein, nucleic acid sequence, DNA, promoter, enhancer, insulator, or general genetic sequence that, once acted upon by the actuator, alters the expression, translation, transcription, or activation level of the effector element. Generally, the element or the genetic sequence encoding the element is delivered cis-directed during treatment or retreatment, except for the actuator, which is delivered during dose adjustment. The sequences of the actuator, effector element, effector sequence, and / or actuator response element may optionally be codon-optimized for the target species.

[0042] In a representative example, an actuator (e.g., tetracycline) induces the expression of the CRISPR-Cas9 protein by activating the Tet-ON operon, thereby driving the expression of both the CRISPR-Cas9 protein and the guide RNA. The CRISPR-Cas9 protein and guide RNA (effect element) target and dissociate the effector sequence of a constitutive promoter that drives the expression of a therapeutic transgene. Therefore, administration of tetracycline enables the expression of a nuclease that separates the promoter from the coding region of the therapeutic transgene, thereby reducing the expression level of the therapeutic transgene in the construct. Because this action is sequence-specific, and because the DNA encoding the effector element, effector sequence, actuator response element, therapeutic transgene, and associated promoter is delivered in cis (or all together in a single genetic construct), initial treatment produces an expression level of L, which is reduced to below L after activation of the dose-reduction system by administration of the actuator (which can be considered a safe exogenous small molecule administration), thus reducing the level of therapeutic transgene expression.

[0043] Another optional actuator may be derived from a class of stop codon readthrough enhancers. An example of a stop codon readthrough enhancer is an aminoglycoside antibiotic. Therapeutic transgenes may be expressed in cis, ending with a stop codon whose readthrough can be promoted by administration of a stop codon readthrough enhancer or by molecules that directly or indirectly enable or enhance stop codon readthrough. For example, the stop codon may be followed by a self-cleaving peptide and a suicide gene, such as diphtheria toxin or a diphtheria toxin subunit. Some representative examples of readthrough enhancers include gentamicin, G418, paromomycin, neomycin, sisomycin, livermycin, or derivatives or combinations thereof. Some examples of stop codons may include UGA, UAG, UAA, which have an optional fourth base to allow inhibition of natural readthrough and / or enhancement or readthrough following activation by a readthrough enhancer. As an example, gentamicin can enhance readthrough when a UGA stop codon is used followed by cytosine (C) or uracil (U) (adenine (A) is less preferred and guanine (G) is least preferred). The system can be used alone or in combination with other systems, such as using a base editor to change one of the bases adjacent to the stop codon in the stop codon, or to convert the codon into a stop codon within the codon itself.

[0044] Generally, the present invention comprises a system for permanently or less preferably and optionally transiently reducing gene expression of a therapeutic construct or multiple constructs within one or more cells comprising at least a few elements. The elements may include: a nucleic acid construct containing a therapeutic transgene, delivered in a formulation capable of entering the cell and stably expressing the therapeutic transgene for at least 4 months; one or more actuators comprising one or more chemical or biomolecules exogenously applicable to a system, cell, tissue, organ, or organism undergoing treatment with the system; one or more actuator-responsive elements and / or actuator-responsive sequences enabling the expression, activation, or stabilization of one or more effector elements or effector sequences; an effector sequence delivered cis-with the therapeutic transgene; and one or more effector elements expressed by the effector sequence, which are capable of permanently reducing the expression of the therapeutic transgene in a manner that causes minimal bystander effect and is therefore safe for surrounding tissues, organs, and organisms, including the organism as a whole.

[0045] The system may be a larger formulation or part of one or more formulations and is administered via a drug delivery system, in a vial for subsequent administration, or in a pre-filled syringe or cartridge. Alternatively, treatment may be administered via an autoinjector or patch injector, guided by ultrasound or other guiding techniques to ensure delivery to the correct location. Alternatively, and optionally, treatment may be delivered via a set of needles or via a set or series of injections to distribute the construct more uniformly at the treatment site. Gene therapy treatments may be delivered via patches, microneedles, a set of microneedles, jet injection, on a carrier, or using other methods to alter the integrity of the tissue through which or into which the delivery is made.

[0046] In some embodiments of the invention, the target cell type of the system may be one or more of the following: adipocytes, preadipocytes, progenitor cells capable of differentiating into adipocytes, stem cells capable of differentiating into adipocytes, resident cell types of subcutaneous tissue, or transient cells or stem cells, but which may be resident cells of subcutaneous tissue. The system is intended to treat or prevent any human or veterinary condition, but in some specific embodiments it may be used to treat single-gene disorders, enzyme deficiencies, protein deficiencies (as a protein replacement therapy), metabolic disorders, autoimmune diseases, tumors, neurological disorders, cardiovascular pathologies, musculoskeletal disorders, hematological disorders, infectious diseases, skin diseases, immune system disorders, pulmonary disorders, kidney or bladder disorders, or disorders of other organs, tissues, or systems, or combinations thereof. In some embodiments of the invention, the system is intended for use in delivering therapeutic or preventative antibodies, therapeutic, preventative, prophylactic proteins, fusion proteins, peptides, immunogens, antigens, enzymes, ribozymes, mRNA, non-coding RNA, miRNA, shRNA or other RNA, or molecules that can act as vaccines or adjuvants by expression from a genetic construct, via expression of a genetic construct.

[0047] Generally, the system can be delivered in one or more ways to subcutaneous tissue, intraperitoneal tissue, directly to adipose tissue, white adipose tissue, or targeted or delivered to adipocytes. Alternatively and optionally in combination with the above-described delivery sites, tissues, and forms, the system can be delivered intramuscularly, intravenously, or directly to the liver via artery or vein. The system can be delivered directly or indirectly to the central nervous system, to the fascia, subarachnoid space, pancreas, epidermis, eye, or generally as part of intraocular or intravitreal delivery to parts of the eye, kidney, bladder, mucous membranes, lung, spleen, heart, or any other cell, tissue, organ, or system of cells, tissues, or organs, or the whole organism.

[0048] The system can be used to treat diseases or in combination with other treatments to manage symptoms or treat diseases or pathological conditions, or to endow organisms, organs, or tissues with function. The system can be used in conjunction with therapeutic genes to treat human, veterinary, or plant diseases, or to enhance the function of humans, veterinarians, or plants.

[0049] The system is designed to contain at least one therapeutic genetic element, which may be a coding gene or a non-coding gene, or a DNA fragment that directly provides the therapeutic element or is transcribed into RNA providing the therapeutic element. Therefore, the system may encode at least a subset of the following: therapeutic antibodies, proteins, enzymes, fusion proteins, peptides, aptamer enzymes, or transfer RNA (tRNA), ribosomal RNA (rRNA), long non-coding RNA (lnc-RNA or ln-RNA or long nc-RNA), short non-coding RNA (short ncRNA or snc-RNA), microRNA (miRNA), messenger RNA (mRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), PIWI-interacting RNA (piRNA), or other RNA, proteins, or combinations thereof. Therapeutic transgenes can directly encode aptamer enzymes, or can be side-linked with one or more aptamer enzymes, ribozymes, or genetic structures that can exert enzymatic activity alone or in combination with proteins or other nucleic acids.

[0050] The system disclosed in this invention comprises nucleic acids, which may be one or more of the following: double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), double-stranded RNA (dsRNA), or single-stranded RNA (ssRNA). Optionally, the construct may comprise a DNA-RNA hybrid, wherein one or more strands are RNA and one or more strands are DNA, or one or more portions of one or more strands are RNA and one or more portions of one or more strands are DNA. The DNA or RNA may be linear, circular, supercoiled, partially supercoiled, or non-supercoiled, partially linear, fragmented, covalently closed, chemically modified (e.g., with a cap and polyA tail), or chemically modified to enhance stability, reduce immunogenicity, or generally to confer any other biological function.

[0051] The system may be used as a protein-nucleic acid complex delivery system, or may generally contain proteins within a nucleic acid-containing formulation, or contain proteins as part of a nucleic acid-containing formulation. The range of proteins can be from DNA-binding proteins to proteins that promote immunogenicity reduction, proteins that promote intracellular nucleic acid transport, proteins that promote nucleic acid protection, and proteins that promote binding to cellular proteins, membranes, organelles, transmembrane proteins, nucleoproteins, or other cellular structures, and generally may exert structural or therapeutic activity, or enhance the structural or therapeutic activity of the system.

[0052] The system can be delivered using formulations containing viral or non-viral vectors, or formulations containing both viral and non-viral vectors with or without targeting or additional targeting. Delivery can be performed in vivo or ex vivo, such as delivery to immune system cells for the purpose of generating chimeric antigen receptor T cells or CAR-T cells. The system can optionally or additionally be delivered in vivo or ex vivo by physical or chemical methods, including one or more of electroporation, nanoparticle bombardment, ultrasound-mediated transfection, or chemically mediated transfection using transfection reagents. Generally, the system is designed to be delivered on its own or via a carrier, delivery agent, formulation, or as part of a larger construct or superstructure to achieve therapeutic effects, targeting, dose adjustment, safety, or durability.

[0053] The system requires the use of an actuator, the delivery of which is performed when the system needs to adjust the therapeutic effect, titrate, or adjust the dosage. The actuator may be an exogenous or endogenous small molecule and may be delivered, or its generation may be triggered by delivery in another manner (physical or chemical). In some embodiments of the invention, the actuator is at least partially composed of: tetracycline or a tetracycline derivative, doxycycline or a doxycycline derivative, an antibiotic or antimicrobial peptide or derivative, rapamycin or a derivative thereof, ganciclovir or a derivative thereof, valganciclovir or a derivative thereof, valacyclovir or a derivative thereof, tamoxifen or a derivative thereof, a hormone or estrogen or a derivative thereof, acetaldehyde or a derivative thereof, L-arginine or a derivative thereof, biotinylated AMP or a derivative thereof, caffeine or a derivative thereof, 2-phenylethylbutyrate or a derivative thereof, 6-hydroxynicotinic acid or a derivative thereof, erythromycin or a derivative thereof. In some embodiments, the actuator may be at least partially composed of: macrolides or derivatives thereof, streptocin or derivatives thereof, theophylline or derivatives thereof, 5-fluorouracil or derivatives thereof, propionate or derivatives thereof, 5-fluorocytosine or derivatives thereof, isopropyl β-D-1-thiogalactopyranoside (IPTG) or derivatives thereof, acetate or derivatives thereof, NADH or derivatives thereof, or generally antibiotics, antimicrobial agents, antiviral agents, anti-inflammatory agents, hormones or pro-inflammatory agents, or generally approved therapeutic agents for the treatment of some other diseases, symptoms or pathological conditions. In some embodiments of the invention, the actuator may be derived from the application of heat or energy, or may be an element of heat, light, magnetic field or other energy or cold or energy loss. In some embodiments, the actuator may be at least partially composed of natural or synthetic peptides or proteins (e.g., SCB1 or derivatives thereof) or chemically modified fusion proteins or peptides. In other embodiments of the invention, the actuator may be at least partially composed of: 3-oxo-C8-HSL or a derivative thereof, phloretin or a derivative thereof, or generally a small molecule, peptide, protein, or nucleic acid capable of enhancing, reducing, or modulating local or systemic inflammation or a partial inflammatory response. In other embodiments, the actuator may be at least partially composed of: cumyl acid salt, cumyl ester, cumic acid or a derivative thereof, or 2,3-dihydroxy-p-cumyl acid salt or a derivative thereof, p-cumyl acid salt or a derivative thereof, or cis-5,6-dihydroxy-4-isopropylcyclohexyl-1,3-dienecarboxylic acid ester or a derivative thereof. In other embodiments of the invention, the actuator may be at least partially composed of at least a portion of: gluconate, maltose, dextrose, idonate, arabinose or a derivative thereof, or generally composed of monosaccharides, disaccharides, polysaccharides, or oligosaccharides. In other embodiments of the invention, the actuator may be at least partially composed of at least a portion of: trimethoprim or a derivative thereof.Finally, for the purposes of this invention, the actuator may be at least partially composed of an exogenously administered small molecule, peptide, protein, RNA, DNA, or pharmacological preparation having or not having additional physical activity or action from within or outside the organism receiving treatment. Furthermore, the actuator may be composed of an effector element, or may serve as both an actuator and an effector in a therapeutic preparation, and may be at least partially composed of endogenously expressed or presented peptides, hormones, proteins, RNA, DNA, enzymes, minerals, or chemical or biochemical substances, and may be capable of interacting with an actuator-response element or actuator-response sequence.

[0054] The system disclosed in this invention utilizes one or more effector elements that, when induced, activated, or subsequently acted upon by an actuator, lead to a reduction in therapeutic dosage, a reduction in therapeutic transgene expression, elimination of cells carrying the system, or a reduction in therapeutic efficacy, either directly or indirectly. The effector elements may be at least partially composed of: inducible cysteine ​​9, native cysteine ​​9, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases or homing endonucleases, deoxycytidine deamination-derived editors, deoxyadenosine deamination-derived base editors, or combinations thereof, and mediate DNA binding, cleavage, single-strand breaks, double-strand breaks, or the death of one or more cells carrying the system or therapeutic payload. Other effectors that can be used for down-titering purposes include caspases 1, 3, 4, 5, 6, 8, 9, 11, and 12; FK506-binding protein 12 (with an F36V mutation) fused to human caspase-8 protein lacking 1–215 AA and linked to an N-myristylation signal from Src kinase; FK506-binding protein 12 (with an F36V mutation) fused to human caspase-9 protein lacking 1–134 AA; bacterial or viral thymidine kinase; bacterial or viral cytosine deaminase; or generally any kinase, caspase, or caspase pathway protein, fragment thereof, or hybrid thereof. In some embodiments of the invention, the effector element may be at least partially composed of: at least a portion of a toxin or diphtheria toxin (DT) or a toxin subunit or diphtheria toxin subunit A (DTA), or at least a portion of diphtheria toxin subunit B (DTB), its homolog, analog, or fragment, or a combination of at least a portion of a toxin or diphtheria toxin (DT) or a toxin subunit or diphtheria toxin subunit A (DTA) with diphtheria toxin subunit B (DTB), its homolog, analog, or fragment, or generally a dimerizable, trimerizable, or monomeric toxin having or lacking stable or unstable or binding domains. Generally, the effector element can mediate down-titration by eliminating the cell carrying the system or by blocking the expression, transcription, translation, or general production of therapeutic proteins.Some specific effectors may consist at least partially of at least one of the following: botulinum toxin, cholera toxin, Clostridium perfringens toxin, Corynebacterium diphtheriae toxin, diphtheria toxin, Shiga toxin of Escherichia coli, heat-labile toxin of Escherichia coli, heat-stable toxin of Escherichia coli, VacA toxin of Helicobacter pylori, pertussis toxin, exotoxin A of Pseudomonas aeruginosa, Staphylococcus aureus enterotoxin, Streptococcus pyogenes hemolysin O, tetanus toxin, Vibrio cholerae cytolysin, adenovirus E1A protein, Ebola virus VP24 protein, Epstein-Barr virus BHRF1 protein, hepatitis C virus NS5A protein, human immunodeficiency virus (HIV) Vpr protein, human T-cell leukemia virus type 1 (HCI-1). The effector may be derived from bacteria, viruses, archaea, or eukaryotes, and may include at least one of the following: Bax, Bak, caspase, Apaf-1, cytochrome c, Fas receptor, Fas ligand, TNF receptor 1, TNF-associated apoptosis-inducing ligand, p53, Bad, Bid, Bim, Noxa, and CD47. It may also bind to external stimuli (e.g., CAR-T cells targeting the CD47 transmembrane protein) to exert its function. Generally, the effector may consist at least in part of a toxin (e.g., diphtheria toxin) or a toxin receptor (e.g., human or simian diphtheria toxin receptor), using the toxin to mediate the action; said toxin or receptor may be of viral, bacterial, archaea, or eukaryotic origin, and optionally be able to inhibit transcription, translation, cell division, mitosis, respiration, oxidative phosphorylation, glycolysis, promote apoptosis, induce senescence, or generally affect one or more cellular functions required for transgene or gene expression, to completely eliminate said function or ideally permanently reduce said function, but optionally temporarily reduce said function.In other embodiments of the invention, the effector element may be a nuclease, with or without targeting, capable of inactivating the therapeutic construct by, for example, dissociating the promoter from the therapeutic transgene, thereby preventing the expression of the therapeutic transgene. Thus, the effector element may consist at least in part of: at least a portion of a nuclease, a nuclease capable of site-specific mutagenesis, at least a portion of the nuclease and the targeting element, a CRISPR-associated protein (Cas) nuclease having or not having a single guide RNA that targets the nuclease to at least a portion of the transgene delivered as part of the system, a nuclease targeting the coding region of the therapeutic transgene, a nuclease targeting the post-translational regulatory element region encoding the therapeutic transgene or a sequence following it, or a recombinase (e.g., Cre recombinase), or other bacterial, viral, archaea, or eukaryotic recombinase or related protein. Effector elements may consist of one or more domains or components, or may be multiple elements in themselves, and may consist at least in part of the following: at least a portion of the Krüppel-associated box domain of human gene ZNF10, or generally a zinc finger protein (ZFN) or ZFN domain, or a portion of a cyclized recombinase having an SV40 nuclear localization signal, or a Cre recombinase having an SV40 T antigen or a fragment thereof, or a Flpe recombinase, or a Flpo recombinase, or an SV40 T antigen sequence or a fragment thereof, or a piggyBac transposase, or generally a transposase, an argonaut protein, a DNA methyltransferase, or a protein constituting or interacting with the chromatin remodeling complex, or a protein constituting or interacting with the DNA methylation pathway. The effector element may act directly on a therapeutic transgene delivered as part of a system, or on adjacent, flanking, or distal sequences; or indirectly reduce gene expression, transcription, or translation, or generally destabilize expression, transcription, or translation or mRNA to reduce the dose of a permanently expressed therapeutic agent, or induce the expression of another protein that can directly or indirectly reduce the expression, transcription, or translation of the therapeutic agent.

[0055] In some embodiments of the invention, the effector element functions through epigenetic silencing or chromatin remodeling, and in other embodiments, the effector element functions by cleaving, editing, inactivating, or generating double-strand or single-strand breaks in therapeutic transgenes or other nucleic acids delivered cis-systemically. For the foregoing or other purposes, the effector element may consist at least in part of at least a portion of: a base editor, a leader editor, a nicking enzyme, a nuclease, a recombinase, a FRT, a transposon, a transposase, a retrotransposon, an integrase, a dihydrofolate reductase, or a reverse transcriptase, or generally a protein capable of modifying DNA, chromatin, or RNA.

[0056] In other embodiments of the invention, the effector element may be composed of: a non-coding gene that produces RNA, such as a non-coding RNA that can interfere with gene expression, transcription, or translation to induce permanent epigenetic silencing (e.g., X inactivation) of a therapeutic construct, said non-coding RNA being at least partially complementary to or antisense with, or not at least partially complementary to, a cis-delivered genetic construct, or producing downstream secondary effects that induce apoptosis, genetic silencing, or recombination or genetic material, thereby achieving a final effect of downregulation titration. The effector element may also be composed of: one or more elements, such as proteins and RNA, for example, it may be at least partially composed of: *Streptococcus pyogenes* Cas9. Cas9 (SpCas9), SpCas9-VQR, SpCas9-VRER, SpCas9-NG, SpCas9-xCas9, SpCas9-Sc++, SpCas9-SpG, SpCas9-SpRY, FnCas9, FnCas9-RHA, SaCas9, SaCas9-KKH, StlCas9, NmCas9, GeoCas9, or catalytically inactivated Cas9, Campylobacter jejuni Cas9 orthologs, or a portion of codon-optimized Cas9, or a Cas9 with the D10A mutation. Partially, CRISPR-related proteins or orthologs, or Cas proteins, such as dCas9, Cas12a, AsCas12a, AsCas12a-RR, AsCas12-RVR, FnCas12a, Cas12b, Cas12e, Cas12f, UnlCas12f1, CnCas12f1, Cas12j, Cas13, Cas13a, or their derived orthologs, homologs, or paralogs fused with the Krüppel-associated box (KRAB) or KRAB-MeCP2 (methyl CpG-binding protein 2).

[0057] Effector elements may have stable or unstable domains, activation or inactivation domains, or dimerization or trimerization domains. These domains may or may not respond to external inducing molecules or inducing molecules that are inherent in the body at certain times or events. Thus, effector elements may be at least partially composed of unstable domains or a portion of a protein, thereby enabling effector degradation in the absence of some exogenous or endogenous inducing-based stimulation.

[0058] In other embodiments of the invention, the effector element may consist at least in part of at least a portion of the following: DNA, RNA-expressing DNA, RNA, protein, or combinations thereof, capable of affecting DNA sequences in a sequence-specific manner to induce double-strand breaks, single-strand breaks, methylation, chromatin modification, or general epigenetic silencing. Some examples of said DNA are mobile transposon elements that can move or insert into the nucleic acids of the system in the presence of endogenous or exogenous proteins, thereby reducing the expression of therapeutic transgenes from said system. In some embodiments of the invention, the effector may consist of triplex-forming oligonucleotides with or without stabilizers to inhibit the expression of therapeutic transgenes.

[0059] In other embodiments of the invention, the effector element can, in the presence of an exogenous inducer or by its own action, reduce transcription, induce apoptosis, induce necrosis, induce pyroptosis, induce ferroptosis, induce senescence, reduce gene expression, reduce secretion, or reduce the translation of therapeutic transgenes or, in general, reduce the translation of some genes in the host cell of systemic nucleic acids, and can be at least partially composed of at least a portion of: a toxin, a toxin derivative, a modified toxin, or a homolog, or a paralog, or a combination thereof. The toxin can be at least a portion of bacterial, fungal, plant, or animal toxins, or particularly at least a portion of: Vibrio cholerae toxin, Shiga toxin, Shigella toxin, ricin, saponins, Staphylococcus aureus enterotoxin H, Clostridium perfringens enterotoxin, streptococcal hemolysin O, ribosome-inactivating protein (RIP) enzyme, immunotoxin, Pseudomonas exotoxin A, diphtheria toxin (DT), or a DT fragment coupled with or without an activating, inactivating, nuclear localization, or signal transduction domain. In some specific embodiments of the present invention, the effector element may be composed at least in part of at least a portion of the following: viral thymidine kinase, cytosine deaminase, D-amino acid oxidase, carboxypeptidase G2, purine nucleoside phosphorylase, thymidine phosphorylase, xanthine-guanine phosphoribosyltransferase, nitroreductase, penicillin-G amidase, multidrug activating enzyme, β-lactamase, horseradish peroxidase, β-galactosidase, deoxyribonucleotide kinase, β-glucuronidase, carboxypeptidase A, cytochrome P450, deoxycytidine kinase of bacterial, viral, archaea or eukaryotic origin, or derivatives thereof.

[0060] In other embodiments of the invention, the effector element can induce programmed cell death, including apoptosis, pyroptosis, ferroptosis, or necrosis, in the presence of an exogenous inducer or spontaneously, and can be at least partially composed of gasdermin A, gasdermin B, gasdermin C, gasdermin D, or gasdermin E, their homologs, analogs, or fragments, or generally an oligomerizable molecule, nucleic acid, protein, or peptide capable of forming pores in various cell membranes. Generally, the effector element can be at least partially composed of a DNA-binding peptide, a viral or bacterial DNA-binding protein, or a protein having catalytic activity capable of chemically modifying nucleotides, nucleosides, or nucleic acids.

[0061] One or more effector elements of the system can act on one or more effector sequences of the system, which themselves can encode at least a portion of the effector element. The effector sequences are intended to mediate dose-down titration activity of the effector element, and can do so directly or indirectly, enhancing, modulating, mediating, or amplifying the effector element activity. The effector sequences can be at least partially composed of at least a portion of: a shared Lox sequence or a sequence capable of mediating the action of a Lox sequence; a shared FRT sequence or a sequence capable of mediating the action of an FRT sequence; a shared Rox sequence or a sequence capable of mediating the action of a Rox sequence; or any sequence that can be targeted by a DNA-binding enzyme, restriction enzyme, DNase, nickase, nuclease, or any other effector element, and can specifically be at least partially composed of at least a portion of:

[0062] Or any other sequence showing low homology to the genome of the targeted species for therapeutic or other benefits of the system. In some other embodiments, the effector sequence may consist of at least a portion of a protospacer adjacent motif (PAM) sequence, such as NGG, NGAG, NGCG, NGN, NRN, NGG, YG, NNGRRT, NNNRRT, NNAGAA, NNNNGATT, NNNNCRAA, TTTV, TYCV, TATV, TTTV, TTN, TTCN, TTTN, or CCN, or a variant thereof that is enzyme-recognizable. In some embodiments, the PAM sequence may be optimized and elongated to reduce off-target effects. In some other embodiments, the effector sequence may consist of at least a portion of attB, attR, attL, or attP sites or other restriction enzyme coding sites, or sites that can generally be safely targeted by integrases, recombinases, nucleases, nickases, chromatin complexes, methyltransferases, or methyltransferases to minimize harmful off-target activities. In some specific embodiments of the present invention, the effector sequence may consist at least in part of at least a portion of a genetic sequence that can be targeted for silencing RNA or protein binding or that can itself be silenced; or serve as a target for guide RNA of CRISPR-related proteins; or be derived from a viral or bacterial genome; or be a sequence that forms a secondary structure in the absence or presence of exogenous or endogenous inducing molecules; or be a sequence comprising at least a portion of: an inflammatory responsive promoter or enhancer, a mammalian promoter or enhancer, or a promoter-enhancer or transactivator capable of forming a secondary structure in the presence or absence of inducing molecules.

[0063] Furthermore, this invention discloses a system that may optionally include an actuator-responsive element, said actuator-responsive element being a nucleic acid sequence or protein that interacts with an actuator to enable activation of an effector element or effector sequence; or being at least partially composed of a tetracycline-controlled transcriptional silencer (tTS); or being at least partially composed of a Kruppel-associated box (KRAB) AB domain of a kid-1 transcriptional repressor; or being at least partially composed of a trans-activator controlled by reverse tetracycline; or being at least partially composed of a Tet repressor (TetR) or a reverse Tet repressor (rTe). The virus may be composed of tR-fused viral proteins such as VP16; or it may be composed of tTS and rtTA driven by the same promoter; or it may be composed of tTS and rtTA driven by different promoters; or it may be composed at least partially of transcriptional repressors; or it may be composed at least partially of transcriptional silencers; or it may be composed at least partially of DNA-binding proteins; or it may be composed at least partially of promoter-binding proteins; or it may be composed at least partially of transcription initiation complex-binding proteins; or it may be composed at least partially of proteins responsible for DNA methylation; or it may be composed at least partially of proteins responsible for histone modification; or it may be composed at least partially of DNA-binding proteins; or it may be composed at least partially of DNA-binding proteins; or it may be composed at least partially of histone ... It is at least partially composed of at least a portion of the Tet-ON system; or at least partially composed of at least a portion of the Tet-OFF system; or at least partially composed of at least a portion of the following: AlcR, ArgR, BirA, EthR, HdnoR, HSP70B (HSPA7), HucR, LgnR, MphR(A), PIP, Rex, RheA, ScbR, TraR, TtgR, or their analogues, homologs, or orthologs, or functionally similar protein fragments, proteins, or fusion proteins; or at least partially composed of the following... A small portion of the composition includes: lacUV5, lac, tac, trc, prpB, aceA, aceB, gntP, gntK, CJ10X2, tacM, malE1, git1, BAD, SPL, P4-N14, cspB, aprE, sod, dapA, porB, ilvC, L10, L26, L16, L51, H30, H36, or natural, synthetic or semi-synthetic analogs, homologs, or orthologs, or functionally similar protein fragments, proteins or fusion proteins, or combinations of one or more of the above elements.

[0064] Furthermore, the actuator response element disclosed in this invention may be composed at least in part of at least a portion of the following: ABI1-Gal4DBD, ABA, PVL1-VP16, PHR-p65, CIB1-TetR, or their analogues, homologs, or orthologs, or functionally similar protein fragments, proteins, or fusion proteins; or it may be composed at least in part of at least a portion of the following: transcription activators, enhancers, derepressors, or proteins, RNA, or DNA capable of directly or indirectly enhancing gene expression in a targeted or generally cis-acting manner.

[0065] The components described in this invention may optionally work synergistically to enable the system to down-titrate and thus reduce the expression levels of one or more therapeutic elements, but may also optionally have additional functions, such as transient down-regulation or transient up-regulation of expression, or may affect other cellular elements or pathways, or interact with other cellular sequences, proteins, organelles or functions, wherein at least one of the primary objectives is to alter the level of therapeutic effect, expression, transcription, translation, benefit or side effect of the whole organism, system, organ, tissue or cell affected by the system, or to substantially achieve dose reduction, or dose titration, or dose down-titration, or dose up-titration, or some similar effect or activity.

[0066] Other implementation plans

[0067] 1. A system for permanently reducing gene expression from one or more intracellular therapeutic constructs or multiple constructs, said system comprising at least the following:

[0068] a. A nucleic acid construct containing a therapeutic transgene, delivered in a formulation capable of entering cells and enabling stable expression of the therapeutic transgene for at least 4 months;

[0069] b. An actuator, which is composed of one or more chemical or biological molecules;

[0070] c. Actuator response elements and / or actuator response sequences that enable the expression, activation, or stabilization of effector elements or effector sequences;

[0071] d. An effector sequence, which is delivered in cis along with the therapeutic transgene; and

[0072] e. An effector element expressed by the effector sequence, the effector element being capable of permanently reducing the expression of the therapeutic transgene in a manner that causes minimal bystander effect, and thus being safe for surrounding tissues, organs and the whole organism.

[0073] 2. The system described in Implementation Scheme 1, which is intended for delivery to subcutaneous tissue.

[0074] 3. The system described in Implementation Scheme 1, which is intended for intraperitoneal delivery.

[0075] 4. The system described in Implementation Scheme 1, which is intended for delivery to adipocytes.

[0076] 5. The system described in Implementation Scheme 1, which is intended for intramuscular delivery.

[0077] 6. The system described in Implementation Scheme 1, which is intended for delivery to the liver.

[0078] 7. The system described in Implementation Scheme 1, which is intended for delivery to the central nervous system.

[0079] 8. The system described in Implementation Scheme 1, which is intended for delivery to the fascia.

[0080] 9. The system described in Implementation Scheme 1, which is intended for delivery into the subarachnoid space.

[0081] 10. The system described in Implementation 1, which is intended for delivery to the pancreas.

[0082] 11. The system described in Implementation 1, which is intended for delivery to the epidermis.

[0083] 12. The system described in Implementation 1, which is intended for transocular delivery.

[0084] 13. The system described in Implementation 1, which is intended for delivery to the kidney.

[0085] 14. The system described in Implementation 1, which is intended for delivery to the bladder.

[0086] 15. The system described in Implementation Scheme 1, which is intended for delivery to mucosa.

[0087] 16. The system described in Implementation 1, which is intended for delivery to the lungs.

[0088] 17. The system described in Implementation 1, which is intended for delivery to the spleen.

[0089] 18. The system described in Implementation Scheme 1, which is intended for intravascular delivery.

[0090] 19. The system described in Implementation 1, which is intended for delivery to the heart.

[0091] 20. The system described in Implementation Scheme 1, which is intended to be used in conjunction with therapeutic gene therapy or delivered in conjunction with therapeutic transgenes for the treatment of human, plant or veterinary diseases or for the enhancement of human, veterinary or plant functions.

[0092] 21. The therapeutic transgene described in Implementation Scheme 1, wherein it encodes at least a portion of a therapeutic antibody.

[0093] 22. The therapeutic transgenic material described in Implementation Scheme 1, which encodes a protein or enzyme that replaces the protein or enzyme required for treatment.

[0094] 23. The therapeutic transgenic material described in Implementation Scheme 1, which encodes a therapeutic peptide.

[0095] 24. The therapeutic transgenic protein or fusion protein described in Implementation Scheme 1.

[0096] 25. The therapeutic transgene described in Implementation Scheme 1 encodes non-coding RNA.

[0097] 26. The therapeutic transgene described in Implementation Scheme 1 encodes an aptamer enzyme, aptamer, ribozyme, or a genetic structure capable of exerting enzymatic activity alone or in combination with a protein.

[0098] 27. The nucleic acid construct described in Implementation Scheme 1 is composed of double-stranded DNA.

[0099] 28. The nucleic acid construct described in Implementation Scheme 1 is composed of single-stranded DNA.

[0100] 29. The nucleic acid construct described in Implementation Scheme 1 is composed of single-stranded RNA.

[0101] 30. The nucleic acid construct described in Implementation Scheme 1 is composed of double-stranded RNA.

[0102] 31. The nucleic acid construct described in Implementation Scheme 1 is composed of single-stranded or double-stranded linear DNA.

[0103] 32. The nucleic acid construct described in Implementation Scheme 1 is composed of single-stranded or double-stranded linear RNA.

[0104] 33. The nucleic acid construct described in Implementation Scheme 1 is composed of single-stranded or double-stranded circular DNA.

[0105] 34. The nucleic acid construct described in Implementation Scheme 1 is composed of single-stranded or double-stranded circular RNA.

[0106] 35. The system described in Implementation Scheme 1, comprising a protein delivered together with the nucleic acid.

[0107] 36. The system described in Implementation Scheme 1, which is delivered in vivo or ex vivo as part of a viral or non-viral agent.

[0108] 37. The system described in Implementation 1, which is delivered by physical methods, such as electroporation, nanoparticle bombardment, ultrasound-mediated transfection, or chemically mediated transfection.

[0109] 38. The actuator described in Implementation Scheme 1, wherein at least a portion is composed of tetracycline or a tetracycline derivative.

[0110] 39. The actuator described in Implementation Scheme 1, wherein at least a portion is composed of doxycycline or a doxycycline derivative.

[0111] 40. The actuator described in Implementation Scheme 1, which is at least partially composed of an antibiotic or an antimicrobial peptide.

[0112] 41. The actuator described in embodiment 1, wherein at least a portion is composed of rapamycin or a derivative thereof.

[0113] 42. The actuator described in embodiment 1, wherein at least a portion is composed of ganciclovir or a derivative thereof.

[0114] 43. The actuator described in embodiment 1, comprising at least a portion of ganciclovir, valganciclovir, valacyclovir, or derivatives thereof.

[0115] 44. The actuator described in embodiment 1, which is at least partially composed of tamoxifen or a derivative thereof.

[0116] 45. The actuator described in Implementation Scheme 1, wherein at least a portion is composed of estrogen or a derivative thereof.

[0117] 46. ​​The actuator described in Implementation Scheme 1, wherein at least a portion is composed of acetaldehyde or a derivative thereof.

[0118] 47. The actuator described in embodiment 1, wherein at least a portion is composed of L-arginine or a derivative thereof.

[0119] 48. The actuator described in embodiment 1, which is at least partially composed of biotinylated AMP or a derivative thereof.

[0120] 49. The actuator described in embodiment 1, wherein at least a portion is composed of 2-phenylethyl butyrate or a derivative thereof.

[0121] 50. The actuator described in embodiment 1, wherein at least a portion is composed of 6-hydroxynicotin or a derivative thereof.

[0122] 51. The actuator described in embodiment 1, wherein at least a portion is composed of erythromycin or a derivative thereof.

[0123] 52. The actuator described in embodiment 1, wherein at least a portion is composed of a macrolide or a derivative thereof.

[0124] 53. The actuator described in embodiment 1, wherein at least a portion is composed of streptomycin or a derivative thereof.

[0125] 54. The actuator described in embodiment 1, which is at least partially composed of NADH or a derivative thereof.

[0126] 55. The actuator described in embodiment 1, which is at least partially composed of elements that cause the generation of increased energy or heat.

[0127] 56. The actuator described in embodiment 1, which is at least partially composed of SCB1 or a derivative thereof.

[0128] 57. The actuator described in embodiment 1, wherein at least a portion is composed of 3-oxo-C8-HSL or a derivative thereof.

[0129] 58. The actuator described in embodiment 1, which is at least partially composed of phloretin or a derivative thereof.

[0130] 59. The actuator described in Implementation Scheme 1 is a small molecule, peptide, protein, or nucleic acid capable of enhancing, reducing, or modulating local or systemic inflammation or a partial inflammatory response.

[0131] 60. The actuator described in Implementation Scheme 1, wherein at least a portion is composed of theophylline or a derivative thereof.

[0132] 61. The actuator described in embodiment 1, which is at least partially composed of IPTG or a derivative thereof.

[0133] 62. The actuator described in embodiment 1, wherein at least a portion is composed of 5-fluorocytosine or a derivative thereof.

[0134] 63. The actuator described in embodiment 1, wherein at least a portion is composed of a propionate ester or a derivative thereof.

[0135] 64. The actuator described in embodiment 1, wherein at least a portion is composed of an acetate or a derivative thereof.

[0136] 65. The actuator described in embodiment 1, wherein at least a portion is composed of cumyl acid salt, cumyl acid ester, or cumic acid or its derivatives.

[0137] 66. The actuator described in embodiment 1, wherein at least a portion is composed of gluconate or a derivative thereof.

[0138] 67. The actuator described in embodiment 1, wherein at least a portion is composed of maltose or a derivative thereof.

[0139] 68. The actuator described in embodiment 1, which is at least partially composed of arabinose or a derivative thereof.

[0140] 69. The actuator described in Implementation Scheme 1, which is at least partially composed of monosaccharides or disaccharides.

[0141] 70. The actuator described in embodiment 1, wherein at least a portion is composed of trimethoprim or a derivative thereof.

[0142] 71. The actuator described in embodiment 1, which is at least partially composed of an exogenously applied small molecule, peptide, protein, RNA, DNA or pharmacological agent.

[0143] 72. The actuator described in embodiment 1, at least a portion of which may serve as at least a portion of the effector element described in embodiment 1.

[0144] 73. The actuator described in any embodiment is at least partially composed of endogenously expressed or presented peptides, hormones, proteins, RNA, DNA, enzymes, minerals, or chemical or biochemical substances, and is capable of interacting with said actuator response element or actuator response sequence.

[0145] 74. The effector element described in Implementation Scheme 1, which is at least partially composed of inducible cysteine ​​9.

[0146] 75. The effector element described in embodiment 1, which is at least partially composed of natural caspase 9.

[0147] 76. The effector element described in Implementation Scheme 1, which is at least partially composed of zinc finger nucleases.

[0148] 77. The effector element described in Implementation Scheme 1, which is at least partially composed of a transcription activator-like effector nuclease (TALEN).

[0149] 78. The effector element described in Implementation Scheme 1, which is at least partially composed of meganuclease.

[0150] 79. The effector element described in Implementation Scheme 1, which is at least partially composed of an editor derived from deoxycytidine deamination.

[0151] 80. The effector element described in Implementation Scheme 1, which is at least partially composed of a base editor derived from deoxyadenosine deamination.

[0152] 81. The effector element described in Implementation Scheme 1, which is at least partially composed of a DNA-binding peptide.

[0153] 82. The effector element described in Implementation Scheme 1, which is at least partially composed of viral or bacterial DNA-binding proteins.

[0154] 83. The effector element described in embodiment 1, which is at least partially composed of natural caspase 8.

[0155] 84. The effector element described in Implementation Scheme 1 is at least partially composed of natural caspase or caspase pathway proteins.

[0156] 85. The effector element described in Embodiment 1 is FK506 binding protein 12 (with an F36V mutation), which is fused to a human caspase-8 protein lacking 1-215 AA and linked to an N-myristylation signal from Src kinase.

[0157] 86. The effector element described in Implementation Scheme 1, wherein a portion thereof is composed of FK506 binding protein 12 (with an F36V mutation) fused to human caspase-9 protein lacking 1-134 AA.

[0158] 87. The effector element described in embodiment 1, which is at least partially composed of at least a portion of viral thymidine kinase.

[0159] 88. The effector element described in Implementation Scheme 1, which is at least partially composed of at least a portion of Escherichia coli cytosine deaminase.

[0160] 89. The effector element described in Implementation Scheme 1 is a protein having catalytic activity capable of chemically modifying nucleotides, nucleosides, or nucleic acids.

[0161] 90. The effector element described in embodiment 1, which is at least partially composed of at least a portion of diphtheria toxin subunit A.

[0162] 91. The effector element described in embodiment 1, which is at least partially composed of at least a portion of diphtheria toxin subunit B.

[0163] 92. The effector element described in Implementation Scheme 1 is a dimerizable or trimerizable toxin, protein, or enzyme.

[0164] 93. The effector element described in Implementation Scheme 1, which is at least partially composed of at least a portion of a human or simian diphtheria toxin receptor.

[0165] 94. The effector element described in embodiment 1 is at least partially composed of at least one of the following: bacteria, viruses, or eukaryotic toxins.

[0166] 95. The effector element described in Embodiment 1, which is at least partially composed of at least a portion of a protein capable of inhibiting transcription.

[0167] 96. The effector element described in embodiment 1, which is at least partially composed of at least a portion of a protein capable of inhibiting translation.

[0168] 97. The effector element described in embodiment 1, which is at least partially composed of at least a portion of a protein capable of inducing apoptosis.

[0169] 98. The effector element described in Implementation Scheme 1, which is at least partially composed of at least a portion of a protein capable of inducing aging.

[0170] 99. The effector element described in Implementation Scheme 1, which is at least partially composed of at least a portion of a protein capable of reducing general gene expression.

[0171] 100. The effector element described in Implementation Scheme 1, which is at least partially composed of at least a portion of a nuclease.

[0172] 101. The effector element described in Implementation Scheme 1, which is at least partially composed of at least a portion of a nuclease capable of site-specific mutagenesis.

[0173] 102. The effector element described in Implementation Scheme 1, which is at least partially composed of at least a portion of a nuclease and at least a portion of a target element.

[0174] 103. The effector element described in Embodiment 1, comprising at least a portion of: a Cas nuclease and a guide RNA, the guide RNA targeting the nuclease to the sequence described in Embodiment 1.

[0175] 104. The effector element described in Embodiment 1, which is at least partially composed of at least a portion of a nuclease targeting the sequence described in Embodiment 1.

[0176] 105. The effector element described in Embodiment 1, which is at least partially composed of at least a portion of a nuclease that targets the promoter region of the sequence described in Embodiment 1.

[0177] 106. The effector element described in Embodiment 1, which is at least partially composed of at least a portion of a nuclease that targets the coding region of the sequence described in Embodiment 1.

[0178] 107. The effector element described in Embodiment 1, which is at least partially composed of at least a portion of a nuclease targeting the post-translational regulatory element region of the sequence described in Embodiment 1.

[0179] 108. The effector element described in Implementation Scheme 1, which is at least partially composed of at least a portion of the recombinase.

[0180] 109. The effector element described in Implementation Scheme 1, which is at least partially composed of at least a portion of Cre recombinase.

[0181] 110. The effector element described in embodiment 1, which is at least partially composed of at least a portion of the Krüppel-associated box domain of the human gene ZNF10.

[0182] 111. The effector element described in Implementation Scheme 1, which is at least partially composed of at least a portion of zinc finger proteins.

[0183] 112. The effector element described in Implementation Scheme 1, which is at least partially composed of at least a portion of a cyclized recombinase having an SV40 nuclear localization signal.

[0184] 113. The effector element described in Embodiment 1, which is at least partially composed of at least a portion of a Cre recombinase having an SV40 T antigen intron inserted into the ORF.

[0185] 114. The effector element described in embodiment 1, which is at least partially composed of at least a portion of the Flpe recombinase.

[0186] 115. The effector element described in embodiment 1, which is at least partially composed of at least a portion of the Flpo recombinase.

[0187] 116. The effector element described in embodiment 1, which is at least partially composed of at least a portion of the Dre recombinase.

[0188] 117. The effector element described in Embodiment 1, which is at least partially composed of at least a portion of the SV40 T antigen sequence.

[0189] 118. The effector element described in embodiment 1, which is at least partially composed of at least a portion of the piggyBac transposase.

[0190] 119. The effector element described in Implementation Scheme 1, which is at least partially composed of at least a portion of a transposase.

[0191] 120. The effector element described in Implementation Scheme 1, which is at least partially composed of at least a portion of Sleeping Beauty transposase.

[0192] 121. The effector element described in embodiment 1, which is at least partially composed of at least a portion of the argonaut protein.

[0193] 122. The effector element described in embodiment 1, which is at least partially composed of at least a portion of a DNA methyltransferase.

[0194] 123. The effector element described in Embodiment 1, which is at least partially composed of at least a portion of a protein constituting or interacting with the chromatin remodeling complex.

[0195] 124. The effector element described in Embodiment 1, which is at least partially composed of at least a portion of a protein constituting or interacting with a DNA methylation pathway.

[0196] 125. The effector element described in embodiment 1, which is at least partially composed of at least a portion of a base editor.

[0197] 126. The effector element described in embodiment 1, which is at least partially composed of at least a portion of the lead editor.

[0198] 127. The effector element described in embodiment 1, which is at least partially composed of at least a portion of the nicking enzyme.

[0199] 128. The effector element described in Implementation Scheme 1, which is at least partially composed of at least a portion of the following: a virus, bacteria, or a eukaryotic gene silencing protein.

[0200] 129. The effector element described in Implementation Scheme 1, which is at least partially composed of at least a portion of siRNA.

[0201] 130. The effector element described in Implementation Scheme 1, which is at least partially composed of at least a portion of miRNA.

[0202] 131. The effector element described in embodiment 1, which is at least partially composed of at least a portion of the FRT.

[0203] 132. The effector element described in embodiment 1, which is at least partially composed of at least a portion of an integrase.

[0204] 133. The effector element described in embodiment 1, which is at least partially composed of at least a portion of RNA capable of reducing gene expression.

[0205] 134. The effector element described in embodiment 1, which is at least partially composed of at least a portion of a protein capable of causing the effector to degrade in the absence of an inducer.

[0206] 135. The effector element described in Implementation Scheme 1, which is at least partially composed of at least a portion of dihydrofolate reductase.

[0207] 136. The effector element described in Embodiment 1, which is at least partially composed of at least a portion of RNA, said at least a portion of said RNA being at least partially complementary to the genetic construct of Embodiment 1.

[0208] 137. The effector element described in embodiment 1, which is at least partially composed of at least a portion of the following: a transposon or a retrotransposon.

[0209] 138. The effector element described in embodiment 1, which is at least partially composed of at least a portion of Streptococcus pyogenes Cas9.

[0210] 139. The effector element described in embodiment 1, which is at least partially composed of at least a portion of Cas9 without catalytic activity.

[0211] 140. The effector element described in embodiment 1, which is at least partially composed of at least a portion of a codon-optimized Cas9.

[0212] 141. The effector element described in embodiment 1, which is at least partially composed of at least a portion of Cas9 having a D10A mutation.

[0213] 142. The effector element described in embodiment 1, which is at least partially composed of at least a portion of dCas9 fused with KRAB.

[0214] 143. The effector element described in embodiment 1, which is at least partially composed of at least a portion of dCas9 fused with a carboxyl-terminal dichotomous repressor domain KRAB-MeCP2 or a derivative thereof.

[0215] 144. The effector element described in Implementation Scheme 1, which is at least partially composed of at least a portion of Campylobacter jejuni Cas9 orthologs.

[0216] 145. The effector element described in Embodiment 1, which is at least partially composed of at least a portion of the following: a CRISPR-related protein or an ortholog.

[0217] 146. The effector element described in Embodiment 1, which is at least partially composed of at least a portion of the following: Cas12a or Cas12b protein.

[0218] 147. The effector element described in embodiment 1, which is at least partially composed of at least a portion of Cas13.

[0219] 148. The effector element described in embodiment 1, which is at least partially composed of at least a portion of the following: Cas12f or Cas13a.

[0220] 149. The effector element described in Embodiment 1, comprising at least a portion of the following: DNA, DNA capable of expressing RNA, RNA, protein, or a combination thereof, capable of affecting a DNA sequence in a sequence-specific manner to induce double-strand breaks, single-strand breaks, methylation, chromatin modification, or general epigenetic silencing.

[0221] 150. The effector element described in embodiment 1 is composed of at least a portion of the following: a toxin, a toxin derivative, a modified toxin or its homolog, direct homolog, or paralog.

[0222] 151. The effector element described in embodiment 1, which is at least partially composed of at least a portion of: bacteria, fungi, plant or animal toxins.

[0223] 152. The effector element described in Implementation Scheme 1, comprising at least a portion of the following: Vibrio cholerae toxin, Shiga toxin, Shigella toxin, ricin, saponin, Staphylococcus aureus enterotoxin H, Clostridium perfringens enterotoxin, streptococcal hemolysin O, ribosome inactivating protein (RIP) enzyme, immunotoxin, Pseudomonas exotoxin A, diphtheria toxin (DT) or DT fragment.

[0224] 153. The effector element described in embodiment 1, which is at least partially composed of at least a portion of: viral thymidine kinase or a derivative thereof.

[0225] 154. The effector element described in embodiment 1, which is at least partially composed of at least a portion of: bacterial cytosine deaminase or a derivative thereof.

[0226] 155. The effector element described in embodiment 1, which is at least partially composed of at least a portion of: D-amino acid oxidase or a derivative thereof.

[0227] 156. The effector element described in embodiment 1, which is at least partially composed of at least a portion of: bacterial carboxypeptidase G2 or a derivative thereof.

[0228] 157. The effector element described in embodiment 1, which is at least partially composed of at least a portion of: purine nucleoside phosphorylase or a derivative thereof.

[0229] 158. The effector element described in embodiment 1, which is at least partially composed of at least a portion of thymidine phosphorylase or a derivative thereof.

[0230] 159. The effector element described in Embodiment 1, which is at least partially composed of at least a portion of: xanthine-guanine phosphoribosyltransferase or a derivative thereof.

[0231] 160. The effector element described in embodiment 1, which is at least partially composed of at least a portion of: nitroreductase or a derivative thereof.

[0232] 161. The effector element described in embodiment 1, which is at least partially composed of at least a portion of: penicillin-G amidase or a derivative thereof.

[0233] 162. The effector element described in embodiment 1, which is at least partially composed of at least a portion of: a multidrug activating enzyme or a derivative thereof.

[0234] 163. The effector element described in embodiment 1, which is at least partially composed of at least a portion of horseradish peroxidase or a derivative thereof.

[0235] 164. The effector element described in embodiment 1, which is at least partially composed of at least a portion of β-galactosidase or a derivative thereof.

[0236] 165. The effector element described in embodiment 1, which is at least partially composed of at least a portion of: deoxyribonucleokinase or a derivative thereof.

[0237] 166. The effector element described in embodiment 1, which is at least partially composed of at least a portion of: β-glucuronidase or a derivative thereof.

[0238] 167. The effector element described in embodiment 1, which is at least partially composed of at least a portion of: carboxypeptidase A or a derivative thereof.

[0239] 168. The effector element described in embodiment 1, which is at least partially composed of at least a portion of cytochrome P450 or a derivative thereof.

[0240] 169. The effector element described in Embodiment 1, which is at least partially composed of at least a portion of: deoxycytidine kinase or a derivative thereof.

[0241] 170. The effector sequence described in Implementation Scheme 1, which at least encodes the effector element.

[0242] 171. The effector sequence described in Implementation Scheme 1, which encodes at least a portion of the following: a shared Lox sequence or a sequence capable of mediating the action of a Lox sequence.

[0243] 172. The effector sequence described in Implementation Scheme 1, which encodes at least a portion of the following: a common FRT sequence or a sequence capable of mediating the action of an FRT sequence.

[0244] 173. The effector sequence described in Implementation Scheme 1, which encodes at least a portion of the following: a shared Rox sequence or a sequence capable of mediating the action of a Rox sequence.

[0245] 174. The effector sequence described in Implementation Scheme 1, which is at least partially composed of constitute.

[0246] 175. The effector sequence described in Implementation Scheme 1, which is at least partially composed of constitute.

[0247] 176. The effector sequence described in Implementation Scheme 1, which is at least partially composed of constitute.

[0248] 177. The effector sequence described in Implementation Scheme 1, which is at least partially composed of constitute.

[0249] 178. The effector sequence described in Implementation Scheme 1, which is at least partially composed of constitute.

[0250] 179. The effector sequence described in Implementation Scheme 1, which is at least partially composed of constitute.

[0251] 180. The effector sequence described in Implementation Scheme 1, which is at least partially composed of constitute.

[0252] 181. The effector sequence described in Implementation Scheme 1, which encodes at least a portion of the following: attB, attR, attL, or attP sites.

[0253] 182. The effector sequence described in Implementation Scheme 1, which is at least partially composed of a genetic sequence that can be targeted by an integrase, recombinase, nuclease, nickase, chromatin complex, or methyltransferase or methyltransferase.

[0254] 183. The effector sequence described in Implementation Scheme 1, which consists at least in part of a genetic sequence that can be targeted to silence RNA or protein binding or that can be silenced itself.

[0255] 184. The effector sequence described in Implementation Scheme 1 is a DNA sequence that can be used as a target of the guide RNA for CRISPR-related proteins.

[0256] 185. The effector sequence described in Implementation Scheme 1 is a DNA sequence derived from the genome of a virus or bacteria.

[0257] 186. The effector sequence described in Implementation Scheme 1, which forms a secondary structure in the absence of an inducing molecule.

[0258] 187. The effector sequence described in Implementation Scheme 1 is an inflammatory responsive promoter.

[0259] 188. The effector sequence described in Implementation Scheme 1 is at least partially derived from a mammalian promoter or enhancer.

[0260] 189. The effector sequence described in Implementation Scheme 1, which forms a secondary structure in the presence of an inducing molecule.

[0261] 190. The actuator response element described in embodiment 1 is a nucleic acid sequence or protein that interacts with the actuator to enable activation of the effector element or effector sequence.

[0262] 191. The actuator response element described in embodiment 1, which is at least partially composed of a tetracycline-controlled transcriptional silencer (tTS).

[0263] 192. The actuator response element described in embodiment 1 is at least partially composed of the Kruppel-associated box (KRAB) AB domain of the kid-1 transcriptional repressor.

[0264] 193. The actuator response element described in embodiment 1 is at least partially composed of an inverse tetracycline-controlled trans activator.

[0265] 194. The actuator response element described in embodiment 1 is at least partially composed of a viral protein, such as VP16, fused with a Tet repressor (TetR) or a reverse Tet repressor (rTetR).

[0266] 195. The actuator response element described in Implementation Scheme 1, which consists of tTS and rtTA driven by the same promoter.

[0267] 196. The actuator response element described in Implementation Scheme 1 is composed of tTS and rtTA driven by different promoters.

[0268] 197. The actuator response element described in embodiment 1 is at least partially composed of a transcriptional repressor.

[0269] 198. The actuator response element described in Implementation Scheme 1, which is at least partially composed of a transcriptional silencer.

[0270] 199. The actuator response element described in embodiment 1 is at least partially composed of a DNA-binding protein.

[0271] 200. The actuator response element described in Implementation Scheme 1 is at least partially composed of a promoter-binding protein.

[0272] 201. The actuator response element described in Implementation Scheme 1 is at least partially composed of a transcription initiation complex binding protein.

[0273] 202. The actuator response element described in Implementation Scheme 1 is at least partially composed of a protein responsible for DNA methylation.

[0274] 203. The actuator response element described in Implementation Scheme 1 is at least partially composed of a protein responsible for histone modification.

[0275] 204. The actuator response element described in embodiment 1 is at least partially constituted by at least a portion of the Tet-ON system.

[0276] 205. The actuator response element described in embodiment 1, which is at least partially constituted by at least a portion of the Tet-OFF system.

[0277] 206. The actuator response element described in embodiment 1 is at least partially composed of at least a portion of the following: AlcR, ArgR, BirA, EthR, HdnoR, HucR, MphR(A), PIP, Rex, RheA, ScbR, TraR, TtgR, or analogues, homologs, or orthologs, or functionally similar protein fragments, proteins, or fusion proteins.

[0278] 207. The actuator response element described in embodiment 1 is at least partially composed of at least a portion of the following: lacUV5, lac, tac, trc, prpB, aceA, aceB, gntP, gntK, CJ10X2, tacM, malE1, git1, BAD, SPL, P4-N14, cspB, aprE, sod, dapA, porB, ilvC, L10, L26, L16, L51, H30, H36, or natural, synthetic or semi-synthetic analogs, homologs, or orthologs, or functionally similar protein fragments, proteins or fusion proteins.

[0279] 208. The actuator response element described in embodiment 1 is composed of at least a portion of the following: ABI1-Gal4DBD, ABA, PVL1-VP16, PHR-p65, CIB1-TetR, or their analogues, homologs, or orthologs, or functionally similar protein fragments, proteins, or fusion proteins.

[0280] 209. The actuator response element described in Embodiment 1 is composed at least in part of at least a portion of: a transcriptional activator, an enhancer, a derepressor, or a protein, RNA, or DNA capable of directly or indirectly enhancing gene expression in a targeted or generally cis-acting manner.

[0281] It should be understood that, within the scope of this invention, those skilled in the art can modify formulations, materials, genetic constructs, sequences, biological and chemical components, or methods of use to the extent that the structures described herein perform the desired functions and remain within the scope of this invention. Those skilled in the art can combine multiple components, elements, or features, with or without modification, to achieve the desired function of the aforementioned formulations.

[0282] Furthermore, all individual features and methods of use described herein, as well as various combinations and each combination of two or more such features and methods of use, are included within the scope of this invention, provided that such features and methods of use are not contradictory in such combinations. It should be understood that those trained in the art can modify certain parts or combinations of such parts while still achieving the main objectives of the invention.

[0283] Finally, it should be understood that the specific scope provided in this invention is not limiting and is merely for illustrative purposes. Values ​​outside the specific scope may be used to achieve the objectives of this invention without modifying the proposed mechanical principles.

[0284] The wording and terminology used in this application are for descriptive purposes and should not be considered restrictive. Terms such as “comprising,” “including,” “having,” “containing,” “involving,” and / or variations thereof, as used in this application, are intended to cover the items listed thereafter and their equivalents, as well as additional items.

[0285] In embodiments of any of the compositions and methods provided herein, "comprising" may be replaced by "consistently consisting of" or "consistent with". The phrase "consistently consisting of" is used herein to claim a specified integer or step, and those that do not materially affect the features or function of the claimed invention. The term "consistent with" as used herein is used to refer to a group of enumerated wholes (e.g., features, elements, characteristics, properties, methods / processing steps, or limitations) or wholes (e.g., features, elements, characteristics, properties, methods / processing steps, or limitations) that are present only.

[0286] The invention is further illustrated by the following examples, which should not be construed as further limitations. The entire contents of all references cited throughout this application (including bibliographic references, granted patents, published patent applications, and co-pending patent applications) are expressly incorporated herein by reference.

[0287] Example

[0288] To provide a fuller understanding of the invention described in this application, the following embodiments are illustrated. The embodiments described in this application are provided to illustrate the systems and methods provided in this disclosure and should not be construed as limiting their scope in any way.

[0289] Example 1

[0290] In the first embodiment, a therapeutic genetic construct with dose-regulating capabilities was created by placing the furin-cleavable insulin gene under the regulation of the EFS promoter to drive basal insulin expression. To enable dose reduction, a tetracycline-inducible promoter and a tetracycline-responsive transcription activator or TetOn operator were added to drive the expression of CRISPR Cas9 protein and sgRNA in the presence of a tetracycline actuator. The sgRNA targets the Cas9 protein to cleave the target sequence that dissociates the EFS promoter from furin-cleavable insulin and eliminates the expression of the furin-cleavable insulin therapeutic transgene. Upon activation by exogenously applied actuator tetracycline, the construct self-inactivates and thus reduces the overall level of therapeutic transgene expression. Figure 1 The plasmid map of the genetic construct described in this embodiment is shown. Actuator: Tetracycline, atomic formula C22H24N2O8, CAS number: 60-54-8 or its equivalent therapeutic agent / USP equivalent CAS RN 64-75-5-. Tetracycline hydrochloride, molecular formula: C22H24N2O8.ClH.

[0291] Nanoparticle framework

[0292]

[0293] Complete sequence (excluding the nanoparticle backbone)

[0294]

[0295]

[0296] Effector elements: Cas9 and sgRNA

[0297] Cas9

[0298] Nucleotide sequence:

[0299]

[0300] amino acid sequence:

[0301]

[0302]

[0303] sgRNA scaffold

[0304]

[0305] U6 Starter

[0306]

[0307] Effector sequences: Cas9 targets corresponding to the above sgRNAs

[0308]

[0309] Proximity Affected Motion (PAM) Sequence

[0310] NGG (N – any nucleotide)

[0311] Actuator response element: Tet repressor / Gen 1 TetOn reverse tetracycline-responsive transcriptional activator M2 (rtTA)

[0312] Nucleotide sequence:

[0313]

[0314] amino acid sequence:

[0315]

[0316] Tetracycline transcription silencer (tTS):

[0317] Nucleotide sequence:

[0318]

[0319] amino acid sequence:

[0320]

[0321] Therapeutic Genetically Modified Insulin: Frin Protease-Cleavageable Human Insulin

[0322] Nucleotide sequence:

[0323]

[0324] amino acid sequence:

[0325]

[0326] Auxiliary sequence:

[0327] Tetracycline-Responsive Element Promoter (TRE Promoter)

[0328]

[0329]

[0330] SV40 late pA

[0331]

[0332] Kozak

[0333]

[0334] Ribosomal jumping 2A peptide from the Thosea Asigna Virus

[0335] Nucleotide sequence:

[0336]

[0337] amino acid sequence:

[0338]

[0339] P2A

[0340] Nucleotide sequence:

[0341]

[0342] amino acid sequence:

[0343]

[0344] EFS promoter

[0345]

[0346] Example 2

[0347] In this embodiment, a doxycycline-inducible self-inactivation construct was delivered cis-together with a therapeutic transgene encoding a secretible anti-TNF-α nanobody (Ozoralizumab). To reduce the expression level of the anti-TNF-α nanobody, the cis-delivered Campylobacter jejuni CRISPR-Cas9 protein was inducibly expressed under the regulation of a third-generation tetracycline repressor / operon (rtTA / TRE3g). Following doxycycline delivery, Cas9 and sgRNA were expressed from the construct and targeted sequences on the construct to segregate the promoter from the therapeutic transgene. Figure 2 The plasmid map of the genetic construct described in this embodiment is shown. The sequence and construct are shown below:

[0348] Complete sequence (excluding the nanoparticle backbone)

[0349]

[0350] Actuator: Doxycycline or doxycycline hydrochloride, CAS RN: 24390-14-5, molecular formula C12H24N2O8.1 / 2C2H6O.ClH.1 / 22H2O.

[0351]

[0352] CBh promoter

[0353]

[0354] Orbital monoclonal antibody

[0355] nucleotide sequence

[0356]

[0357] amino acid sequence

[0358]

[0359] furin cleavage site

[0360] nucleotide sequence

[0361]

[0362] amino acid sequence

[0363]

[0364] P2A

[0365] nucleotide sequence

[0366]

[0367] amino acid sequence

[0368]

[0369] Effector element: Cas9

[0370]

[0371] amino acid sequence

[0372]

[0373] Effector sequence: target of cjCas9

[0374]

[0375] PAM

[0376] NNNNACAC (N – any nucleotide)

[0377] U6 Starter

[0378]

[0379] CjCas9 gRNA scaffold

[0380]

[0381] Actuator response element: Tet inhibitor / Gen 3 TetOn, third-generation rtTA / TRE3G

[0382] nucleotide sequence

[0383]

[0384] amino acid sequence

[0385]

[0386] TRE3G Starter

[0387]

[0388] bGH poly(A) sequence

[0389]

[0390] SV40 poly(A) sequence

[0391]

[0392] The activity of a suicide gene-based downregulation titration mechanism was qualified using nLuc bioluminescent reporters. Figures 3A to 3B The expression of the reporter transgene is permanently reduced to approximately 1 / 2 × 1 / 3 × 10⁻⁶ by the induction of a self-silencing signal activated by the delivery of the operon. Figures 3A to 3B "Suicide gene-strong promoter" and "suicide gene-weak promoter"), and are the functions of promoters that drive the expression of suicide genes. When a strong promoter is used to drive reporter transgenes and activated suicide genes, the strong promoter produces a strong initial signal ( Figure 3A "Suicide gene - strong promoter" and more pronounced downregulation titration ( Figure 3B "Suicide gene - strong promoter"). Conversely, low-strength promoters produce lower expression signals ( Figure 3A "Suicide gene - weak promoter" and lower levels of downregulation titration after operon delivery ( Figure 3B "Suicide gene - weak promoter").

[0393] Example 3

[0394] In this embodiment, a secretible α-glucosidase is delivered under the regulation of a CMV promoter as an enzyme replacement therapy for Pompe disease. To ensure that the enzyme level does not exceed the therapeutic limit, the genetic construct is delivered in cis along with a toxic subunit, which destabilizes and inhibits elongation factor 2 (EF2), thereby preventing elongation and inhibiting protein synthesis. Figure 4 The plasmid map of the genetic construct described in this embodiment is shown.

[0395] Complete sequence (excluding the nanoparticle backbone)

[0396]

[0397]

[0398] CMV promoter

[0399]

[0400] secGAA (sp7-δ8-hGAA)

[0401] nucleotide sequence

[0402]

[0403] amino acid sequence

[0404]

[0405] P2A

[0406] nucleotide sequence

[0407]

[0408] amino acid sequence

[0409]

[0410] Tet-on 3G

[0411] nucleotide sequence

[0412]

[0413] amino acid sequence

[0414]

[0415] TRE3G Starter

[0416]

[0417] Diphtheria toxin subunit A (DTA):

[0418] nucleotide sequence

[0419]

[0420] amino acid sequence

[0421]

[0422] K19 aptamerase

[0423]

[0424] Insulator sequence

[0425]

[0426] bGH poly(A) sequence

[0427]

[0428] SV40 poly(A) sequence

[0429]

[0430] Example 4

[0431] In this embodiment, the GLP-1 receptor antagonist analog exenatide is delivered under the regulation of the CBh promoter. The cis-construct of the exenatide sequence cDNA contains a K19 aptamer and an unstable Cre recombinase, the exenatide sequence cDNA being coupled to a secreted peptide. Following tetracycline administration, the K19 aptamer stabilizes the Cre transcript, and its coupling with trimethoprim activates the recombinase activity, thereby targeting a sequence within the promoter region of the construct to eliminate the expression of the therapeutic transgene (exenatide). Figure 5 The plasmid map of the genetic construct described in this embodiment is shown. The construct composition, actuator, effector, and overall sequence are shown below:

[0432] Actuators: Tetracycline (for K19 aptamer enzyme) and trimethoprim CAS RN 738-70-5, molecular formula C14H18N4O3 (for unstable Cre).

[0433] Complete sequence (excluding the nanoparticle backbone)

[0434]

[0435]

[0436] loxP site

[0437]

[0438] CBh promoter

[0439]

[0440] Exenatide-4

[0441] nucleotide sequence

[0442]

[0443] amino acid sequence

[0444]

[0445] EFS promoter

[0446]

[0447] Unstable Cre recombinase (DD-CRE):

[0448] nucleotide sequence

[0449]

[0450] amino acid sequence

[0451]

[0452] K19 aptamerase

[0453]

[0454] Insulator sequence

[0455]

[0456] bGH poly(A) sequence

[0457]

[0458] SV40 poly(A) sequence

[0459]

[0460] The activity of the self-inactivation Cre-based downregulation titration mechanism was identified using nLuc bioluminescent reporters. Figure 6 The expression of the reporter transgene is permanently reduced to about 1 / 3 × 10⁻⁶ by inducing a self-silencing signal activated by the delivery of the operon. Figure 6 ; "A").

[0461] Example 5

[0462] In this embodiment, enzyme replacement therapy is delivered under the regulation of a constitutive promoter. Under the regulation of the third-generation TetOn promoter / operon and the K19 aptamer enzyme, down-titering is mediated by Abrin-A, a ribosome-inhibiting protein (RIP) derived from Abrus precatorius L. Administration of tetracycline or doxycycline activates the TetOn operator / promoter that drives RIP protein expression, which inhibits ribosomes and reduces overall gene expression in the therapeutic construct. Figure 7 The plasmid map of the genetic construct described in this embodiment is shown.

[0463] Complete sequence (excluding the nanoparticle backbone)

[0464]

[0465]

[0466] CBh promoter

[0467]

[0468] secGAA (sp7-δ8-hGAA)

[0469] nucleotide sequence

[0470]

[0471] amino acid sequence

[0472]

[0473] P2A

[0474] nucleotide sequence

[0475]

[0476] amino acid sequence

[0477]

[0478] Tet-on 3G

[0479] nucleotide sequence

[0480]

[0481] amino acid sequence

[0482]

[0483] TRE3G Starter

[0484]

[0485] Arbin A chain

[0486] nucleotide sequence

[0487]

[0488] amino acid sequence

[0489]

[0490] K19 aptamerase

[0491]

[0492] Insulator sequence

[0493]

[0494] bGH poly(A) sequence

[0495]

[0496] SV40 poly(A) sequence

[0497]

[0498] Example 6

[0499] In this embodiment, a glucagon-like peptide-1 receptor agonist (GLP-1RA) for managing type 2 diabetes is delivered. The GLP-1RA peptide is delivered along with a secretion signal under the regulation of a constitutive promoter. Dose-down titration can be achieved through self-directed, small molecule-induced epigenetic silencing in the construct promoter region. Figure 8 The plasmid map of the genetic construct described in this embodiment is shown.

[0500] Complete sequence (excluding the nanoparticle backbone)

[0501]

[0502]

[0503] CBh promoter

[0504]

[0505] Zinc finger target site

[0506]

[0507] GLP-1 RA

[0508] nucleotide sequence

[0509]

[0510] amino acid sequence

[0511]

[0512] bGH poly(A) sequence

[0513]

[0514] SV40 poly(A) sequence

[0515]

[0516] K19 aptamerase

[0517]

[0518] Insulator sequence

[0519]

[0520] Epigenetic effector (hD3A-mD3L-ZF-ZN627)

[0521] nucleotide sequence

[0522]

[0523] amino acid sequence

[0524]

[0525] EFS promoter

[0526]

[0527] The activity of self-silencing epigenetic regulators was identified using nLuc bioluminescent reporters. Figure 9 The expression of reporter transgenes was reduced to approximately 1 / 2× by delivering a self-silencing signal activated by the operon.

[0528] Example 7

[0529] In this embodiment, a glucagon-like peptide-1 and gastric inhibitor peptide dual receptor agonist (GLP / GIP) for the treatment of type 2 diabetes or obesity is delivered under the regulation of the EFS promoter. To enable dose-down titration when the therapeutic agent achieves optimal therapeutic effect, or to reduce the dose when the therapeutic agent exceeds tolerance limits, a small molecule actuator (cumulate) is used to activate the cis-delivered epigenetic effector element to silence the expression of the therapeutic transgene. Figure 10 The plasmid map of the genetic construct described in this embodiment is shown. The specific components of the construct and the complete construct design are shown below.

[0530] Actuator: water-insoluble cumyl acid salt, 4-isopropylbenzoic acid (Sigma 268402) or its USP equivalent.

[0531] Complete sequence (excluding the nanoparticle backbone)

[0532]

[0533] Effector element: The standard epigenetic silencing subsystem of this invention

[0534] hD3A-mD3L-ZF-ZN627

[0535] nucleotide sequence

[0536]

[0537] amino acid sequence

[0538]

[0539] Effector sequence: The target DNA sequence of the standard epigenetic silencing system of this invention.

[0540] ZF target sequence

[0541]

[0542] Actuator response elements: cumyl acid salt-induced promoters, repressors, and promoter sequences

[0543] Culurate-inducible promoter (CMV5+CuO)

[0544]

[0545] CuO sequence

[0546]

[0547] CymR inhibitors

[0548] nucleotide sequence

[0549]

[0550] amino acid sequence

[0551]

[0552] Therapeutic transgenic GIP / GLP dual receptor agonists:

[0553] GIP / GLP-1 RA

[0554] nucleotide sequence

[0555]

[0556] amino acid sequence

[0557]

[0558]

[0559] EFS promoter

[0560]

[0561] P2A

[0562] nucleotide sequence

[0563]

[0564] amino acid sequence

[0565]

[0566] bGH poly(A) sequence

[0567]

[0568] SV40 poly(A) sequence

[0569]

[0570] Example 8

[0571] In this embodiment, the system comprising a dual-actuator-regulated effector element is designed to deliver a biologically active recombinant acid α-glucosidase for the treatment of type II glycogen storage disease (Pompe disease). The first actuator is a tetracycline or a tetracycline derivative that acts on the TetOn inducible promoter and binds to the TetOn operon to activate the expression of rapamycin-inducible caspase 9. The second actuator is rapamycin or a rapamycin analog or derivative that activates inducible caspase 9 to promote the initiation of apoptosis in cells carrying the therapeutic construct. Figure 11 The plasmid map of the genetic construct described in this embodiment is shown.

[0572] Complete sequence (excluding the nanoparticle backbone)

[0573]

[0574]

[0575] CBh promoter

[0576]

[0577] secGAA (sp7-δ8-hGAA)

[0578] nucleotide sequence

[0579]

[0580] amino acid sequence

[0581]

[0582] P2A

[0583] nucleotide sequence

[0584]

[0585] amino acid sequence

[0586]

[0587] tTS

[0588] nucleotide sequence

[0589]

[0590] amino acid sequence

[0591]

[0592] T2A

[0593] nucleotide sequence

[0594]

[0595] amino acid sequence

[0596]

[0597] rtTA

[0598] nucleotide sequence

[0599]

[0600] amino acid sequence

[0601]

[0602] TRE promoter

[0603]

[0604] RapaCasp9

[0605] nucleotide sequence

[0606]

[0607] amino acid sequence

[0608]

[0609] bGH poly(A) sequence

[0610]

[0611] SV40 poly(A) sequence

[0612]

[0613] The system was tested with reporter genes of rapamycin at three concentrations (from 1 nM to 0.01 nM) and at a constant concentration (0.1 nM) of tetracycline, and promoted a decrease in nLuc expression (as measured by the intensity of the bioluminescent signal). Figure 12 Two promoters were used to drive initial transgene expression: the stronger CBh promoter and the weaker EFS promoter. The construct without downregulated titration genetic elements (LNP CBh-nLuc) was used as a reference control.

[0614] Compared to the "untreated" control, no expression changes were observed in the LNP-CBh-nLuc construct. On the other hand, the construct containing i-cysteine ​​9 showed a dose-dependent reduction in gene expression of rapamycin after co-activation with tetracycline. Appropriate concentrations of rapamycin and tetracycline were obtained during treatment, after treatment with the therapeutic construct, and after local or systemic administration of the actuator small molecule.

[0615] Example 9

[0616] In this embodiment, a single actuator-regulated effector element is designed to deliver a biologically active recombinant insulin-like growth factor 2 (IGF-2) for the treatment of Silver-Russell syndrome. The actuator is a tetracycline or a tetracycline derivative that acts on a third-generation TetOn inducible promoter and binds to the TetOn operon to activate the expression of the N-terminal domain of gasdermin D. This leads to the oligomerization of gasdermin D to form pores in the cell membrane and initiate pyroptosis. Figure 13 The plasmid map of the genetic construct described in this embodiment is shown.

[0617] Complete sequence (excluding the nanoparticle backbone)

[0618]

[0619] Transgenic: Insulin-like growth factor 2 (IGF2)

[0620] nucleotide sequence

[0621]

[0622] amino acid sequence

[0623]

[0624] Actuator: Tetracycline or its derivatives

[0625] Actuator response element: Tet inhibitor / Gen 3 TetOn, third-generation rtTA / TRE3G

[0626] TRE3G Starter

[0627]

[0628] TetOn 3G

[0629] nucleotide sequence

[0630]

[0631] amino acid sequence

[0632]

[0633] Effector element: N-terminal domain of gasdermin D

[0634] nucleotide sequence

[0635]

[0636] amino acid sequence

[0637]

[0638] Auxiliary sequence:

[0639] CBh promoter

[0640]

[0641] Self-cleaving 2A peptide from porcine teschovirus-1

[0642] nucleotide sequence

[0643]

[0644] amino acid sequence

[0645]

[0646] Kozak

[0647]

[0648]

[0649] Example 10

[0650] In this embodiment, the effector element regulated by a single actuator is designed to deliver a biologically active β-glucocerebrosidase for the treatment of Gaucher's disease. β-glucocerebrosidase is expressed under the control of a CMV promoter. The actuator is a tetracycline or a tetracycline derivative that acts on a third-generation TetOn inducible promoter. This promoter binds to the TetOn operon and activates the expression of a viral DNA-binding protein (IE2 from human cytomegalovirus), which binds to the CMV promoter and inhibits the expression of β-glucocerebrosidase. Tetracycline and the TetOn operon further activate the expression of a transcriptional transactivator (tTA), which continues to activate the TetOn operon after tetracycline withdrawal. Figure 14 The plasmid map of the genetic construct described in this embodiment is shown.

[0651] Complete sequence (excluding the nanoparticle backbone)

[0652]

[0653] Transgenic: β-glucocerebroside lipase

[0654] nucleotide sequence

[0655]

[0656] amino acid sequence

[0657]

[0658] Actuator: Tetracycline or its derivatives, Enhanced tTA (Advanced tTA)

[0659] Enhanced tTA

[0660] nucleotide sequence

[0661]

[0662] amino acid sequence

[0663]

[0664] Actuator response element: Tet inhibitor / Gen 3 TetOn, third-generation rtTA / TRE3G

[0665] TRE3G

[0666]

[0667] TetOn 3G

[0668] nucleotide sequence

[0669]

[0670] amino acid sequence

[0671]

[0672] Effector element: IE2 from human cytomegalovirus

[0673] nucleotide sequence

[0674]

[0675] amino acid sequence

[0676]

[0677] Auxiliary sequence:

[0678] CMV promoter

[0679]

[0680] Self-cleaving 2A peptide from porcine chezinvirus-1

[0681] nucleotide sequence

[0682]

[0683] amino acid sequence

[0684]

[0685] Kozak

[0686]

[0687]

[0688] Example 11

[0689] In this embodiment, a single actuator-regulated effector element is designed to deliver bioactive lamin A for the treatment of progeria. Lamin A is expressed under the control of a constitutive CBh promoter, and the gene is flanked by one of two serine recombinase recognition sequences (attP and attB). The actuator is a tetracycline or a tetracycline derivative that acts on a tetracycline-inducible promoter and binds to the tet operon to activate the expression of a serine integrase derived from Bxb1 phage. The integrase cleaves the genetic sequence of lamin A, thereby shutting down gene expression. Figure 15 The plasmid map of the genetic construct described in this embodiment is shown.

[0690] Complete sequence (excluding the nanoparticle backbone)

[0691]

[0692] Genetically modified: Lamin A

[0693] nucleotide sequence

[0694]

[0695] amino acid sequence

[0696]

[0697] Actuator: Tetracycline or its derivatives

[0698] Actuator response element: Tet inhibitor / Gen 3 TetOn, third-generation rtTA / TRE3G

[0699] TRE3G

[0700]

[0701] TetOn3G

[0702] nucleotide sequence

[0703]

[0704] amino acid sequence

[0705]

[0706] Effector elements: Bxb1 serine integrase, associated attB and attP sites

[0707] attP site

[0708]

[0709] attB site

[0710]

[0711] Bxb1 serine integrase

[0712] nucleotide sequence

[0713]

[0714] amino acid sequence

[0715]

[0716] Auxiliary sequence:

[0717] CBh promoter

[0718]

[0719] Self-cleaving 2A peptide from porcine chezinvirus-1

[0720] nucleotide sequence

[0721]

[0722] amino acid sequence

[0723]

[0724] Kozak

[0725]

[0726]

[0727] Example 12

[0728] In this embodiment, a single actuator regulates the delivery of a functional copy of the cystic fibrosis transmembrane conductance regulator (CFTR) for the treatment of cystic fibrosis. The CBh promoter controls the expression of CFTR and inducible caspase 9 (iCasp9) linked by a self-cleaving peptide (P2A). The actuator is AP20187, a ligand that induces caspase 9 dimerization and apoptosis. Figure 16 The plasmid map of the genetic construct described in this embodiment is shown.

[0729] Complete sequence (excluding the nanoparticle backbone)

[0730]

[0731] Transgenic: Cystic fibrosis conduction regulator protein

[0732] nucleotide sequence

[0733]

[0734] amino acid sequence

[0735]

[0736] Actuator: AP20187 or other dimer capable of dimerizing iCasp9

[0737] Effector element: iCasp9

[0738] nucleotide sequence

[0739]

[0740] amino acid sequence

[0741]

[0742] Auxiliary sequence:

[0743] CBh promoter

[0744]

[0745] Self-cleaving 2A peptide from porcine chezinvirus-1

[0746] nucleotide sequence

[0747]

[0748] amino acid sequence

[0749]

[0750] Kozak

[0751]

[0752] bGH polyA

[0753]

[0754] Example 13

[0755] In this embodiment, a single actuator regulates the delivery of a functional copy of hexosaminidase subunit alpha (HexA) intended for the treatment of Tay-Sachs disease. The CBh promoter controls the expression of HexA and inducible caspase 8 (iCasp8) linked by a self-cleaving peptide (P2A). The actuator is AP20187, a ligand that induces caspase 8 dimerization and apoptosis. Figure 17 The plasmid map of the genetic construct described in this embodiment is shown.

[0756] Complete sequence (excluding the nanoparticle backbone)

[0757]

[0758] Transgenic: Aminohexosidase A

[0759] nucleotide sequence

[0760]

[0761] amino acid sequence

[0762]

[0763] Actuator: AP20187 or other dimer capable of dimerizing iCasp8

[0764] Effector element: iCasp8

[0765] nucleotide sequence

[0766]

[0767] amino acid sequence

[0768]

[0769] Auxiliary sequence:

[0770] CBh promoter

[0771]

[0772] Self-cleaving 2A peptide from porcine chezinvirus-1

[0773] nucleotide sequence

[0774]

[0775] amino acid sequence

[0776]

[0777] Kozak

[0778]

[0779]

[0780] Example 14

[0781] In this embodiment, a system comprising a toggle-regulated effector element is designed to deliver bioactive fibroblast growth factor 18 for the treatment of osteoarthritis. The system consists of a constitutive CMV promoter followed by several TetO sequences, driving the expression of both fibroblast growth factor 18 and a lac repressor protein fused to KRAB. A second promoter followed by a LacO or CuO sequence drives the expression of the Tet repressor protein fused to KRAB (tet transcription silencer / tTS). Upon addition of tetracycline, fibroblast growth factor 18 and the lac repressor / cuminate repressor protein are expressed, achieving a stable equilibrium where the lac / cuminate-responsive second promoter is continuously repressed, and fibroblast growth factor 18 is continuously produced. Upon addition of IPTG or cuminate, the lac / cuminate repressor protein no longer binds to the LacO / CuO sequence, thereby enabling the expression of the TetR-KRAB fusion protein. The switch reaches a new stable equilibrium in which the TetR-KRAB fusion protein is continuously expressed, inhibiting the expression of fibroblast growth factor 18. Figure 18 The plasmid map of the genetic construct described in this embodiment is shown.

[0782] Complete sequence (excluding the nanoparticle backbone)

[0783]

[0784] Genetically modified: fibroblast growth factor 18

[0785] nucleotide sequence

[0786]

[0787] amino acid sequence

[0788]

[0789] Actuator: Tetracycline or its derivatives, IPTG or cumyl sulfate

[0790] Actuator response elements: Tet repressor-KRAB(tTS) / TRE3G, Lac repressor-KRAB / CMV-7x LacO, or cumyl acid salt repressor / CuOn promoter

[0791] tTS

[0792] nucleotide sequence

[0793]

[0794] amino acid sequence

[0795]

[0796] 7x LacO promoter

[0797]

[0798] Lac repressor-KRAB

[0799] nucleotide sequence

[0800]

[0801] amino acid sequence

[0802]

[0803] 7x CuO promoter

[0804]

[0805] cumyl salt repressor

[0806] nucleotide sequence

[0807]

[0808] amino acid sequence

[0809]

[0810] Auxiliary sequence:

[0811] Self-cleaving 2A peptide from porcine chezinvirus-1

[0812] nucleotide sequence

[0813]

[0814] amino acid sequence

[0815]

[0816] Kozak

[0817]

[0818]

[0819] Equivalent scheme

[0820] Those skilled in the art will recognize or be able to identify many equivalents of the specific embodiments of the invention described in this application using only conventional methods. Such equivalents are intended to be covered by the following claims.

[0821] All references (including patent documents) are incorporated in their entirety by way of citation.

Claims

1. A system for permanently reducing gene expression from one or more intracellular therapeutic constructs or multiple constructs, said system comprising at least the following: (a) A nucleic acid construct containing a therapeutic transgene, delivered in a formulation capable of entering cells and enabling stable expression of the therapeutic transgene for at least 4 months; (b) An actuator which is composed of one or more chemical or biological molecules; (c) Actuator response elements and / or actuator response sequences that enable the expression, activation or stabilization of effector elements or effector sequences; (d) The effector sequence, which is delivered in cis along with the therapeutic transgene; and (e) An effector element expressed by the effector sequence, the effector element being capable of permanently reducing the expression of the therapeutic transgene in a manner that causes minimal bystander effect and is therefore safe for surrounding tissues, organs and the whole organism.

2. The system of claim 1, wherein the system is intended for delivery to subcutaneous tissue.

3. The system of claim 1 or 2, wherein the system is intended for intraperitoneal delivery.

4. The system of any one of the preceding claims, wherein the system is intended for delivery to adipocytes.

5. The system of any one of the preceding claims, wherein the system is intended for intramuscular delivery.

6. The system of any one of the preceding claims, wherein the system is intended for delivery to the liver.

7. The system of any one of the preceding claims, wherein the system is intended for delivery to the central nervous system.

8. The system of any one of the preceding claims, wherein the system is intended for delivery to the fascia.

9. The system of any one of the preceding claims, wherein the system is intended for delivery into the subarachnoid space.

10. The system of any one of the preceding claims, wherein the system is intended for delivery to the pancreas.

11. The system of any one of the preceding claims, wherein the system is intended for delivery to the epidermis.

12. The system of any one of the preceding claims, wherein the system is intended for transocular delivery.

13. The system of any one of the preceding claims, wherein the system is intended for delivery to the kidney.

14. The system of any one of the preceding claims, wherein the system is intended for delivery to the bladder.

15. The system of any one of the preceding claims, wherein the system is intended for delivery to a mucosa.

16. The system of any one of the preceding claims, wherein the system is intended for delivery to the lungs.

17. The system of any one of the preceding claims, wherein the system is intended for delivery to the spleen.

18. The system of any one of the preceding claims, wherein the system is intended for intravascular delivery.

19. The system of any one of the preceding claims, wherein the system is intended for delivery to the heart.

20. The system of any one of the preceding claims, wherein the system is intended for use in conjunction with therapeutic gene therapy or for delivery in conjunction with therapeutic transgenes for the treatment of human, plant, or veterinary diseases or for the enhancement of human, veterinary, or plant functions.

21. The system of any one of the preceding claims, wherein the therapeutic transgene encodes at least a portion of a therapeutic antibody.

22. The system of any of the preceding claims, wherein the therapeutic transgene encodes a protein or enzyme that replaces the protein or enzyme required for treatment.

23. The system of any one of the preceding claims, wherein the therapeutic transgene encodes a therapeutic peptide.

24. The system of any one of the preceding claims, wherein the therapeutic transgene encodes a protein or fusion protein.

25. The system of any one of the preceding claims, wherein the therapeutic transgene encodes a non-coding RNA.

26. The system of any one of the preceding claims, wherein the therapeutic transgene encodes an aptamer enzyme, aptamer, ribozyme, or genetic structure capable of exerting enzymatic activity alone or in combination with a protein.

27. The system of any one of the preceding claims, wherein the nucleic acid construct is composed of double-stranded DNA.

28. The system of any one of the preceding claims, wherein the nucleic acid construct is composed of single-stranded DNA.

29. The system of any one of the preceding claims, wherein the nucleic acid construct is composed of single-stranded RNA.

30. The system of any one of the preceding claims, wherein the nucleic acid construct is composed of double-stranded RNA.

31. The system of any one of the preceding claims, wherein the nucleic acid construct is composed of single-stranded or double-stranded linear DNA.

32. The system of any one of the preceding claims, wherein the nucleic acid construct is composed of single-stranded or double-stranded linear RNA.

33. The system of any one of the preceding claims, wherein the nucleic acid construct is composed of single-stranded or double-stranded circular DNA.

34. The system of any one of the preceding claims, wherein the nucleic acid construct is composed of single-stranded or double-stranded circular RNA.

35. The system of any one of the preceding claims, wherein the system comprises a protein delivered together with the nucleic acid.

36. The system of any one of the preceding claims, wherein the system is delivered in vivo or ex vivo as part of a viral or non-viral preparation.

37. The system of any one of the preceding claims, wherein the system is delivered by physical methods, such as electroporation, nanoparticle bombardment, ultrasound-mediated transfection, or chemically mediated transfection.

38. The system of any one of the preceding claims, wherein the actuator is at least partially composed of tetracycline or a tetracycline derivative.

39. The system of any one of the preceding claims, wherein the actuator is at least partially composed of doxycycline or a doxycycline derivative.

40. The system of any one of the preceding claims, wherein the actuator is at least partially composed of an antibiotic or an antimicrobial peptide.

41. The system of any one of the preceding claims, wherein the actuator is at least partially composed of rapamycin or a derivative thereof.

42. The system of any one of the preceding claims, wherein the actuator is at least partially composed of ganciclovir or a derivative thereof.

43. The system of any of the preceding claims, wherein the actuator is at least partially composed of ganciclovir, valganciclovir, valacyclovir, or derivatives thereof.

44. The system of any of the preceding claims, wherein the actuator is at least partially composed of tamoxifen or a derivative thereof.

45. The system of any one of the preceding claims, wherein the actuator is at least partially composed of estrogen or a derivative thereof.

46. ​​The system of any one of the preceding claims, wherein the actuator is at least partially composed of acetaldehyde or a derivative thereof.

47. The system of any one of the preceding claims, wherein the actuator is at least partially composed of L-arginine or a derivative thereof.

48. The system of any one of the preceding claims, wherein the actuator is at least partially composed of biotinylated AMP or a derivative thereof.

49. The system of any one of the preceding claims, wherein the actuator is at least partially composed of 2-phenylethyl butyrate or a derivative thereof.

50. The system of any one of the preceding claims, wherein the actuator is at least partially composed of 6-hydroxynicotin or a derivative thereof.

51. The system of any one of the preceding claims, wherein the actuator is at least partially composed of erythromycin or a derivative thereof.

52. The system of any one of the preceding claims, wherein the actuator is at least partially composed of a macrolide or a derivative thereof.

53. The system of any one of the preceding claims, wherein the actuator is at least partially composed of streptomycin or a derivative thereof.

54. The system of any one of the preceding claims, wherein the actuator is at least partially composed of NADH or a derivative thereof.

55. The system of any of the preceding claims, wherein the actuator is at least partially composed of elements that cause the generation of increased energy or heat.

56. The system of any of the preceding claims, wherein the actuator is at least partially composed of SCB1 or a derivative thereof.

57. The system of any of the preceding claims, wherein the actuator is at least partially composed of 3-oxo-C8-HSL or a derivative thereof.

58. The system of any one of the preceding claims, wherein the actuator is at least partially composed of phloretin or a derivative thereof.

59. The system of any one of the preceding claims, wherein the actuator is a small molecule, peptide, protein, or nucleic acid capable of enhancing, reducing, or modulating local or systemic inflammation or a partial inflammatory response.

60. The system of any one of the preceding claims, wherein the actuator is at least partially composed of theophylline or a derivative thereof.

61. The system of any one of the preceding claims, wherein the actuator is at least partially composed of IPTG or a derivative thereof.

62. The system of any of the preceding claims, wherein the actuator is at least partially composed of 5-fluorocytosine or a derivative thereof.

63. The system of any one of the preceding claims, wherein the actuator is at least partially composed of a propionate ester or a derivative thereof.

64. The system of any one of the preceding claims, wherein the actuator is at least partially composed of an acetate or a derivative thereof.

65. The system of any one of the preceding claims, wherein the actuator is at least partially composed of cumyl acid salt, cumyl ester, or cumic acid or its derivatives.

66. The system of any one of the preceding claims, wherein the actuator is at least partially composed of gluconate or a derivative thereof.

67. The system of any one of the preceding claims, wherein the actuator is at least partially composed of maltose or a derivative thereof.

68. The system of any one of the preceding claims, wherein the actuator is at least partially composed of arabinose or a derivative thereof.

69. The system of any one of the preceding claims, wherein the actuator is at least partially composed of a monosaccharide or a disaccharide.

70. The system of any one of the preceding claims, wherein the actuator is at least partially composed of trimethoprim or a derivative thereof.

71. The system of any of the preceding claims, wherein the actuator is at least partially composed of an exogenously applied small molecule, peptide, protein, RNA, DNA, or pharmacological agent.

72. The system of any one of the preceding claims, wherein the actuator is at least a part of the effector element of any one of the preceding claims.

73. The system of any one of the preceding claims, wherein the actuator is at least partially composed of endogenously expressed or presented peptides, hormones, proteins, RNA, DNA, enzymes, minerals, or chemical or biochemical substances, and is capable of interacting with the actuator response element or actuator response sequence.

74. The system of any of the preceding claims, wherein the effector element is at least partially composed of inducible cysteine ​​9.

75. The system of any of the preceding claims, wherein the effector element is at least partially composed of natural cysteine ​​9.

76. The system of any one of the preceding claims, wherein the effector element is at least partially composed of a zinc finger nuclease.

77. The system of any of the preceding claims, wherein the effector element is at least partially composed of a transcription activator-like effector nuclease (TALEN).

78. The system of any one of the preceding claims, wherein the effector element is at least partially composed of a meganuclease.

79. The system of any of the preceding claims, wherein the effector element is at least partially composed of an editor derived from deoxycytidine deamination.

80. The system of any of the preceding claims, wherein the effector element is at least partially composed of a base editor derived from deoxyadenosine deamination.

81. The system of any of the preceding claims, wherein the effector element is at least partially composed of a DNA-binding peptide.

82. The system of any of the preceding claims, wherein the effector element is at least partially composed of a viral or bacterial DNA-binding protein.

83. The system of any one of the preceding claims, wherein the effector element is at least partially composed of natural caspase 8.

84. The system of any of the preceding claims, wherein the effector element is at least partially composed of natural caspase or caspase pathway proteins.

85. The system of any one of the preceding claims, wherein the effector element is FK506 binding protein 12 (with an F36V mutation), which is fused to a human caspase-8 protein lacking 1-215 AA and linked to an N-myristylation signal from Src kinase.

86. The system of any one of the preceding claims, wherein the effector element portion comprises an FK506 binding protein 12 (with an F36V mutation) fused to a human caspase-9 protein lacking 1-134 AA.

87. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of viral thymidine kinase.

88. The system of any one of the preceding claims, wherein the effector element is at least partially composed of at least a portion of Escherichia coli cytosine deaminase.

89. The system of any one of the preceding claims, wherein the effector element is a protein having catalytic activity capable of chemically modifying nucleotides, nucleosides or nucleic acids.

90. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of diphtheria toxin subunit A.

91. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of diphtheria toxin subunit B.

92. The system of any one of the preceding claims, wherein the effector element is a dimerizable or trimerizable toxin, protein, or enzyme.

93. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of a human or simian diphtheria toxin receptor.

94. The system of any of the preceding claims, wherein the effector element is composed of at least a portion of bacteria, viruses or eukaryotic toxins.

95. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of a protein capable of inhibiting transcription.

96. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of a protein capable of inhibiting translation.

97. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of a protein capable of inducing apoptosis.

98. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of a protein capable of inducing aging.

99. The system of any of the preceding claims, wherein the effector element comprises at least a portion of a protein capable of reducing general gene expression.

100. The system of any one of the preceding claims, wherein the effector element is at least partially composed of at least a portion of a nuclease.

101. The system of any one of the preceding claims, wherein the effector element is at least partially composed of at least a portion of a nuclease capable of site-specific mutagenesis.

102. The system of any one of the preceding claims, wherein the effector element is at least partially composed of at least a portion of a nuclease and at least a portion of a target element.

103. The system of any one of the preceding claims, wherein the effector element comprises at least a portion of: a Cas nuclease and a guide RNA, the guide RNA targeting the nuclease to the sequence of any one of the preceding claims.

104. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of a nuclease targeting the sequence of any of the preceding claims.

105. The system of any one of the preceding claims, wherein the effector element is at least a portion of a nuclease that targets the promoter region of the sequence of any one of the preceding claims.

106. The system of any of the preceding claims, wherein the effector element is at least a portion thereof composed of at least a portion of a nuclease targeting the coding region of the sequence of any of the preceding claims.

107. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of a nuclease targeting the post-translational regulatory element region of the sequence of any of the preceding claims.

108. The system of any one of the preceding claims, wherein the effector element is at least partially composed of at least a portion of the recombinase.

109. The system of any one of the preceding claims, wherein the effector element is at least partially composed of at least a portion of Cre recombinase.

110. The system of any of the preceding claims, wherein the effector element is at least partially constituted by at least a portion of the Krüppel-associated box domain of the human gene ZNF10.

111. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of a zinc finger protein.

112. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of a cyclized recombinase having an SV40 nuclear localization signal.

113. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of a Cre recombinase having an SV40 T antigen intron inserted into the ORF.

114. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of the Flpe recombinase.

115. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of the Flpo recombinase.

116. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of the Dre recombinase.

117. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of the SV40 T antigen sequence.

118. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of the piggyBac transposase.

119. The system of any one of the preceding claims, wherein the effector element is at least partially composed of at least a portion of a transposase.

120. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of Sleeping Beauty transposase.

121. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of the argonaut protein.

122. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of a DNA methyltransferase.

123. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of a protein constituting or interacting with the chromatin remodeling complex.

124. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of a protein constituting or interacting with a DNA methylation pathway.

125. The system of any of the preceding claims, wherein the effector element is at least partially constituted by at least a portion of a base editor.

126. The system of any of the preceding claims, wherein the effector element is at least partially constituted by at least a portion of the lead editor.

127. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of the nicking enzyme.

128. The system of any of the preceding claims, wherein the effector element comprises at least a portion of a virus, bacteria, or eukaryotic gene silencing protein.

129. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of siRNA.

130. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of the miRNA.

131. The system of any of the preceding claims, wherein the effector element is at least partially constituted by at least a portion of the FRT.

132. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of an integrase.

133. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of RNA capable of reducing gene expression.

134. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of a protein capable of causing the effector to degrade in the absence of an inducer.

135. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of dihydrofolate reductase.

136. The system of any one of the preceding claims, wherein the effector element is at least partially composed of at least a portion of RNA, the at least portion of which is at least partially complementary to the genetic construct of any one of the preceding claims.

137. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of: a transposon or a retrotransposon.

138. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of Streptococcus pyogenes Cas9.

139. The system of any one of the preceding claims, wherein the effector element is at least partially composed of at least a portion of catalytically inactive Cas9.

140. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of a codon-optimized Cas9.

141. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of Cas9 having a D10A mutation.

142. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of the dCas9 fused with KRAB.

143. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of dCas9 fused with a carboxyl-terminal dichotomous repressor domain KRAB-MeCP2 or a derivative thereof.

144. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of an ortholog of Campylobacter jujeni Cas9.

145. The system of any one of the preceding claims, wherein the effector element comprises at least a portion of: a CRISPR-related protein or an ortholog.

146. The system of any of the preceding claims, wherein the effector element is composed at least in part of at least a portion of the following: Cas12a or Cas12b protein.

147. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of Cas13.

148. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of: Cas12f or Cas13a.

149. The system of any one of the preceding claims, wherein the effector element comprises at least a portion of the following: DNA, DNA capable of expressing RNA, RNA, protein, or a combination thereof, capable of affecting a DNA sequence in a sequence-specific manner to induce double-strand breaks, single-strand breaks, methylation, chromatin modification, or general epigenetic silencing.

150. The system of any one of the preceding claims, wherein the effector element comprises at least a portion of: a toxin, a toxin derivative, a modified toxin or its homolog, direct homolog, or paralog.

151. The system of any of the preceding claims, wherein the effector element is composed of at least a portion of: bacteria, fungi, plant or animal toxins.

152. The system of any one of the preceding claims, wherein the effector element comprises at least a portion of: Vibrio cholerae toxin, Shiga toxin, Shigella toxin, ricin, saponin, Staphylococcus aureus enterotoxin H, Clostridium perfringens enterotoxin, streptococcal hemolysin O, ribosome inactivating protein (RIP) enzyme, immunotoxin, Pseudomonas exotoxin A, diphtheria toxin (DT), or DT fragment.

153. The system of any one of the preceding claims, wherein the effector element comprises at least a portion of: viral thymidine kinase or a derivative thereof.

154. The system of any one of the preceding claims, wherein the effector element is composed of at least a portion of: bacterial cytosine deaminase or a derivative thereof.

155. The system of any one of the preceding claims, wherein the effector element is composed of at least a portion of: D-amino acid oxidase or a derivative thereof.

156. The system of any one of the preceding claims, wherein the effector element is at least partially composed of at least a portion of bacterial carboxypeptidase G2 or a derivative thereof.

157. The system of any one of the preceding claims, wherein the effector element is at least partially composed of at least a portion of: purine nucleoside phosphorylase or a derivative thereof.

158. The system of any one of the preceding claims, wherein the effector element is at least partially composed of at least a portion of thymidine phosphorylase or a derivative thereof.

159. The system of any one of the preceding claims, wherein the effector element is at least partially composed of at least a portion of xanthine-guanine phosphoribosyltransferase or a derivative thereof.

160. The system of any one of the preceding claims, wherein the effector element is at least partially composed of at least a portion of: nitroreductase or a derivative thereof.

161. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of: penicillin-G amidase or a derivative thereof.

162. The system of any one of the preceding claims, wherein the effector element comprises at least a portion of a multidrug activating enzyme or a derivative thereof.

163. The system of any one of the preceding claims, wherein the effector element is at least partially composed of at least a portion of horseradish peroxidase or a derivative thereof.

164. The system of any one of the preceding claims, wherein the effector element is at least partially composed of at least a portion of β-galactosidase or a derivative thereof.

165. The system of any one of the preceding claims, wherein the effector element is composed of at least a portion of: deoxyribonucleokinase or a derivative thereof.

166. The system of any one of the preceding claims, wherein the effector element is at least partially composed of at least a portion of: β-glucuronidase or a derivative thereof.

167. The system of any of the preceding claims, wherein the effector element is at least partially composed of at least a portion of carboxypeptidase A or a derivative thereof.

168. The system of any one of the preceding claims, wherein the effector element is composed of at least a portion of cytochrome P450 or a derivative thereof.

169. The system of any one of the preceding claims, wherein the effector element is at least partially composed of at least a portion of: deoxycytidine kinase or a derivative thereof.

170. The system of any of the preceding claims, wherein the effector sequence at least encodes the effector element.

171. The system of any of the preceding claims, wherein the effector sequence encodes at least a portion of the following: a common Lox sequence or a sequence capable of mediating the action of a Lox sequence.

172. The system of any of the preceding claims, wherein the effector sequence encodes at least a portion of the following: a common FRT sequence or a sequence capable of mediating the action of the FRT sequence.

173. The system of any of the preceding claims, wherein the effector sequence encodes at least a portion of the following: a shared Rox sequence or a sequence capable of mediating the action of a Rox sequence.

174. The system of any one of the preceding claims, wherein the effector sequence is at least partially composed of constitute.

175. The system of any one of the preceding claims, wherein the effector sequence is at least partially composed of constitute.

176. The system of any one of the preceding claims, wherein the effector sequence is at least partially composed of constitute.

177. The system of any one of the preceding claims, wherein the effector sequence is at least partially composed of constitute.

178. The system of any one of the preceding claims, wherein the effector sequence is at least partially composed of constitute.

179. The system of any one of the preceding claims, wherein the effector sequence is at least partially composed of constitute.

180. The system of any of the foregoing claims, wherein the effector sequence is at least partially comprised of Gly-Ser.

181. The system of any of the preceding claims, wherein the effector sequence encodes at least a portion of the following: attB, attR, attL, or attP sites.

182. The system of any one of the preceding claims, wherein the effector sequence comprises at least a portion of a genetic sequence that can be targeted by an integrase, recombinase, nuclease, nickase, chromatin complex, or methyltransferase.

183. The system of any of the preceding claims, wherein the effector sequence comprises at least a portion of a genetic sequence that can be targeted to silence RNA or protein binding or that can itself be silenced.

184. The system of any one of the preceding claims, wherein the effector sequence is a DNA sequence that can be used as a target of a guide RNA for a CRISPR-related protein.

185. The system of any one of the preceding claims, wherein the effector sequence is a DNA sequence derived from the genome of a virus or bacteria.

186. The system of any of the preceding claims, wherein the effector sequence forms a secondary structure in the absence of an inducing molecule.

187. The system of any one of the preceding claims, wherein the effector sequence is an inflammatory responsive promoter.

188. The system of any of the preceding claims, wherein the effector sequence is at least partially derived from a mammalian promoter or enhancer.

189. The system of any of the preceding claims, wherein the effector sequence forms a secondary structure in the presence of an inducing molecule.

190. The system of any one of the preceding claims, wherein the actuator response element is a nucleic acid sequence or protein that interacts with the actuator to enable activation of the effector element or effector sequence.

191. The system of any of the preceding claims, wherein the actuator response element is at least partially composed of a tetracycline-controlled transcriptional silencer (tTS).

192. The system of any one of the preceding claims, wherein the actuator response element is at least partially composed of the Kruppel-associated box (KRAB) AB domain of the kid-1 transcriptional repressor.

193. The system of any one of the preceding claims, wherein the actuator response element is at least partially composed of an inverse tetracycline-controlled trans activator.

194. The system of any one of the preceding claims, wherein the actuator response element is at least partially composed of a viral protein, such as VP16, fused with a Tet repressor (TetR) or a reverse Tet repressor (rTetR).

195. The system of any one of the preceding claims, wherein the actuator response element is composed of tTS and rtTA driven by the same promoter.

196. The system of any one of the preceding claims, wherein the actuator response element comprises tTS and rtTA driven by different promoters.

197. The system of any one of the preceding claims, wherein the actuator response element is at least partially composed of a transcriptional repressor.

198. The system of any one of the preceding claims, wherein the actuator response element is at least partially composed of a transcriptional silencer.

199. The system of any one of the preceding claims, wherein the actuator response element is at least partially composed of a DNA-binding protein.

200. The system of any one of the preceding claims, wherein the actuator response element is at least partially composed of a promoter-binding protein.

201. The system of any of the preceding claims, wherein the actuator response element is at least partially composed of a transcription initiation complex binding protein.

202. The system of any of the preceding claims, wherein the actuator response element is at least partially composed of a protein responsible for DNA methylation.

203. The system of any of the preceding claims, wherein the actuator response element is at least partially composed of a protein responsible for histone modification.

204. The system of any of the preceding claims, wherein the actuator response element is at least partially constituted by at least a portion of the Tet-ON system.

205. The system of any one of the preceding claims, wherein the actuator response element is at least partially constituted by at least a portion of the Tet-OFF system.

206. The system of any one of the preceding claims, wherein the actuator response element is composed of at least a portion of: AlcR, ArgR, BirA, EthR, HdnoR, HucR, MphR(A), PIP, Rex, RheA, ScbR, TraR, TtgR, or analogs, homologs, or orthologs thereof, or functionally similar protein fragments, proteins, or fusion proteins.

207. The system of any one of the preceding claims, wherein the actuator response element is at least partially composed of at least a portion of: lacUV5, lac, tac, trc, prpB, aceA, aceB, gntP, gntK, CJ10X2, tacM, malE1, git1, BAD, SPL, P4-N14, cspB, aprE, sod, dapA, porB, ilvC, L10, L26, L16, L51, H30, H36, or natural, synthetic or semi-synthetic analogs, homologs, or orthologs, or functionally similar protein fragments, proteins or fusion proteins.

208. The system of any one of the preceding claims, wherein the actuator response element is composed of at least a portion of: ABI1-Gal4DBD, ABA, PVL1-VP16, PHR-p65, CIB1-TetR, or analogs, homologs, or orthologs thereof, or functionally similar protein fragments, proteins or fusion proteins.

209. The system of any one of the preceding claims, wherein the actuator response element comprises at least a portion of: a transcriptional activator, an enhancer, a derepressor, or a protein, RNA, or DNA capable of directly or indirectly enhancing gene expression in a targeted or generally cis-acting manner.

Images