GENETIC FACTORS REGULATE THE LEVEL OF EXPRESSION

Abstract

Most therapeutic treatments require dose adjustments after the initial administration of the prescribed dose. Dose adjustment is crucial to ensuring optimal safety and efficacy profiles of the treatment. Gene therapy is inherently limited by the lack of dose adjustment capabilities after the initial dose. This invention reveals a genetic element that enables dose adjustment after the initial dose in gene therapy. This dose adjustment capability allows single-injection gene therapy to potentially replace multi-injection subcutaneous protein regimens, improving pharmacokinetics, safety / efficacy profiles, treatment compliance, and reducing healthcare costs.

Description

[0001] GENETIC ELEMENTS FOR AD JUSTMENT OF EXPRESSION

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 536,530, filed September 5, 2023, entitled “GENETIC ELEMENTS FOR ADJUSTMENT OF EXPRESSION,” the disclosure of which is hereby incorporated by reference in its entirety.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (R087270004WO00-SEQ-KVC.xml; Size: 246,308 bytes; and Date of Creation: September 4, 2024) is herein incorporated by reference in its entirety.

[0006] FIELD

[0007] The present invention generally relates to human and veterinary therapeutics and more specifically to gene therapy treatments of conditions that may require the adjustment of the dose following initial administration.

[0008] SUMMARY

[0009] Effects of pharmacological treatments are not homogeneous nor consistent due to the diversity of human biology. As a result, most treatments could benefit from the adjustment of the dosing regimen following the initial treatment based on the physician assessment of the treatment’s effect. Dose adjustment is important to ensure an optimal safety and efficacy profile of a treatment. Should the initial treatment prove insufficiently effective at managing the pathology or symptoms and the side effects optimally tolerable, the treatment dose may be increased. Conversely, should the treatment dose be such that the side effects are excessive or cannot be tolerated by the patient, the treatment dose should be decreased. Similarly, if a patient is cured of a condition, the dose should be reduced to a maintenance level or completely to zero, depending on the pathology. Gene therapy, however, is inherently limited by its inability to adjust the dose following initial administration as a therapeutic modality.

[0010] When genetic cargo is delivered by a viral or non- viral vector the genetic cargo either integrates into the host cell genome and resides within the cell permanently and becomes passed on to progeny, or the genetic cargo enters the nucleus and forms an episome expressing for years at a level defined by the promoter, chromatic, epigenetics, or generally the natural intracellular environment and extracellular signals. As an example, AAV-delivered non-integrating gene therapies have demonstrated stable transgene expression over a period of multiple years in preclinical and clinical studies. Similarly, lentivirally delivered therapeutic transgenes integrate permanently into the host cell genome and provide multiyear stable expression of the therapeutic transgene. Currently gene expression of gene therapy treatments cannot be adjusted up or down in a manner that would enable therapeutic treatment of conditions that require dose adjustment with gene therapies. While transcriptional control with inducible promoters is possible, this control is transient and does not provide for stable up or down titration. Similarly, suicide genes can be used to completely turn off gene expression in cells such as CAR-T cells by fully eliminating the cell via induction of apoptosis. The latter approach, however, is incompatible with protein replacement therapies as creating a protein replacement factory in vivo and eliminating the target cells creates significant toxicity in the case of all secretory cell types targeted by previous gene therapies. On top of the unacceptable toxicity to the presently targeted cell types, this approach does not allow for adjustment of the dose as the suicide genes are universally eliminated in a manner that cannot control the resulting level of therapeutic transgene expression.

[0011] This invention provides at least in part a combination of target cell types, genetic elements, activators and effectors that enable safe and effective, permanent dose adjustment following initial dosing with a gene therapy. The invention provides at least in part dose adjustment of gene therapy, which allows single-injection gene therapy to replace multiinjection subcutaneous protein regiments while improving pharmacokinetics, safety / efficacy profile, medication adherence, and reducing healthcare costs.

[0012] Accordingly, aspects of the present disclosure relate to a system for permanently reducing gene expression from a therapeutic construct or a plethora of constructs within one or more cells comprised of at least: (a) a therapeutic transgene containing nucleic acid construct delivered in a formulation capable of entry into cells and enablement of stable therapeutic transgene expression for a period of at least 4 months, (b) an actuator comprised of one or more chemical or biological molecules, (c) an actuator response element and / or actuator response sequence that enables expression, activation, or stabilizes the effector element or effector sequence, (d) an effector sequence delivered in cis with the therapeutic transgene, (e) an effector element expressed from said effector sequence capable of permanently reducing said therapeutic transgene expression in a manner that elicits minimal bystander effect and may be therefore safe to surrounding tissues, organs, and organism, including in its entirety.

[0013] In some embodiments, the system is intended for delivery to subcutaneous tissues. 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 ophthalmic delivery. In some embodiments, the system is intended for delivery to the kidneys. In some embodiments, the system is intended for delivery to the bladder. In some embodiments, the system is intended for delivery to the mucous membranes. 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 embodiments, the system is intended to be used in conjunction with therapeutic gene therapy or delivered in conjunction with a therapeutic transgene for the treatment of human, plant, or veterinary diseases or for the augmentation of human, veterinary, or plant function.

[0014] In some embodiments, the therapeutic transgene encodes at least a part of a therapeutic antibody. In some embodiments, the therapeutic transgene encodes a protein or enzyme required for protein or enzyme 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 a non-coding RNA. In some embodiments, the therapeutic transgene encodes an aptazyme, aptamer, a ribozyme, or a genetic structure that is capable of enzymatic activity alone or in conjunction with a protein.

[0015] In some embodiments, the nucleic acid construct is comprised of double stranded DNA. In some embodiments, the nucleic acid construct is comprised of single stranded DNA. In some embodiments, the nucleic acid construct is comprised of single stranded RNA. In some embodiments, the nucleic acid construct is comprised of double stranded RNA. In some embodiments, the nucleic acid construct is comprised of single stranded or double stranded linear DNA. In some embodiments, the nucleic acid construct is comprised of single stranded or double stranded linear RNA. In some embodiments, the nucleic acid construct is comprised of single stranded or double stranded circular DNA. In some embodiments, the nucleic acid construct is comprised of single stranded or double stranded circular RNA.

[0016] In some embodiments, the system includes proteins delivered with the nucleic acid. In some embodiments, the system is delivered as part of a viral or non-viral formulation, in vivo or ex vivo. In some embodiments, the system is delivered by physical methods such as electroporation, nanoparticle bombardment, ultrasound mediated transfection, or chemically mediated transfection.

[0017] In some embodiments, the actuator is comprised at least in part from a tetracycline or tetracycline derivative. In some embodiments, the actuator is comprised at least in part from a doxycycline or doxycycline derivative. In some embodiments, the actuator is comprised at least in part from an antibiotic or antimicrobial peptide. In some embodiments, the actuator is comprised at least in part from a rapamycin or derivative thereof. In some embodiments, the actuator is comprised at least in part from a ganciclovir or derivative thereof. In some embodiments, the actuator is comprised at least in part from a ganciclovir, valganciclovir, valaciclovir, or derivative thereof. In some embodiments, the actuator is comprised at least in part from a tamoxifen or derivative thereof. In some embodiments, the actuator is comprised at least in part from an estrogen or derivative thereof. In some embodiments, the actuator is comprised at least in part from an acetaldehyde or derivative thereof. In some embodiments, the actuator is comprised at least in part from an L-arginine or derivative thereof. In some embodiments, the actuator is comprised at least in part from a Biotinyl- AMP or derivative thereof. In some embodiments, the actuator is comprised at least in part from a 2-phenylethyl butyrate or derivative thereof. In some embodiments, the actuator is comprised at least in part from a 6-hydroxy nicotine or derivative thereof. In some embodiments, the actuator is comprised at least in part from an erythromycin or derivative thereof. In some embodiments, the actuator is comprised at least in part from a macrolide or derivative thereof. In some embodiments, the actuator is comprised at least in part from a streptogramin or derivative thereof. In some embodiments, the actuator is comprised at least in part from NADH or derivative thereof. In some embodiments, the actuator is comprised at least in part from an element resulting in the formation of increased energy or heat. In some embodiments, the actuator is comprised at least in part from SCB1 or derivative thereof. In some embodiments, the actuator is comprised at least in part from 3-oxo-C8-HSL or derivative thereof. In some embodiments, the actuator is comprised at least in part from Phloretin or derivative thereof. In some embodiments, the actuator is a small molecule, peptide, protein or nucleic acid that is capable of increasing, decreasing, or modulating local or systemic inflammation, or a part of the inflammatory response. In some embodiments, the actuator is comprised at least in part from theophylline or derivative thereof. In some embodiments, the actuator is comprised at least in part from IPTG or derivative thereof. In some embodiments, the actuator is comprised at least in part from 5-fluorocytosine or derivative thereof. In some embodiments, the actuator is comprised at least in part from propionate or derivative thereof. In some embodiments, the actuator is comprised at least in part from acetate or derivative thereof. In some embodiments, the actuator is comprised at least in part from cumate, cumate ester, or cumic acid, or derivative thereof. In some embodiments, the actuator is comprised at least in part from gluconate or derivative thereof. In some embodiments, the actuator is comprised at least in part from maltose or derivative thereof. In some embodiments, the actuator is comprised at least in part from arabinose or derivative thereof. In some embodiments, the actuator is comprised at least in part from mono- or di- saccharide. In some embodiments, the actuator is comprised at least in part from a trimethoprim or derivative thereof. In some embodiments, the actuator is comprised at least in part from an exogenously administered small molecule, peptide, protein, RNA, DNA, or pharmacological formulation. In some embodiments, at least a part of the actuator can act as at least a part of the effector element. In some embodiments, the actuator is comprised at least in part from an endogenously expressed or presented peptide, hormone, protein, RNA, DNA, enzyme, mineral or chemical or biochemical substance and is capable of interacting with the actuator response element or actuator response sequence.

[0018] In some embodiments, the effector element is comprised at least in part from an inducible Caspase 9. In some embodiments, the effector element is comprised at least in part from a natural Caspase 9. In some embodiments, the effector element is comprised at least in part from a Zinc-Finger Nuclease. In some embodiments, the effector element is comprised at least in part from a transcription activator-like effector nucleases (TALEN). In some embodiments, the effector element is comprised at least in part from a meganuclease. In some embodiments, the effector element is comprised at least in part from a deoxycytidine deamination-derived editor. In some embodiments, the effector element is comprised at least in part from a deoxyadenosine deamination-derived base editor. In some embodiments, the effector element is comprised at least in part from a DNA-binding peptide. In some embodiments, the effector element is comprised at least in part from a viral or bacterial DNA- binding protein. In some embodiments, the effector element is comprised at least in part from a natural Caspase 8. In some embodiments, the effector element is comprised at least in part from a natural Caspase or a Caspase pathway protein. In some embodiments, the effector element is a FK506-binding protein 12 (e.g., with F36V mutation) fused with human caspase- 8 protein deleted for 1-215 AA and linked to the N-myristoylation signal from Src kinase. In some embodiments, the effector element is comprised in part from an FK506-binding protein 12 (e.g., with F36V mutation) fused with human caspase-9 protein deleted for 1-134 AA. In some embodiments, the effector element is comprised at least in part from at least a part of a viral thymidine kinase. In some embodiments, the effector element is comprised at least in part from at least a part of an Escherichia Coli cytosine deaminase. In some embodiments, the effector element is a protein with catalytic activity capable of chemically modifying a nucleotide, nucleoside, or nucleic acid. In some embodiments, the effector element is comprised at least a part from at least a part of a diphtheria toxin subunit A. In some embodiments, the effector element is comprised at least a part from at least a part of a 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 comprised at least in part from, at least a part of the human or simian diphtheria toxin receptor. In some embodiments, the effector element is comprised at least a part from at least a part of a bacterial, viral, or eukaryotic toxin. In some embodiments, the effector element is comprised at least a part from at least a part of a protein capable of inhibiting transcription. In some embodiments, the effector element is comprised at least a part from at least a part of a protein capable of inhibiting translation. In some embodiments, the effector element is comprised at least a part from at least a part of a protein capable of inducing apoptosis. In some embodiments, the effector element is comprised at least a part from at least a part of a protein capable of inducing senescence. In some embodiments, the effector element is comprised at least a part from at least a part of a protein capable of reducing general gene expression. In some embodiments, the effector element is comprised at least a part from at least a part of a nuclease. In some embodiments, the effector element is comprised at least a part from at least a part of a nuclease capable of site specific mutagenesis. In some embodiments, the effector element is comprised at least a part from at least a part of a nuclease and at least a part of a targeting element. In some embodiments, the effector element is comprised at least a part from at least a part of a Cas nuclease and guide RNA targeting the nuclease to the sequence. In some embodiments, the effector element is comprised at least a part from at least a part of a nuclease targeting the sequence. In some embodiments, the effector element is comprised at least a part from at least a part of a nuclease targeting a promoter region of the sequence. In some embodiments, the effector element is comprised at least a part from at least a part of a nuclease targeting a coding region of the sequence. In some embodiments, the effector element is comprised at least a part from at least a part of a nuclease targeting a post translational regulation element region of the sequence. In some embodiments, the effector element is comprised at least a part from at least a part of a recombinase. In some embodiments, the effector element is comprised at least a part from at least a part of a Cre recombinase. In some embodiments, the effector element is comprised at least a part from at least a part of a Kriippel-associated box domain from human gene ZNF10. In some embodiments, the effector element is comprised at least a part from at least a part of a zinc finger protein. In some embodiments, the effector element is comprised at least a part from at least a part of a cyclization recombinase with SV40 nuclear localization signal. In some embodiments, the effector element is comprised at least a part from at least a part of a Cre recombinase with SV40 T-antigen intron inserted in the ORE In some embodiments, the effector element is comprised at least a part from at least a part of a Flpe recombinase. In some embodiments, the effector element is comprised at least a part from at least a part of a Flpo recombinase. In some embodiments, the effector element is comprised at least a part from at least a part of a Dre recombinase. In some embodiments, the effector element is comprised at least a part from at least a part of an SV40 T-antigen sequence. In some embodiments, the effector element is comprised at least a part from at least a part of a piggyBac transposase. In some embodiments, the effector element is comprised at least a part from at least a part of a transposase. In some embodiments, the effector element is comprised at least a part from at least a part of a sleeping beauty transposase. In some embodiments, the effector element is comprised at least a part from at least a part of an argonaut protein. In some embodiments, the effector element is comprised at least a part from at least a part of a DNA methyltransferase. In some embodiments, the effector element is comprised at least a part from at least a part of a protein which makes up or interacts with chromatin remodeling complex. In some embodiments, the effector element is comprised at least a part from at least a part of a protein that makes up or interacts with the DNA methylation pathway. In some embodiments, the effector element is comprised at least a part from at least a part of a base editor. In some embodiments, the effector element is comprised at least a part from at least a part of a prime editor. In some embodiments, the effector element is comprised at least a part from at least a part of a nickase. In some embodiments, the effector element is comprised at least a part from at least a part of a viral, bacterial, or eukaryotic gene silencing protein. In some embodiments, the effector element is comprised at least a part from at least a part of a siRNA. In some embodiments, the effector element is comprised at least a part from at least a part of a miRNA. In some embodiments, the effector element is comprised at least a part from at least a part of FRT. In some embodiments, the effector element is comprised at least a part from at least a part of an integrase. In some embodiments, the effector element is comprised at least a part from at least a part of an RNA capable of reducing gene expression. In some embodiments, the effector element is comprised at least a part from at least a part of a protein enabling the effector degradation in the absence of an inducer. In some embodiments, the effector element is comprised at least a part from at least a part of a dihydrofolate reductase. In some embodiments, the effector element is comprised at least a part from at least a part of RNA, which is at least partially complementary to the genetic construct. In some embodiments, the effector element is comprised at least a part from at least a part of transposon or retrotransposon. In some embodiments, the effector element is comprised at least a part from at least a part of a streptococcus pyogenes Cas9. In some embodiments, the effector element is comprised at least a part from at least a part of a catalytically inactive Cas9. In some embodiments, the effector element is comprised at least a part from at least a part of a codon optimized Cas9. In some embodiments, the effector element is comprised at least a part from at least a part of Cas9 with a D10A mutation. In some embodiments, the effector element is comprised at least a part from at least a part of a dCas9 fused with KRAB. In some embodiments, the effector element is comprised at least a part from at least a part of dCas9 fused with a carboxy terminal bipartite repressor domain, KRAB-MeCP2 or derivative. In some embodiments, the effector element is comprised at least a part from at least a part of a Campylobacter jujeni Cas9 ortholog. In some embodiments, the effector element is comprised at least a part from at least a part of a CRISPR associated protein or an ortholog. In some embodiments, the effector element is comprised at least a part from at least a part of a Casl2a or Casl2b protein. In some embodiments, the effector element is comprised at least a part from at least a part of Casl3. In some embodiments, the effector element is comprised at least a part from at least a part of Casl2f or Casl3a. In some embodiments, the effector element is comprised at least a part from at least a part of a DNA, DNA capable of expressing, RNA, RNA, protein, or combination thereof capable of effecting DNA sequences in a sequence specific manner to induce double stranded breaks, single stranded breaks, methylation, chromatin modification, or generally epigenetic silencing. In some embodiments, the effector element is comprised at least in part from at least a part of a toxin, toxin derivative, modified toxin, or a homolog, ortholog, or paralog thereof. In some embodiments, the effector element is comprised at least in part from at least a part of a bacterial, fungal, plant, or animal toxin. In some embodiments, the effector element is comprised at least in part from at least a part of a vibrio cholerae toxin, shiga toxin, shigella toxin, ricin, saporin, staphylococcus aureus enterotoxin H, Clostridium perfringens enterotoxin, streptolysin O, ribosome inactivating protein (RIP) enzyme, immunotoxin, pseudomonas exotoxin A, diphtheria toxin (DT), or DT fragment. In some embodiments, the effector element is comprised at least in part from at least a part of a viral thymidine kinase or derivative thereof. In some embodiments, the effector element is comprised at least in part from at least a part of a bacterial cytosine deaminase or derivative thereof. In some embodiments, the effector element is comprised at least in part from at least a part of a D-amino-acid oxidase or derivative thereof. In some embodiments, the effector element is comprised at least in part from at least a part of a bacterial carboxypeptidase G2 or derivative thereof. In some embodiments, the effector element is comprised at least in part from at least a part of a purine nucleoside phosphorylase or derivative thereof. In some embodiments, the effector element is comprised at least in part from at least a part of a thymidine phosphorylase or derivative thereof. In some embodiments, the effector element is comprised at least in part from at least a part of a xanthine-guanine phosphoribosyl transferase or derivative thereof. In some embodiments, the effector element is comprised at least in part from at least a part of a nitroreductase or derivative thereof. In some embodiments, the effector element is comprised at least in part from at least a part of a penicillin-G amidase or derivative thereof. In some embodiments, the effector element is comprised at least in part from at least a part of a multi-drug activation enzyme or derivative thereof. In some embodiments, the effector element is comprised at least in part from at least a part of a horseradish peroxidase or derivative thereof. In some embodiments, the effector element is comprised at least in part from at least a part of a Beta-galactosidase or derivative thereof. In some embodiments, the effector element is comprised at least in part from at least a part of a deoxyribonucleotide kinase or derivative thereof. In some embodiments, the effector element is comprised at least in part from at least a part of a Beta-glucoronidase or derivative thereof. In some embodiments, the effector element is comprised at least in part from at least a part of a carboxypeptidase A or derivative thereof. In some embodiments, the effector element is comprised at least in part from at least a part of a cytochrome P450 or derivative thereof. In some embodiments, the effector element is comprised at least in part from at least a part of a deoxycytidine kinase or derivative thereof.

[0019] In some embodiments, the effector sequence encodes at least the effector element. In some embodiments, the effector sequence encodes at least a part of the consensus Lox sequence or a sequence capable of mediating the effect of a Lox sequence. In some embodiments, the effector sequence encodes at least a part of the consensus FRT sequence or a sequence capable of mediating the effect of an FRT sequence. In some embodiments, the effector sequence encodes at least a part of the consensus Rox sequence or a sequence capable of mediating the effect of a Rox sequence. In some embodiments, the effector sequence is comprised at least in part from GTGTAGTTCGACCATTCGTG (SEQ ID NO: 1). In some embodiments, the effector sequence is comprised at least in part from GTTCAGGATCACGTTACCGC (SEQ ID NO: 2). In some embodiments, the effector sequence is comprised at least in part from GGGCGAGGAGCTGTTCACCG (SEQ ID NO: 3). In some embodiments, the effector sequence is comprised at least in part from GAAGTTCGAGGGCGACACCC (SEQ ID NO: 4). In some embodiments, the effector sequence is comprised at least in part from GCAGATGTAGTGTTTCCACA (SEQ ID NO: 5). In some embodiments, the effector sequence is comprised at least in part from CCTAAGGTTAAGTCGCCCTCG (SEQ ID NO: 6). In some embodiments, the effector sequence is comprised at least in part from CAACAAGATGAAGAGCACCAA (SEQ ID NO: 7). In some embodiments, the effector sequence encodes at least a part of attB, attR, attL, or attP sites. In some embodiments, the effector sequence is comprised at least in part from genetic sequences, which can be targeted by integrases, recombinases, nucleases, nickases, chromatin complexes, or methylases or methyltransferases. In some embodiments, the effector sequence is comprised at least in part from genetic sequences, which can be targeted for silencing RNA or protein binding or can be itself silenced. In some embodiments, the effector sequence is a DNA sequence that can serve as a target of a guide RNA for a CRISPR associated protein. In some embodiments, the effector sequence is a DNA sequence derived from viral or bacterial genomes. In some embodiments, the effector sequence forms a secondary structure in the absence of an inducer molecule. In some embodiments, the effector sequence is an inflammation responsive promoter. In some embodiments, the effector sequence is derived at least in part from a mammalian promoter or enhancer. In some embodiments, the effector sequence forms a secondary structure in the presence of an inducer molecule.

[0020] In some embodiments, the actuator response element is a nucleic acid sequence or a protein that interacts with an actuator to enable activity of the effector element or effector sequence. In some embodiments, the actuator response element is comprised at least in part from a tetracycline-controlled transcriptional silencer (tTS). In some embodiments, the actuator response element is comprised at least in part from a Kruppel associated box (KRAB) AB domain of the kid-1 transcriptional repressor. In some embodiments, the actuator response element is composed at least in part of a reverse tetracycline-controlled transactivator. In some embodiments, the actuator response element is comprised at least in part of a viral protein such as VP 16 fused with a Tet-Repressor (TetR) or a reverse Tet- Repressor (rTetR). In some embodiments, the actuator response element is comprised of a tTS and rtTA driven by the same promoter. In some embodiments, the actuator response element is comprised of tTS and rtTA driven by different promoters. In some embodiments, the actuator response element is comprised at least in part from a transcriptional repressor. In some embodiments, the actuator response element is comprised at least in part from a transcriptional silencer. In some embodiments, the actuator response element is comprised at least in part from a DNA binding protein. In some embodiments, the actuator response element is comprised at least in part from a promoter binding protein. In some embodiments, the actuator response element is comprised at least in part from a transcription initiation complex binding protein. In some embodiments, the actuator response element is comprised at least in part of a protein responsible for DNA methylation. In some embodiments, the actuator response element is comprised at least in part of a protein responsible for histone modification. In some embodiments, the actuator response element is comprised at least in part of at least a part of a Tet-ON system. In some embodiments, the actuator response element is comprised at least in part of at least a part of a Tet-OFF system.

[0021] In some embodiments, the actuator response element is comprised at least in part of at least a part of AlcR, ArgR, BirA, EthR, HdnoR, HucR, MphR(A), PIP, Rex, RheA, ScbR, TraR, TtgR or their analog, homolog, ortholog or protein fragment, protein, or fusion protein of similar functionality. In some embodiments, the actuator response element is comprised at least in part from at least a part of lacUV5, lac, tac, trc, prpB, aceA, aceB, gntP, gntK, CJ10X2, tacM, malEl, gitl, BAD, SPL, P4-N14, cspB, aprE, sod, dapA, porB, ilvC, LIO, L26, L16, L51, H30, H36 or natural, synthetic or semi-synthetic analogs, homologs, orthologs or protein fragments, proteins, or fusion proteins of similar functionality. In some embodiments, the actuator response element is comprised at least in part from at least a part of ABIl-Gal4DBD, ABA, PVL1-VP16, PHR-p65, CIBl-TetR, or their analog, homolog, ortholog or protein fragment, protein, or fusion protein of similar functionality. In some embodiments, the actuator response element is comprised at least in part from at least a part of a transcriptional activator, enhancer, de-repressor, or protein, RNA, or DNA capable of increasing gene expression directly or indirectly in a targeted or generally cis-acting manner. Aspects of the present disclosure relate, at least in part, to methods of using any one of the systems described herein. In some embodiments, any one of the systems described herein is used in a method of reducing gene expression in a cell. In some embodiments, the cell is a liver cell, a muscle cell, a central nervous system cell, a pancreatic cell, a skin cell, a kidney cell, a bladder cell, a lung cell, a cardiac cell, an adipocyte, or a spleen cell. In some embodiments, any one of the systems described herein is used in a method of reducing gene expression in an organ. In some embodiments, the organ is liver, pancreas, skin, brain, eye, spleen, kidney, heart, or bladder.

[0022] Aspects of the present disclosure relate, at least in part, to methods of delivering any one of the systems described herein to a cell, tissue, or organ of a subject. Aspects of the present 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 macaque. 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 canine. In some embodiments, the mammalian subject is a cat.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented in this disclosure. The accompanying drawings are not intended to be drawn to scale. The drawings are illustrative only. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

[0025] FIG. 1 shows an npRB2.29vl plasmid map.

[0026] FIG. 2 shows an npRB2.28vl plasmid map.

[0027] FIGs. 3A-3B are bar graphs showing effective expression of reporter genes. FIG. 3A show multiple systems demonstrating effective expression of a reporter gene. FIG. 3B shows relative reduction in expression following equal dose of inducer.

[0028] FIG. 4 shows an npRB2.31vl plasmid map.

[0029] FIG. 5 shows an npRB2.33vl plasmid map. FIG. 6 is a bar graph showing relative bioluminescence of primary human adipocytes treated with liquid nanoparticles (LNP).

[0030] FIG. 7 shows an npRB2.32vl plasmid map.

[0031] FIG. 8 shows an npRB2.30vl plasmid map.

[0032] FIG. 9 is a bar graph showing relative bioluminescence of primary human adipocytes treated with liquid nanoparticles (LNP).

[0033] FIG. 10 shows an npRB2.35vl plasmid map.

[0034] FIG. 11 shows an npRB2.34vlT plasmid map.

[0035] FIG. 12 is a bar graph showing relative expression in primary human adipocytes treated with liquid nanoparticles (LNP).

[0036] FIG. 13 shows an npRB2.22vl (CBh-IGF2-P2A-Tet3G+TRE3G-GSDMD-NT) plasmid map.

[0037] FIG. 14 shows an npRB2.23vl (CMV-GBAl-P2A-Tet3G+TRE3G-IE2-P2A-tTA) plasmid map.

[0038] FIG. 15 shows an npRB2.24vl (attP-CBh-attB-LMNA-P2A-Tet3G+ TRE3G-Bxbl) plasmid map.

[0039] FIG. 16 shows an npRB2.25vl (CBh-CFTR-P2A-iCasp9) plasmid map.

[0040] FIG. 17 shows an npRB2.26vl (CBh-HEXA-P2A-iCasp8) plasmid map.

[0041] FIG. 18 shows an npRB2.27v2 (TRE3G-FGF18-P2A-EacI-NES- KRAB+7xLacOPromoter-tTS) plasmid map.

[0042] DETAILED DESCRIPTION

[0043] The present invention generally describes a combination of target cell types and anatomical regions, genetic elements, actuators, effector elements, effector sequences, and actuator response elements that enable safe and effective, permanent dose adjustment following initial dosing with a gene therapy. The invention for the first time enables dose adjustment of gene therapy in a permanent, durable, safe and effective manner. The invention therefore for the first time allows for the use of gene therapy to replace protein replacement, enzyme replacement, or general protein injection treatments with a single injection adjustable gene therapy approach.

[0044] In general, the invention describes a genetic construct for the delivery to a broad range of cell types, but most ideally adipocytes or other non-life sustaining or organ sustaining cell type that includes an actuator, effector element, effector sequence, and actuator response element. For the purpose of this invention an actuator can be a small molecule, large molecule, peptide, protein, nucleic acid, physical or chemical stimulus that enables, increases, or activates the expression or activity of an effector element by acting directly on the effector element or on the actuator response element or both. Similarly, for the purpose of this invention, an 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 aforementioned elements which can target therapeutic genetic sequences or more specifically effector sequences to enable general dose reduction or specifically reduction in the level of transcription, expression, translation, or functionality of the therapeutic genetic cargo. In line with this, effector sequences are targets, or downstream mediators of activity or recognition sites, or sites of activity of, or are generally associated with the effector element to enable down-titration or dose reduction of the gene therapy treatment. Finally, actuator response elements are proteins, nucleic acid sequences, DNA, promoters, enhancers, insulators, or generally genetic sequences, that once acted upon by the actuator change the level of expression, translation, transcription, or activation of the effector element. In general, said elements or genetic sequences encoding said elements are delivered in cis at the time of treatment or re-treatment, with the exception of the actuator, which is delivered at the time of dose adjustment. The sequences of the actuator, effector element, effector sequence, and / or actuator response element may be optionally codon-optimized for the species of interest.

[0045] In one representative example, an actuator such as tetracycline induces expression of a CRISPR-Cas9 protein by activating a Tet-ON operator, which drives expression of a CRISPR-Cas9 protein and guide RNA. The CRISPR-Cas9 protein and guide RNA, which are the effector elements, target the effector sequence which separates a constitutive promoter that drives therapeutic transgene expression. As a result, administration of tetracycline, enables expression of a nuclease that separates the promoter from the coding region of the therapeutic transgene reducing the level of expression of the therapeutic transgene from this construct. Since the effect is sequence specific and the DNA encoding the effector element, effector sequence, actuator response element, therapeutic transgene, and associated promoters are delivered in cis (or all together in one genetic construct), the initial treatment produces expression at a level L, which is reduced to a level less than L after activation of the dose reduction system by administration of an actuator, which can be dosed as a safe exogenous small molecule to reduce the level of therapeutic transgene expression.

[0046] Another optional actuator can be derived from a class of Stop-Codon readthrough enhancers. One example of Stop-Codon readthrough enhancers are aminoglycoside antibiotics. The therapeutic transgene can be expressed in cis ending with a stop codon, the read-through of which can be facilitated by the administration of a Stop-Codon readthrough enhancer or a molecule enabling or enhancing Stop-Codon readthrough directly or indirectly. The Stop-Codon, may for example be followed by a self-cleaving peptide and a suicide gene such as for example Diphtheria Toxin or Diphtheria Toxin subunit. Some representative examples of readthrough enhancers include Gentamicin, G418, Paromomycin, Neomycin, Sisomycin, Lividomycin or derivatives or combinations thereof. Some examples of stop codons may include UGA, UAG, UAA, with optional 4thbase allowing for suppression of natural read-through and / or enhancement or readthrough following activation by a readthrough enhancer. As an example, Gentamicin can enhance readthrough when an UGA stop codon is used and then followed by a Cytosine (C) or Uracil (U), less preferably Adenine (A) and least preferably Guanine (G). Said system may be used alone or in conjunction with other systems such as for example the use of a base editor to change one of the bases neighboring the stop codon in the stop codon, or in a codon to turn the said codon into a stop codon.

[0047] In general, the invention is comprised of a system for permanently or less preferably and optionally transiently reducing gene expression from a therapeutic construct or a plethora of constructs within one or more cells comprised of at least several elements. Said elements can include a therapeutic transgene containing nucleic acid construct delivered in a formulation capable of entry into cells and enablement of stable therapeutic transgene expression for a period of at least 4 months, one or more actuators comprised of one or more chemical or biological molecules that can be administered exogenously to the system, cell, tissue, organ, or organism undergoing treatment with said system, one or more actuator response elements and / or actuator response sequences that enable expression, activation, or stabilize one or more effector element or effector sequence, an effector sequence delivered in cis with the therapeutic transgene, and one or more effector elements expressed from said effector sequence(s) capable of permanently reducing said therapeutic transgene expression in a manner that elicits minimal bystander effect and is therefore safe to surrounding tissues, organs, and organism, including in its entirety.

[0048] The system may be part of a larger formulation or one or more formulations and be administered via drug delivery system or delivered in a vial for subsequent administration, or a prefilled syringe or cartridge. Alternatively, the treatment can be administered via an autoinjector or a patch injector, via ultrasonic guidance, or via other guidance techniques to ensure delivery to the correct location. Alternatively, and optionally the treatment can be delivered by a set of needles or via a set or series of injections to more evenly distribute the constructs in the treatment site. The gene therapy treatment can be delivered via a patch, microneedles, a set of microneedles, jet injection, on a carrier, or using other means to alter the integrity of the tissues through which or into which it is being delivered.

[0049] In some embodiments of the present invention the target cell type of said system may be one or more of an adipocyte, pre-adipocyte, progenitor capable of differentiating into an adipocyte, stem cell capable of differentiating into an adipocyte, a resident cell type of the subcutaneous tissues, or a transient cell or stem cell, but one that can become a resident cell of the subcutaneous tissues. The system is intended to treat or prevent any human or veterinary disorder, but may be used in some specific embodiments to treat monogenic disorders, enzyme insufficiency disorders, protein insufficiency disorders (as protein replacement therapy), metabolic disorders, autoimmune disorders, oncologies, neurologic disorders, cardiovascular pathologies, musculoskeletal disorders, hematologic disorders, infectious disorders, dermatologic disorders, immune system disorders, pulmonary system disorders, kidney or bladder disorders, or disorders of other organs, tissues, systems, or combinations thereof. In some embodiments of the present invention the system is intended for the delivery of therapeutic or prophylactic antibodies via expression from the genetic construct, therapeutic, preventative, augmentative, or 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 via expression from the genetic construct.

[0050] Generally, the system may be delivered via one or more means to the subcutaneous tissues, intraperitoneally, delivered directly to adipose tissues, to white adipose tissues, or targeted or delivered to adipocytes. Alternatively and optionally in conjunction with the aforementioned delivery locations, tissues, and forms, the system can be delivered via intramuscular delivery, dosed intravenously or directly to the liver via an artery or a vein, the system can be used in the central nervous system via direct or indirect delivery, delivered to the fascia, subarachnoid space, the pancreas, epidermis, the eye or generally to parts of the eye as part of ophthalmic or intravitreous delivery, the kidneys, bladder, mucous membranes, lung, spleen, heart, or any other cell, tissue, organ, or system of cells, tissues, organs, or the entire organism.

[0051] Said system can be used to treat disease or in conjunction with other therapeutics to manage symptoms or treat a disease or a pathology, or to bestow function upon an organism, organ, or tissue. The system may be used in conjunction with a therapeutic gene to treat human, veterinary, or plant diseases or augment human, veterinary, or plant function. The system is intended to contain at least one therapeutic genetic element which can be a coding gene or a non-coding gene, or a DNA segment that directly provides a therapeutic element or is transcribed into an RNA that provides a therapeutic element. Said system may therefore encode at least a part of a therapeutic antibody, protein, enzyme, fusion protein, peptide, aptazyme, or transfer RNA (tRNA), ribosomal RNA (rRNA), long non-coding RNA (Inc-RNA or In-RNA or long nc-RNA), short non-coding RNA (short ncRNA or snc-RNA), micro RNA (miRNA), messenger RNA (mRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), PlWI-interacting RNA (piRNA) or other RNA, protein, or combination thereof. The therapeutic transgene may directly encode an aptazyme or maybe flanked by an aptazyme or multiple aptazymes, ribozymes, or genetic structures capable of enzymatic activity alone or in conjunction with a protein or other nucleic acids.

[0052] The system disclosed in this invention contains a nucleic acid, which may be one or more of a double stranded DNA (dsDNA), single stranded DNA (ssDNA), double stranded RNA (dsRNA), or single stranded RNA (ssRNA). Optionally, the construct may contain a DNA-RNA hybrid where one or more strands are RNA and one or more strands are DNA or one or more parts of one or more strands are RNA and one or more parts of one or more strands are DNA. Said DNA or RNA may be linear, circular, supercoiled, partially supercoiled, or not-supercoiled, partially linear, segmented, covalently closed, modified with chemical modifications such as caps and polyA tails, or chemically modified for enhanced stability, decreased immunogenicity, or generally to bestow any other biological function.

[0053] The system may be delivered as a protein-nucleic acid complex or may generally contain proteins inside the formulation that contains the nucleic acid or as part of the formulation that contains the nucleic acid. Said proteins can range from DNA binding proteins, to proteins promoting decreased immunogenicity, to proteins promoting nucleic acid transport inside a cell, to proteins promoting protection of the nucleic acid, to proteins promoting binding to cell proteins, membranes, organelles, transmembrane proteins, nuclear proteins, or other cellular structures and may generally serve structural or therapeutic activity or augment structural or therapeutic activity of the system.

[0054] The system can be delivered using a viral or non- viral vector containing formulation, or a formulation that contains both viral and non-viral vectors with or without targeting or additional targeting. Said delivery can be performed in vivo or ex vivo, as in for example the delivery to immune system cells for the purposes of production of Chimeric Antigen Receptor T Cells or CAR-T cells. The system can optionally or additionally be delivered by physical or chemical methods including one or more of electroporation, nanoparticle bombardment, ultrasound mediated transfection, or chemically mediated transfection using a transfection reagent in vivo or ex vivo. Generally, the system is intended to be delivered on its own or via a carrier, vehicle, formulation, or as part of greater constructs or superstructures to enable therapeutic effect, targeting, dose adjustment, safety, or durability.

[0055] The system entails the use of an actuator the delivery of which is performed at a time when the system requires adjustment of therapeutic effect, titration, or dose adjustment. Said actuator may be an exogenous or endogenous small molecule and may be delivered or the production of said actuator may be elicited by the delivery of another means, physical or chemical. In some embodiments of the present invention said actuator is comprised at least in part from a tetracycline or tetracycline derivative, a doxycycline or doxycycline derivative, an antibiotic or antimicrobial peptide or derivative, a rapamycin or derivative thereof, a ganciclovir or derivative thereof, a valganciclovir or derivative thereof, a valaciclovir or derivative thereof, a tamoxifen or derivative thereof, a hormone or an estrogen or derivative thereof, an acetaldehyde or derivative thereof, an L-arginine or derivative thereof, a Biotinyl- AMP or derivative thereof, caffeine or derivative thereof, a 2-phenylethyl butyrate or derivative thereof, a 6-hydroxy nicotine or derivative thereof, an erythromycin or derivative thereof. In some embodiments the actuator may be comprised at least in part of a macrolide or derivative thereof, a streptogramin or derivative thereof, theophylline or derivative thereof, 5-fluorouracile or derivative thereof, propionate or derivative thereof, 5-fluorocytosine or derivative thereof, Isopropyl P-D-l -thiogalactopyranoside (IPTG) or derivative thereof, acetate or derivative thereof, NADH or derivative thereof, or generally an antibiotic, antimicrobial, antiviral, anti-inflammatory, hormone, or pro-inflammatory agent, or generally an approved therapeutic agent for the treatment of some other disease, symptom, or pathology. In some embodiments of the present 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 comprised at least in part of a natural or synthetic peptide or protein such as SCB 1 or derivative thereof or a fusion protein or peptide that is chemically modified. In yet other embodiments of the present invention the actuator may be comprised at least in part from 3-oxo-C8-HSL or derivative thereof, phloretin or derivative thereof, or generally a small molecule, peptide, protein or nucleic acid that is capable of increasing, decreasing, or modulating local or systemic inflammation, or a part of the inflammatory response. In yet other embodiments the actuator is comprised at least in part form a cumate, cumate ester, cumic acid, or derivative thereof, or 2,3-dihydroxy-p-cumate or derivative thereof, p-cumate or derivative thereof, or cis-5,6- dihydroxy-4-isopropylcyclohexa-l,3-dienecarboxylate or derivative thereof. In other embodiments of the present invention the actuator may be comprised at least in part from at least a part of gluconate, maltose, dextrose, idonate, arabinose or derivative thereof, or be generally comprised from a monosaccharide, disaccharide, polysaccharide, or oligosaccharide. In yet other embodiments of the present invention the actuator may be comprised at least in part from at least a part of trimethoprim or derivative thereof. Finally, for the purposes of this invention the actuator can be comprised at least in part from an exogenously administered small molecule, peptide, protein, RNA, DNA, or pharmacological formulation with or without the additional physical activity or action from within or outside of the treated organism. Moreover, an actuator may be comprised from the effector element or serve the purpose of the actuator and effector in the therapeutic formulation and may be comprised at least in part of an endogenously expressed or presented peptide, hormone, protein, RNA, DNA, enzyme, mineral or chemical or biochemical substance and is capable of interacting with the actuator response element or actuator response sequence.

[0056] The system disclosed in the present invention utilizes one or more effector elements, which when induced, activated, or produced following action by an actuator cause by direct or indirect action the reduction of the therapeutic dose, therapeutic transgene expression, elimination of the cell carrying the system, or reduction in the production of therapeutic effect. Said effector elements may be comprised at least in part from an inducible Caspase 9, natural Caspase 9, Zinc-Finger Nuclease, transcription activator-like effector nucleases (TALEN), meganuclease or homing endonuclease, deoxycytidine deamination-derived editor, deoxyadenosine deamination-derived base editor, or combination thereof and mediate DNA- binding, cleavage, single stranded breaks, double stranded breaks, or death of the cell or cells carrying the system or therapeutic cargo. Other effectors, which may be used for the purpose of down-titration include Caspase 1, 3, 4, 5, 6, 8, 9, 11, and 12, FK506-binding protein 12 (with F36V mutation) fused with human caspase-8 protein deleted for 1-215 AA and linked to the N-myristoylation signal from Src kinase, FK506-binding protein 12 (with F36V mutation) fused with human caspase-9 protein deleted for 1-134 AA, bacterial or viral thymidine kinase, bacterial or viral cytosine deaminase, or generally any kinase, Caspase, or Caspase pathway protein a fragment or hybrid thereof. In some embodiments of the present invention the effector element may be comprised at last in part from at least a part of a toxin or diphtheria toxin (DT) or a toxin subunit or diphtheria toxin subunit A (DTA) or at least a part of or in combination with a diphtheria toxin subunit B (DTB), homologs, analogs, or fragments thereof or generally dimerizable, trimerizable, or monomeric toxins with or without stabilization or destabilization or binding domains. In general, the effector element may mediate down titration by eliminating the cell carrying the system or block expression, transcription, translation, or general production of the therapeutic protein. Some specific effectors may be comprised at least a part from at least a part of Botulinum toxin, Cholera toxin, Clostridium perfringens toxin, Corynebacterium diphtheriae toxin, Diphtheria toxin, E. coli Shiga toxin, E. coli heat-labile toxin, E. coli heat-stable toxin, Helicobacter pylori VacA toxin, Pertussis toxin, Pseudomonas aeruginosa exotoxin A, Staphylococcus aureus enterotoxin, Streptococcus pyogenes streptolysin 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 (HTLV-1) Tax protein, Influenza A virus PB 1-F2 protein, Poxvirus F1L protein, Rabies virus P protein, Rotavirus NSP4 protein, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N protein, Vaccinia virus F1L protein, West Nile virus NS3 protein or related protein. The origin of the effector may be bacterial, viral, archaeal or eukaryotic and may include at least a part of Bax, Bak, Caspases, Apaf-1, Cytochrome c, Fas receptor, Fas ligand, TNF receptor 1, TNF-related apoptosis-inducing ligand, p53, Bad, Bid, Bim, Noxa, CD47 and may function in conjunction with external stimuli, such as for example CAR-T cells targeting CD47 transmembrane proteins. In general, the effector may be comprised at least in part from a toxin, such as a diphtheria toxin or a toxin receptor such as a human or a simian diphtheria toxin receptor with the use of a toxin to mediate effect; said toxin or receptor may be viral, bacterial, archaeal or eukaryotic origin and optionally able to inhibit transcription, translation, cell division, mitosis, respiration, oxidative phosphorylation, glycolysis, promote apoptosis, induce senescence or generally one or more cellular functions required for transgene or gene expression to completely eliminate said function or reduce it ideally permanently, but optionally temporarily. In other embodiments of the present invention the effector element may be a nuclease, with or without targeting, capable of inactivating the therapeutic construct by for example separating the promoter from the therapeutic transgene to prevent therapeutic transgene expression. As such, the effector element may be comprised at least in part of at least a part of a nuclease, nuclease capable of site specific mutagenesis, nuclease and at least a part of a targeting element, a CRIS PR-associated protein (Cas) nuclease with or without a single guide RNA targeting the nuclease to at least a part of the transgene delivered as part of the system, nuclease targeting a coding region of the therapeutic transgene, nuclease targeting a post translational regulation element region of the sequence the encodes or follows the therapeutic transgene, or a recombinase, such as a Cre recombinase, or other bacterial, viral, archaeal, or eukaryotic recombinase or related protein. The effector element may be comprised of one or more domains or components or itself be a plethora of elements and may be comprised at least in part of at least a part of a Kruppel-associated box domain from human gene ZNF10, or generally a zinc finger protein (ZFN) or ZFN domain, or a part of a cyclization recombinase with SV40 nuclear localization signal, or a Cre recombinase with SV40 T-antigen or a fragment thereof, or a Flpe recombinase, or a Flpo recombinase, or an SV40 T-antigen sequence or fragment thereof or a piggyBac transposase, or generally a transposases, an argonaut protein, a DNA methyltransferase, or a protein which makes up or interacts with chromatin remodeling complex, makes up or interacts with the DNA methylation pathway. Said effector elements may act directly on the therapeutic transgene delivered as part of the system or on proximal, neighboring, flanking, or distal sequences, or indirectly to reduce gene expression, transcription, translation, or generally destabilize expression, transcription, translation, or mRNA to reduce the dose of the therapeutic expressed permanently or induce expression of another protein that can reduce the expression, transcription, or translation of the therapeutic directly or indirectly.

[0057] In some embodiments of the present invention the effector element acts by epigenetic silencing or chromatin remodeling, and in other embodiments of the present invention the effector element acts to cut, edit, deactivate, or create double or single stranded breaks in the therapeutic transgene or other nucleic acids delivered in cis with the system. For the aforementioned purpose or other purposes the effector element may be comprised at least in part from at least a part of a base editor, prime editor, nickase, nuclease, recombinase, FRT, transposon, transposase, retrotransposon, integrase, dihydrofolate reductase, or reverse transcriptase, or generally a protein with the ability to modify DNA, chromatins, or RNA.

[0058] In other embodiments of the present invention the effector element may be comprised of a non-coding gene that produces an RNA, such as a non-coding RNA that can interfere with gene expression, transcription, translation to cause permanent epigenetic silencing of the therapeutic construct as in for example X-inactivation, which may or may not be at least partially complementary or antisense to the genetic construct delivered in cis, or to create a downstream secondary effect that induces apoptosis, genetic silencing, or recombination or genetic material to ultimate effect down-titration. The effector element may be comprised of one or more elements such as a protein and an RNA, as an example, it can be comprised at least in part of a streptococcus pyogenes 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 inactive Cas9, Campylobacter jujeni Cas9 ortholog, or a part of a codon optimized Cas9, or a part of Cas9 with a D10A mutation, CRISPR associated protein or an ortholog, or a Cas protein such as dCas9 fused with Kruppel associated box (KRAB), or a KRAB-MeCP2 (methyl-CpG binding protein 2), Cas 12a, AsCasl2a, AsCasl2a-RR, AsCasl2a-RVR, FnCasl2a, Cas 12b, Casl2e, Casl2f, UnlCasl2fl, CnCasl2fl, Casl2j, Casl3, Casl3a or derivative ortholog, homolog, or paralog thereof.

[0059] The effector element may have stabilizing or destabilizing domains, activating or inactivating domains, or dimerizing or trimerizing domains, which may or may not be responsive to an external inducer molecule or an inducer that is inherently present at certain times or in certain events in vivo, as such, the effector element may be comprised at least in part of a destabilization domain or a part of a protein that enables effector degradation in the absence of some exogenous or endogenous inducer-based stimulus.

[0060] In other embodiments of the present invention the effector element may be comprised at least a part from at least a part of a DNA, DNA capable of expressing, RNA, RNA, protein, or combination thereof capable of effecting DNA sequences in a sequence specific manner to induce double stranded breaks, single stranded breaks, methylation, chromatin modification, or generally epigenetic silencing. Some examples of said DNA are mobile transposable elements, which in the presence of endogenous or exogenous proteins can move or insert into the system’s nucleic acid in such a way as to decrease the expression of the therapeutic transgene from said system. In some embodiments of the present invention, the effector may be comprised of a triplex-forming oligonucleotide, with or without a stabilizer, to inhibit expression of the therapeutic transgene.

[0061] In yet other embodiments of the present invention the effector element in the presence of an exogenous inducer or on its own may be capable of reducing transcription, inducing apoptosis, inducing necrosis, inducing pyroptosis, inducing ferroptosis, inducing senescence, reducing gene expression, reducing secretion, or reducing translation of the therapeutic transgene or generally of some of the genes in the cell that hosts the system’s nucleic acid and may be comprised from at least a part of at least a part of a toxin, toxin derivative, modified toxin, or a homolog, ortholog, or paralog thereof or a combination thereof. Said toxins may be at least a part of a bacterial, fungal, plant, or animal toxin, or specifically at least a part of a vibrio cholerae toxin, shiga toxin, shigella toxin, ricin, saporin, staphylococcus aureus enterotoxin H, Clostridium perfringens enterotoxin, streptolysin O, ribosome inactivating protein (RIP) enzyme, immunotoxin, pseudomonas exotoxin A, diphtheria toxin (DT), or DT fragment with or without coupling to activation, inactivation, nuclear localization, or signaling domains. In some specific emdobiments of the present invention the effector element may be comprised at least in part from at least a part of a viral thymidine kinase, a cytosine deaminase, D-amino-acid oxidase, carboxypeptidase G2, purine nucleoside phosphorylase, thymidine phosphorylase, xanthine-guanine phosphoribosyl transferase, nitroreductase, penicillin-G amidase, multi-drug activation enzyme, Beta-lactamase, horseradish peroxidase, Beta-galactosidase, deoxyribonucleotide kinase, Beta-glucoronidase, carboxypeptidase A, cytochrome P450, deoxycytidine kinase of bacterial, viral, archaeal or eukaryotic origin or derivative thereof.

[0062] In yet other embodiments of the present invention the effector element in the presence of an exogenous inducer or on its own may be capable of inducing programmed cell death, including apoptosis, pyroptosis, ferroptosis, or necrosis and may be comprised at least in part of gasdermin A, gasdermin B, gasdermin C, gasdermin D, or gasdermin E, homologs, analogs, or fragments thereof or generally oligomerizable molecules, nucleic acids, proteins or peptides capable of forming pores in various cellular membranes. In general, the effector may be comprised at least in part of a DNA-binding peptide, viral or bacterial DNA-binding protein, a protein with catalytic activity capable of chemically modifying a nucleotide, nucleoside, or nucleic acid.

[0063] One or more effector elements of the system may act on one or more effector sequences of the system, which may themselves encode at least a portion of the effector element. Said effector sequences are intended to mediate the dose down-titration activity of the effector element and may do it directly, indirectly, enhance activity, moderate activity, mediate activity, or amplify the activity of the effector element. Said effector sequences may be comprised at least a part from at least a part of the consensus Lox sequence or a sequence capable of mediating the effect of a Lox sequence, the consensus ERT sequence or a sequence capable of mediating the effect of an ERT sequence, the consensus Rox sequence or a sequence capable of mediating the effect of a Rox sequence, or be any sequence that can be targeted by a DNA-binding enzyme, restriction enzyme, DNase, nickase, nuclease, or any other effector element and may be specifically comprised at least a part from at least a part of degree of homology with the genome of the species being targeted for the therapeutic or any other benefit of said system. In other specific embodiments the effector sequence can be comprised from at least a part 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 recognizable by an enzyme. In some embodiments a PAM sequence may be optimized and lengthened to reduce off-target effects. In other specific embodiments the effector sequence can be comprised from at least a part of the attB, attR, attL, or attP sites or other restriction enzyme coding sites, or generally sites that can be safely targeted by integrases, recombinases, nucleases, nickases, chromatin complexes, or methylases or methyltransferases with minimum deleterious off-target activity. In some specific embodiments of the present invention said effector sequences can be at least in part comprised of at least a part of genetic sequences, which can be targeted for silencing RNA or protein binding or can be itself silenced, or serve as a target of a guide RNA for a CRISPR associated protein, or be derived from viral or bacterial genomes, or sequences that form secondary structures in the absence or presence of an exogenous or endogenous inducer molecule, or sequences that comprise at least a part of an inflammation responsive promoter or enhancer, a mammalian promoter or enhancer, or promoters enhancers or trans-activators capable of forming secondary structures in the presence or absence of inducer molecules.

[0064] Moreover, the present invention discloses a system, which may optionally contain an actuator response element, which is a nucleic acid sequence or a protein that interacts with an actuator to enable activity of the effector element or effector sequence, or which is comprised at least in part from a tetracycline-controlled transcriptional silencer (tTS), or which is comprised at least in part from a Kruppel associated box (KRAB) AB domain of the kid-1 transcriptional repressor, or which is composed at least in part of a reverse tetracycline- controlled transactivator, or which is comprised at least in part of a viral protein such as VP 16 fused with a Tet-Repressor (TetR) or a reverse Tet-Repressor (rTetR), or which is comprised of a tTS and rtTA driven by the same promoter, or which is comprised of tTS and rtTA driven by different promoters, or which is comprised at least in part from a transcriptional repressor, or which is comprised at least in part from a transcriptional silencer, or which is comprised at least in part from a DNA binding protein, or which is comprised at least in part from a promoter binding protein, or which is comprised at least in part from a transcription initiation complex binding protein, or which is comprised at least in part of a protein responsible for DNA methylation, or which is comprised at least in part of a protein responsible for histone modification, or which is comprised at least in part of at least a part of a Tet-ON system, or which is comprised at least in part of at least a part of a Tet-OFF system, or which is comprised at least in part of at least a part of AlcR, ArgR, BirA, EthR, HdnoR, HSP70B (HSPA7), HucR, LgnR, MphR(A), PIP, Rex, RheA, ScbR, TraR, TtgR or their analog, homolog, ortholog or protein fragment, protein, or fusion protein of similar functionality, or which is comprised at least in part from at least a part of lacUV5, lac, tac, trc, prpB, aceA, aceB, gntP, gntK, CJ10X2, tacM, malEl, gitl, BAD, SPL, P4-N14, cspB, aprE, sod, dapA, porB, ilvC, LIO, L26, L16, L51, H30, H36 or natural, synthetic or semi- synthetic analogs, homologs, orthologs or protein fragments, proteins, or fusion proteins of similar functionality or combination of one or more of the aforementioned elements.

[0065] Moreover, the actuator response element disclosed in this invention may be comprised at least in part from at least a part of ABIl-Gal4DBD, ABA, PVL1-VP16, PHR-p65, CIB1- TetR, or their analog, homolog, ortholog or protein fragment, protein, or fusion protein of similar functionality, or which is comprised at least in part from at least a part of a transcriptional activator, enhancer, de-repressor, or protein, RNA, or DNA capable of increasing gene expression directly or indirectly in a targeted or generally cis-acting manner.

[0066] The components described in this invention can optionally work in concert to enable down-titration of the system and therefore a reduction in the level of expression of one or more therapeutic elements, but may also optionally have additional functionality such as transient down-regulation of expression or transient upregulation of expression, or may impact other cellular elements or pathways, or interact with other cellular sequences, proteins, organelles, or function, with at least one of the main goals being changing the level of therapeutic effect, expression, transcription, translation, benefit, or side-effect of the overall organism, system, organ, tissue, or cell effected by the system, or in essence dose reduction, or dose titration, or dose down-titration, or dose up-titration, or some similar action or activity.

[0067] ADDITIONAL EMBODIMENTS

[0068] 1. A system for permanently reducing gene expression from a therapeutic construct or a plethora of constructs within one or more cells comprised of at least: a. A therapeutic transgene containing nucleic acid construct delivered in a formulation capable of entry into cells and enablement of stable therapeutic transgene expression for a period of at least 4 months, b. An actuator comprised of one or more chemical or biological molecules, c. An actuator response element and / or actuator response sequence that enables expression, activation, or stabilizes the effector element or effector sequence, d. An effector sequence delivered in cis with the therapeutic transgene, e. An effector element expressed from said effector sequence capable of permanently reducing said therapeutic transgene expression in a manner that elicits minimal bystander effect and is therefore safe to surrounding tissues, organs, and organism in its entirety.

[0069] 2. A system in Embodiment 1 intended for delivery to subcutaneous tissues.

[0070] 3. A system in Embodiment 1 intended for intraperitoneal delivery.

[0071] 4. A system in Embodiment 1 intended for delivery to adipocytes.

[0072] 5. A system in Embodiment 1 intended for intramuscular delivery.

[0073] 6. A system in Embodiment 1 intended for delivery to the liver.

[0074] 7. A system in Embodiment 1 intended for delivery to the central nervous system.

[0075] 8. A system in Embodiment 1 intended for delivery to the fascia.

[0076] 9. A system in Embodiment 1 intended for delivery to the subarachnoid space.

[0077] 10. A system in Embodiment 1 intended for delivery to the pancreas.

[0078] 11. A system in Embodiment 1 intended for delivery to the epidermis.

[0079] 12. A system in Embodiment 1 intended for ophthalmic delivery.

[0080] 13. A system in Embodiment 1 intended for delivery to the kidneys.

[0081] 14. A system in Embodiment 1 intended for delivery to the bladder.

[0082] 15. A system in Embodiment 1 intended for delivery to the mucous membranes.

[0083] 16. A system in Embodiment 1 intended for delivery to the lung.

[0084] 17. A system in Embodiment 1 intended for delivery to the spleen.

[0085] 18. A system in Embodiment 1 intended for intravascular delivery.

[0086] 19. A system in Embodiment 1 intended for delivery to the heart.

[0087] 20. A system in Embodiment 1 intended to be used in conjunction with therapeutic gene therapy or delivered in conjunction with a therapeutic transgene for the treatment of human, plant, or veterinary diseases or for the augmentation of human, veterinary, or plant function.

[0088] 21. A therapeutic transgene in Embodiment 1, which encodes at least a part of a therapeutic antibody.

[0089] 22. A therapeutic transgene in Embodiment 1, which encodes a protein or enzyme required for protein or enzyme replacement therapy.

[0090] 23. A therapeutic transgene in Embodiment 1, which encodes a therapeutic peptide. 24. A therapeutic transgene in Embodiment 1, which encodes a protein or fusion protein.

[0091] 25. A therapeutic transgene in Embodiment 1, which encodes a non-coding RNA.

[0092] 26. A therapeutic transgene in Embodiment 1, which encodes an aptazyme, aptamer, a ribozyme, or a genetic structure that is capable of enzymatic activity alone or in conjunction with a protein.

[0093] 27. A nucleic acid construct in Embodiment 1, which is comprised of double stranded DNA.

[0094] 28. A nucleic acid construct in Embodiment 1, which is comprised of single stranded DNA.

[0095] 29. A nucleic acid construct in Embodiment 1, which is comprised of single stranded RNA.

[0096] 30. A nucleic acid construct in Embodiment 1, which is comprised of double stranded RNA.

[0097] 31. A nucleic acid construct in Embodiment 1, which is comprised of single stranded or double stranded linear DNA.

[0098] 32. A nucleic acid construct in Embodiment 1, which is comprised of single stranded or double stranded linear RNA.

[0099] 33. A nucleic acid construct in Embodiment 1, which is comprised of single stranded or double stranded circular DNA.

[0100] 34. A nucleic acid construct in Embodiment 1, which is comprised of single stranded or double stranded circular RNA.

[0101] 35. A system in Embodiment 1 which includes proteins delivered with the nucleic acid.

[0102] 36. A system in Embodiment 1, which is delivered as part of a viral or non-viral formulation, in vivo or ex vivo.

[0103] 37. A system in Embodiment 1, which is delivered by physical methods such as electroporation, nanoparticle bombardment, ultrasound mediated transfection, or chemically mediated transfection.

[0104] 38. An actuator in Embodiment 1, which is comprised at least in part from a tetracycline or tetracycline derivative.

[0105] 39. An actuator in Embodiment 1, which is comprised at least in part from a doxycycline or doxycycline derivative.

[0106] 40. An actuator in Embodiment 1, which is comprised at least in part from an antibiotic or antimicrobial peptide. 41. An actuator in Embodiment 1, which is comprised at least in part from a rapamycin or derivative thereof.

[0107] 42. An actuator in Embodiment 1, which is comprised at least in part from a ganciclovir or derivative thereof.

[0108] 43. An actuator in Embodiment 1, which is comprised at least in part from a ganciclovir, valganciclovir, valaciclovir, or derivative thereof.

[0109] 44. An actuator in Embodiment 1, which is comprised at least in part from a tamoxifen or derivative thereof.

[0110] 45. An actuator in Embodiment 1, which is comprised at least in part from an estrogen or derivative thereof.

[0111] 46. An actuator in Embodiment 1, which is comprised at least in part from an acetaldehyde or derivative thereof.

[0112] 47. An actuator in Embodiment 1, which is comprised at least in part from an L-arginine or derivative thereof.

[0113] 48. An actuator in Embodiment 1, which is comprised at least in part from a Biotinyl- AMP or derivative thereof.

[0114] 49. An actuator in Embodiment 1, which is comprised at least in part from a 2- phenylethyl butyrate or derivative thereof.

[0115] 50. An actuator in Embodiment 1, which is comprised at least in part from a 6-hydroxy nicotine or derivative thereof.

[0116] 51. An actuator in Embodiment 1, which is comprised at least in part from an erythromycin or derivative thereof.

[0117] 52. An actuator in Embodiment 1, which is comprised at least in part from a macrolide or derivative thereof.

[0118] 53. An actuator in Embodiment 1, which is comprised at least in part from a streptogramin or derivative thereof.

[0119] 54. An actuator in Embodiment 1, which is comprised at least in part from NADH or derivative thereof.

[0120] 55. An actuator in Embodiment 1, which is comprised at least in part from an element resulting in the formation of increased energy or heat.

[0121] 56. An actuator in Embodiment 1, which is comprised at least in part from SCB1 or derivative thereof.

[0122] 57. An actuator in Embodiment 1, which is comprised at least in part from 3-oxo-C8- HSL or derivative thereof. 58. An actuator in Embodiment 1, which is comprised at least in part from Phloretin or derivative thereof.

[0123] 59. An actuator in Embodiment 1, which is a small molecule, peptide, protein or nucleic acid that is capable of increasing, decreasing, or modulating local or systemic inflammation, or a part of the inflammatory response.

[0124] 60. An actuator in Embodiment 1, which is comprised at least in part from theophylline or derivative thereof.

[0125] 61. An actuator in Embodiment 1, which is comprised at least in part from IPTG or derivative thereof.

[0126] 62. An actuator in Embodiment 1, which is comprised at least in part from 5- fluorocytosine or derivative thereof.

[0127] 63. An actuator in Embodiment 1, which is comprised at least in part from propionate or derivative thereof.

[0128] 64. An actuator in Embodiment 1, which is comprised at least in part from acetate or derivative thereof.

[0129] 65. An actuator in Embodiment 1, which is comprised at least in part from cumate, cumate ester, or cumic acid, or derivative thereof.

[0130] 66. An actuator in Embodiment 1, which is comprised at least in part from gluconate or derivative thereof.

[0131] 67. An actuator in Embodiment 1, which is comprised at least in part from maltose or derivative thereof.

[0132] 68. An actuator in Embodiment 1, which is comprised at least in part from arabinose or derivative thereof.

[0133] 69. An actuator in Embodiment 1, which is comprised at least in part from mono- or disaccharide.

[0134] 70. An actuator in Embodiment 1, which is comprised at least in part from a trimethoprim or derivative thereof.

[0135] 71. An actuator in Embodiment 1, which is comprised at least in part from an exogenously administered small molecule, peptide, protein, RNA, DNA, or pharmacological formulation.

[0136] 72. An actuator in Embodiment 1, at least a part of which can act as at least a part of the effector element in Embodiment 1.

[0137] 73. An actuator in any Embodiment which is comprised at least in part from an endogenously expressed or presented peptide, hormone, protein, RNA, DNA, enzyme, mineral or chemical or biochemical substance and is capable of interacting with the actuator response element or actuator response sequence.

[0138] 74. An effector element in Embodiment 1, which is comprised at least in part from an inducible Caspase 9.

[0139] 75. An effector element in Embodiment 1, which is comprised at least in part from a natural Caspase 9.

[0140] 76. An effector element in Embodiment 1, which is comprised at least in part from a Zinc-Finger Nuclease.

[0141] 77. An effector element in Embodiment 1, which is comprised at least in part from a transcription activator- like effector nucleases (TALEN).

[0142] 78. An effector element in Embodiment 1, which is comprised at least in part from a meganuclease.

[0143] 79. An effector element in Embodiment 1, which is comprised at least in part from a deoxycytidine deamination-derived editor

[0144] 80. An effector element in Embodiment 1, which is comprised at least in part from a deoxyadenosine deamination-derived base editor.

[0145] 81. An effector element in Embodiment 1, which is comprised at least in part from a DNA-binding peptide.

[0146] 82. An effector element in Embodiment 1, which is comprised at least in part from a viral or bacterial DNA-binding protein.

[0147] 83. An effector element in Embodiment 1, which is comprised at least in part from a natural Caspase 8.

[0148] 84. An effector element in Embodiment 1, which is comprised at least in part from a natural Caspase or a Caspase pathway protein.

[0149] 85. An effector element in Embodiment 1, which is a FK506-binding protein 12 (with F36V mutation) fused with human caspase-8 protein deleted for 1-215 AA and linked to the N-myristoylation signal from Src kinase.

[0150] 86. An effector element in Embodiment 1, which is comprised in part from an FK506- binding protein 12 (with F36V mutation) fused with human caspase-9 protein deleted for 1-134 AA.

[0151] 87. An effector element in Embodiment 1, which is comprised at least in part from at least a part of a viral thymidine kinase.

[0152] 88. An effector element in Embodiment 1, which is comprised at least in part from at least a part of an Escherichia Coli cytosine deaminase. 89. An effector element in Embodiment 1, which is a protein with catalytic activity capable of chemically modifying a nucleotide, nucleoside, or nucleic acid.

[0153] 90. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a diphtheria toxin subunit A.

[0154] 91. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a diphtheria toxin subunit B.

[0155] 92. An effector element in Embodiment 1, which is a dimerizable or trimerizable toxin, protein, or enzyme.

[0156] 93. An effector element in Embodiment 1, which is comprised at least in part from, at least a part of the human or simian diphtheria toxin receptor.

[0157] 94. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a bacterial, viral, or eukaryotic toxin.

[0158] 95. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a protein capable of inhibiting transcription.

[0159] 96. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a protein capable of inhibiting translation.

[0160] 97. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a protein capable of inducing apoptosis.

[0161] 98. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a protein capable of inducing senescence.

[0162] 99. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a protein capable of reducing general gene expression.

[0163] 100. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a nuclease.

[0164] 101. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a nuclease capable of site specific mutagenesis.

[0165] 102. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a nuclease and at least a part of a targeting element.

[0166] 103. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a Cas nuclease and guide RNA targeting the nuclease to the sequence in Embodiment 1.

[0167] 104. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a nuclease targeting the sequence in Embodiment 1. 105. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a nuclease targeting a promoter region of the sequence in Embodiment 1.

[0168] 106. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a nuclease targeting a coding region of the sequence in Embodiment 1.

[0169] 107. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a nuclease targeting a post translational regulation element region of the sequence in Embodiment 1.

[0170] 108. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a recombinase.

[0171] 109. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a Cre recombinase.

[0172] 110. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a Kriippel-associated box domain from human gene ZNF10.

[0173] 111. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a zinc finger protein.

[0174] 112. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a cyclization recombinase with SV40 nuclear localization signal.

[0175] 113. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a Cre recombinase with SV40 T-antigen intron inserted in the ORE

[0176] 114. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a Flpe recombinase.

[0177] 115. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a Flpo recombinase.

[0178] 116. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a Dre recombinase.

[0179] 117. An effector element in Embodiment 1, which is comprised at least a part from at least a part of an SV40 T-antigen sequence.

[0180] 118. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a piggyBac transposase.

[0181] 119. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a transposase. 120. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a sleeping beauty transposase.

[0182] 121. An effector element in Embodiment 1, which is comprised at least a part from at least a part of an argonaut protein.

[0183] 122. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a DNA methyltransferase.

[0184] 123. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a protein which makes up or interacts with chromatin remodeling complex.

[0185] 124. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a protein that makes up or interacts with the DNA methylation pathway.

[0186] 125. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a base editor.

[0187] 126. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a prime editor.

[0188] 127. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a nickase.

[0189] 128. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a viral, bacterial, or eukaryotic gene silencing protein.

[0190] 129. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a siRNA.

[0191] 130. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a miRNA.

[0192] 131. An effector element in Embodiment 1, which is comprised at least a part from at least a part of FRT.

[0193] 132. An effector element in Embodiment 1, which is comprised at least a part from at least a part of an integrase.

[0194] 133. An effector element in Embodiment 1, which is comprised at least a part from at least a part of an RNA capable of reducing gene expression.

[0195] 134. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a protein enabling the effector degradation in the absence of an inducer. 135. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a dihydrofolate reductase.

[0196] 136. An effector element in Embodiment 1, which is comprised at least a part from at least a part of RNA, which is at least partially complementary to the genetic construct in Embodiment 1.

[0197] 137. An effector element in Embodiment 1, which is comprised at least a part from at least a part of transposon or retrotransposon.

[0198] 138. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a streptococcus pyogenes Cas9.

[0199] 139. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a catalytically inactive Cas9.

[0200] 140. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a codon optimized Cas9.

[0201] 141. An effector element in Embodiment 1, which is comprised at least a part from at least a part of Cas9 with a D10A mutation.

[0202] 142. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a dCas9 fused with KRAB.

[0203] 143. An effector element in Embodiment 1, which is comprised at least a part from at least a part of dCas9 fused with a carboxy terminal bipartite repressor domain, KRAB-MeCP2 or derivative.

[0204] 144. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a Campylobacter jujeni Cas9 ortholog.

[0205] 145. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a CRISPR associated protein or an ortholog.

[0206] 146. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a Casl2a or Casl2b protein.

[0207] 147. An effector element in Embodiment 1, which is comprised at least a part from at least a part of Casl3.

[0208] 148. An effector element in Embodiment 1, which is comprised at least a part from at least a part of Casl2f or Casl3a.

[0209] 149. An effector element in Embodiment 1, which is comprised at least a part from at least a part of a DNA, DNA capable of expressing, RNA, RNA, protein, or combination thereof capable of effecting DNA sequences in a sequence specific manner to induce double stranded breaks, single stranded breaks, methylation, chromatin modification, or generally epigenetic silencing.

[0210] 150. An effector element in Embodiment 1, which is comprised at least in part from at least a part of a toxin, toxin derivative, modified toxin, or a homolog, ortholog, or paralog thereof.

[0211] 151. An effector element in Embodiment 1, which is comprised at least in part from at least a part of a bacterial, fungal, plant, or animal toxin.

[0212] 152. An effector element in Embodiment 1, which is comprised at least in part from at least a part of a vibrio cholerae toxin, shiga toxin, shigella toxin, ricin, saporin, staphylococcus aureus enterotoxin H, Clostridium perfringens enterotoxin, streptolysin O, ribosome inactivating protein (RIP) enzyme, immunotoxin, pseudomonas exotoxin A, diphtheria toxin (DT), or DT fragment.

[0213] 153. An effector element in Embodiment 1, which is comprised at least in part from at least a part of a viral thymidine kinase or derivative thereof.

[0214] 154. An effector element in Embodiment 1, which is comprised at least in part from at least a part of a bacterial cytosine deaminase or derivative thereof.

[0215] 155. An effector element in Embodiment 1, which is comprised at least in part from at least a part of a D-amino-acid oxidase or derivative thereof.

[0216] 156. An effector element in Embodiment 1, which is comprised at least in part from at least a part of a bacterial carboxypeptidase G2 or derivative thereof.

[0217] 157. An effector element in Embodiment 1, which is comprised at least in part from at least a part of a purine nucleoside phosphorylase or derivative thereof.

[0218] 158. An effector element in Embodiment 1, which is comprised at least in part from at least a part of a thymidine phosphorylase or derivative thereof.

[0219] 159. An effector element in Embodiment 1, which is comprised at least in part from at least a part of a xanthine-guanine phosphoribosyl transferase or derivative thereof.

[0220] 160. An effector element in Embodiment 1, which is comprised at least in part from at least a part of a nitroreductase or derivative thereof.

[0221] 161. An effector element in Embodiment 1, which is comprised at least in part from at least a part of a penicillin-G amidase or derivative thereof.

[0222] 162. An effector element in Embodiment 1, which is comprised at least in part from at least a part of a multi-drug activation enzyme or derivative thereof.

[0223] 163. An effector element in Embodiment 1, which is comprised at least in part from at least a part of a horseradish peroxidase or derivative thereof. 164. An effector element in Embodiment 1, which is comprised at least in part from at least a part of a Beta-galactosidase or derivative thereof.

[0224] 165. An effector element in Embodiment 1, which is comprised at least in part from at least a part of a deoxyribonucleotide kinase or derivative thereof.

[0225] 166. An effector element in Embodiment 1, which is comprised at least in part from at least a part of a Beta-glucoronidase or derivative thereof.

[0226] 167. An effector element in Embodiment 1, which is comprised at least in part from at least a part of a carboxypeptidase A or derivative thereof.

[0227] 168. An effector element in Embodiment 1, which is comprised at least in part from at least a part of a cytochrome P450 or derivative thereof.

[0228] 169. An effector element in Embodiment 1, which is comprised at least in part from at least a part of a deoxycytidine kinase or derivative thereof.

[0229] 170. An effector sequence in Embodiment 1, which encodes at least the effector element.

[0230] 171. An effector sequence in Embodiment 1, which encodes at least a part of the consensus Lox sequence or a sequence capable of mediating the effect of a Lox sequence.

[0231] 172. An effector sequence in Embodiment 1, which encodes at least a part of the consensus FRT sequence or a sequence capable of mediating the effect of an FRT sequence.

[0232] 173. An effector sequence in Embodiment 1, which encodes at least a part of the consensus Rox sequence or a sequence capable of mediating the effect of a Rox sequence.

[0233] 174. An effector sequence in Embodiment 1, which is comprised at least in part from GTGTAGTTCGACCATTCGTG (SEQ ID NO: 1).

[0234] 175. An effector sequence in Embodiment 1, which is comprised at least in part from GTTCAGGATCACGTTACCGC (SEQ ID NO: 2).

[0235] 176. An effector sequence in Embodiment 1, which is comprised at least in part from GGGCGAGGAGCTGTTCACCG (SEQ ID NO: 3).

[0236] 177. An effector sequence in Embodiment 1, which is comprised at least in part from GAAGTTCGAGGGCGACACCC (SEQ ID NO: 4).

[0237] 178. An effector sequence in Embodiment 1, which is comprised at least in part from GCAGATGTAGTGTTTCCACA (SEQ ID NO: 5). 179. An effector sequence in Embodiment 1, which is comprised at least in part from CCTAAGGTTAAGTCGCCCTCG (SEQ ID NO: 6).

[0238] 180. An effector sequence in Embodiment 1, which is comprised at least in part from CAACAAGATGAAGAGCACCAA (SEQ ID NO: 7).

[0239] 181. An effector sequence in Embodiment 1, which encodes at least a part of attB, attR, attL, or attP sites.

[0240] 182. An effector sequence in Embodiment 1, which is comprised at least in part from genetic sequences, which can be targeted by integrases, recombinases, nucleases, nickases, chromatin complexes, or methylases or methyltransferases.

[0241] 183. An effector sequence in Embodiment 1, which is comprised at least in part from genetic sequences, which can be targeted for silencing RNA or protein binding or can be itself silenced.

[0242] 184. An effector sequence in Embodiment 1, which is a DNA sequence that can serve as a target of a guide RNA for a CRISPR associated protein.

[0243] 185. An effector sequence in Embodiment 1, which is a DNA sequence derived from viral or bacterial genomes.

[0244] 186. An effector sequence in Embodiment 1, which forms a secondary structure in the absence of an inducer molecule.

[0245] 187. An effector sequence in Embodiment 1, which is an inflammation responsive promoter.

[0246] 188. An effector sequence in Embodiment 1, which is derived at least in part from a mammalian promoter or enhancer.

[0247] 189. An effector sequence in Embodiment 1, which forms a secondary structure in the presence of an inducer molecule.

[0248] 190. An actuator response element in Embodiment 1, which is a nucleic acid sequence or a protein that interacts with an actuator to enable activity of the effector element or effector sequence.

[0249] 191. An actuator response element in Embodiment 1, which is comprised at least in part from a tetracycline-controlled transcriptional silencer (tTS).

[0250] 192. An actuator response element in Embodiment 1, which is comprised at least in part from a Kruppel associated box (KRAB) AB domain of the kid-1 transcriptional repressor.

[0251] 193. An actuator response element in Embodiment 1, which is composed at least in part of a reverse tetracycline-controlled transactivator. 194. An actuator response element in Embodiment 1, which is comprised at least in part of a viral protein such as VP 16 fused with a Tet- Repressor (TetR) or a reverse Tet- Repressor (rTetR).

[0252] 195. An actuator response element in Embodiment 1, which is comprised of a tTS and rtTA driven by the same promoter.

[0253] 196. An actuator response element in Embodiment 1, which is comprised of tTS and rtTA driven by different promoters.

[0254] 197. An actuator response element in Embodiment 1, which is comprised at least in part from a transcriptional repressor.

[0255] 198. An actuator response element in Embodiment 1, which is comprised at least in part from a transcriptional silencer.

[0256] 199. An actuator response element in Embodiment 1, which is comprised at least in part from a DNA binding protein.

[0257] 200. An actuator response element in Embodiment 1, which is comprised at least in part from a promoter binding protein.

[0258] 201. An actuator response element in Embodiment 1, which is comprised at least in part from a transcription initiation complex binding protein.

[0259] 202. An actuator response element in Embodiment 1, which is comprised at least in part of a protein responsible for DNA methylation.

[0260] 203. An actuator response element in Embodiment 1, which is comprised at least in part of a protein responsible for histone modification.

[0261] 204. An actuator response element in Embodiment 1, which is comprised at least in part of at least a part of a Tet-ON system.

[0262] 205. An actuator response element in Embodiment 1, which is comprised at least in part of at least a part of a Tet-OFF system.

[0263] 206. An actuator response element in Embodiment 1, which is comprised at least in part of at least a part of AlcR, ArgR, BirA, EthR, HdnoR, HucR, MphR(A), PIP, Rex, RheA, ScbR, TraR, TtgR or their analog, homolog, ortholog or protein fragment, protein, or fusion protein of similar functionality.

[0264] 207. An actuator response element in Embodiment 1, which is comprised at least in part from at least a part of lacUV5, lac, tac, trc, prpB, aceA, aceB, gntP, gntK, CJ10X2, tacM, malEl, gitl, BAD, SPL, P4-N14, cspB, aprE, sod, dapA, porB, ilvC, L10, L26, L16, L51, H30, H36 or natural, synthetic or semi-synthetic analogs, homologs, orthologs or protein fragments, proteins, or fusion proteins of similar functionality.

[0265] 208. An actuator response element in Embodiment 1, which is comprised at least in part from at least a part of ABIl-Gal4DBD, ABA, PVL1-VP16, PHR-p65, CIB1- TetR, or their analog, homolog, ortholog or protein fragment, protein, or fusion protein of similar functionality.

[0266] 209. An actuator response element in Embodiment 1, which is comprised at least in part from at least a part of a transcriptional activator, enhancer, de-repressor, or protein, RNA, or DNA capable of increasing gene expression directly or indirectly in a targeted or generally cis-acting manner.

[0267] It should be understood that within the scope of this invention the formulation, materials, genetic constructs, sequences, biological and chemical compositions, or methods of use may be varied by one skilled in the art, to the extent that the structures described herewithin perform the desired function and remain within the scope of the present invention. Various parts, components or characteristics may be used in combination, with or without modification by someone skilled in the art to achieve the desired functionality of the aforedescribed formulation.

[0268] Moreover, all individual features and methods of use described herein, and each and every combination of two or more of such features and methods of use, are included within the scope of the present invention provided that these features and methods of use in such a combination are not mutually inconsistent. It is understood that certain portions or combinations of such portions can be varied by someone trained in the art while still achieving the main goal of the invention.

[0269] Finally, it is understood that the specific ranges provided in the current invention are not restrictive and are for example purposes only, values outside of the specified ranges may be used to achieve the goal of the invention without modification to the proposed mechanistic principals.

[0270] The phraseology and terminology used in this application is for the purpose of description and should not be regarded as limiting. The use of terms such as “including,” “comprising,” “having,” “containing,” “involving,” and / or variations thereof in this application, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of’ or “consisting of’. The phrase “consisting essentially of’ is used herein to require the specified integer(s) or steps as well as those which do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g. a feature, element, characteristic, property, method / process step or limitation) or group of integers (e.g. features, elements, characteristics, properties, method / process steps or limitations) alone.

[0271] The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent application) cited throughout this application are hereby expressly incorporated by reference.

[0272] EXAMPLES

[0273] In order that the invention described in the present application may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the systems and methods provided in this disclosure and are not to be construed in any way as limiting their scope.

[0274] Example 1

[0275] In the first example, a therapeutic genetic construct with dose adjustment functionality is created by placing a furin-cleavable insulin gene under the regulation of an EFS promoter to drive basal insulin expression. To enable dose reduction a tetracycline inducible promoter and tetracycline responsive transcriptional activator or TetOn operator are added to drive expression of a CRISPR Cas9 protein and sgRNA in the presence of the tetracycline actuator. The sgRNA targets the Cas9 protein to cleave a target sequence separating the EFS promoter from the furin-cleavable insulin and eliminating expression of the furin-cleavable insulin therapeutic transgene. The construct self-inactivates and therefore reduces the overall level of therapeutic transgene expression following activation by an exogenously administered actuator - tetracycline. FIG. 1 shows a plasmid map for the genetic construct described in this example. Actuator: tetracycline, atomic formula C22H24N2O8, CAS Number: 60-54-8 or equivalent therapeutic / USP equivalent thereof CAS RN 64-75-5 -. Tetracycline Hydrochloride, Molecular Formula: C22H24N2O8.C1H. Nanoplasmid backbone 3NF1

[0276] 3NF2

[0277] Effector element: Cas9 and sgRNA

[0278] Cas9

[0279] Nucleotide sequence: CCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGG

[0280] ACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCC

[0281] ACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCT

[0282] GAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTAC

[0283] GTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGC

[0284] GAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCC

[0285] GCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGA

[0286] AGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCT

[0287] GACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGG

[0288] CGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGAC

[0289] CGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGT

[0290] GGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGAT

[0291] CTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGAC

[0292] ATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCG

[0293] AGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGC

[0294] TGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACG

[0295] GCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGG

[0296] CTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAA

[0297] GAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCAC

[0298] ATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGA

[0299] AGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCG

[0300] TGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGC

[0301] CGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATC

[0302] CTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTG

[0303] TACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACC

[0304] GGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGA

[0305] CTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCG

[0306] ACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGC

[0307] TGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGA

[0308] GAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGT

[0309] GGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAA

[0310] CACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTG

[0311] AAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCG

[0312] AGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAAC sgRNA sgRNA scaffold

[0313] U6 promoter

[0314] Effector sequence: target of Cas9 corresponding to above sgRNA Protospacer adjacent motif (PAM sequence)

[0315] NGG (N - any nucleotide)

[0316] Actuator response element: TetRepressor / Gen 1 TetOn Reverse tetracycline responsive transcriptional activator M2 (rtTA)

[0317] Nucleotide sequence:

[0318] Amino acid sequence:

[0319] Tetracycline transcriptional silencer (tTS):

[0320] Nucleotide sequence: ID NO: 20)

[0321] Therapeutic transgene: furin-cleavable human insulin

[0322] Nucleotide sequence: 21)

[0323] Amino acid sequence:

[0324] Auxiliary Sequences:

[0325] Tetracycline-Responsive Element Promoter (TRE promoter)

[0326] BGH pA

[0327] SV40 late pA

[0328] (SEQ ID NO: 25) Kozak

[0329] GCCACC

[0330] Ribosome-skipping 2A peptide from Thosea Asigna Virus

[0331] Nucleotide sequence:

[0332] (SEQ ID NO: 26)

[0333] Amino acid sequence:

[0334] EGRGSLLTCGDVEENPGP (SEQ ID NO: 27)

[0335] P2A

[0336] Nucleotide sequence

[0337] Amino acid sequence

[0338] ATNFSLLKQAGDVEENPGP (SEQ ID NO: 29)

[0339] EFS promoter

[0340] Example 2

[0341] In the present example, a doxycycline-inducible, self-inactivating construct is delivered in cis with a therapeutic transgene encoding a secretable anti-TNF-alpha nanobody (Ozoralizumab). To reduce the level of anti-TNF-alpha nanobody expression, an in-cis delivered Campylobacter jejuni CRISPR Cas9 protein is inducibly expressed under the regulation of a third generation Tetracycline repressor / operator (rtTA / TRE3g). Upon delivery of doxycycline, the Cas9 and sgRNA are expressed from the construct and target a sequence on the construct to separate the promoter from the therapeutic transgene. FIG. 2 shows a plasmid map for the genetic construct described in this example. The sequences and constructs are shown below:

[0342] Full sequence (except nanoplasmid backbone)

[0343] Actuator: doxycycline or Doxycycline Hyclate, CAS RN: 24390-14-5, molecular formula

[0344] 3NF1

[0345] Ctggggactttccagcctggggactttccagctgggactttccagg (SEQ ID NO: 10)

[0346] 3NF2

[0347] Ctggggactttccagctggggactttccagctgggactttccaggag (SEQ ID NO: 11)

[0348] CBh promoter Amino acid sequence

[0349] Furin cleavage site

[0350] Nucleotide sequence

[0351] Amino acid sequene

[0352] RKRR (SEQ ID NO: 36)

[0353] P2A

[0354] Nucleotide sequence

[0355] Amino acid sequence

[0356] GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 38)

[0357] Effector element: Cas9 GCGGAGAGGCTACGACGACATCAAGAACAGCGACGACAAAGAGAAGGGCGCCA

[0358] TCCTGAAGGCCATCAAGCAGAACGAGGAAAAGCTGGCCAACTACCAGTCCGTGG

[0359] GCGAGTACCTGTACAAAGAGTACTTCCAGAAGTTCAAAGAGAACAGCAAAGAAT

[0360] TCACCAACGTGCGGAACAAGAAAGAAAGCTACGAGCGGTGTATCGCCCAGAGCT

[0361] TCCTGAAGGATGAGCTGAAGCTGATCTTCAAGAAGCAGAGAGAGTTCGGCTTCA

[0362] GCTTCAGCAAGAAATTCGAGGAAGAGGTGCTGAGCGTCGCCTTCTACAAGAGAG

[0363] CCCTGAAGGACTTCAGCCACCTCGTGGGCAACTGCAGCTTCTTCACCGACGAGAA

[0364] GAGAGCCCCCAAGAACAGCCCCCTGGCCTTCATGTTCGTGGCCCTGACCCGGATC

[0365] ATCAACCTGCTGAACAATCTGAAGAACACCGAGGGCATCCTGTACACCAAGGACG

[0366] ACCTGAACGCCCTGCTGAATGAGGTGCTGAAGAACGGCACCCTGACCTACAAGC

[0367] AGACCAAGAAGCTGCTGGGCCTGAGCGACGACTACGAGTTTAAGGGCGAGAAGG

[0368] GCACCTACTTCATCGAGTTCAAGAAGTACAAAGAGTTCATCAAGGCCCTGGGCGA

[0369] GCACAACCTGAGCCAGGACGATCTGAATGAGATCGCCAAGGACATCACCCTGATC

[0370] AAGGACGAGATTAAGCTGAAGAAGGCCCTGGCCAAATACGACCTGAATCAGAAC

[0371] CAGATCGACAGCCTGAGCAAGCTGGAATTCAAGGATCACCTGAACATCAGCTTCA

[0372] AGGCTCTGAAGCTGGTCACCCCCCTGATGCTGGAAGGCAAGAAGTACGACGAGG

[0373] CCTGCAACGAGCTGAACCTGAAGGTGGCCATCAACGAGGACAAGAAGGACTTCC

[0374] TGCCCGCCTTCAACGAAACCTACTACAAGGACGAAGTGACCAACCCCGTGGTGCT

[0375] GCGGGCCATCAAAGAATACCGGAAGGTGCTGAATGCCCTGCTCAAGAAATACGGC

[0376] AAGGTGCACAAGATCAACATCGAGCTGGCCCGGGAAGTGGGCAAGAACCACAGC

[0377] CAGCGGGCCAAGATCGAGAAAGAGCAGAACGAAAACTACAAGGCCAAGAAGGA

[0378] CGCTGAGCTGGAATGCGAGAAGCTGGGACTGAAGATCAACAGCAAGAACATCCT

[0379] GAAGCTGCGGCTGTTCAAAGAACAGAAAGAGTTCTGCGCCTACAGCGGCGAGAA

[0380] GATCAAGATCAGCGATCTGCAGGACGAGAAGATGCTGGAAATCGACCACATCTAC

[0381] CCCTACAGCCGGTCCTTCGACGACAGCTACATGAACAAGGTGCTGGTGTTCACCA

[0382] AACAGAACCAGGAAAAACTGAACCAGACCCCCTTCGAGGCCTTCGGCAACGACA

[0383] GCGCCAAGTGGCAGAAAATCGAGGTGCTGGCCAAGAACCTGCCCACCAAGAAAC

[0384] AGAAGAGAATCCTGGACAAGAATTACAAGGACAAAGAGCAGAAGAACTTCAAGG

[0385] ACCGGAACCTGAACGACACCCGGTATATCGCCCGGCTGGTGCTGAACTACACAAA

[0386] GGACTACCTGGATTTCCTGCCCCTGTCCGACGACGAGAACACCAAGCTGAACGAT

[0387] ACCCAGAAAGGCTCCAAGGTGCACGTGGAAGCCAAGAGCGGCATGCTGACCAGC

[0388] GCCCTGAGACACACCTGGGGCTTCAGCGCCAAGGATCGGAACAACCATCTGCAC

[0389] CACGCCATCGACGCCGTGATCATTGCCTACGCCAACAACAGCATCGTGAAGGCCT

[0390] TCTCCGACTTCAAGAAAGAACAGGAAAGCAACAGCGCCGAGCTGTACGCCAAGA

[0391] Effector sequence: target of cjCas9

[0392] PAM

[0393] NNNNACAC (N - any nucleotide)

[0394] U6 promoter gRNA scaffold for CjCas9

[0395] Actuator response element: TetRepressor / Gen 3 TetOn, third generation rtTA / TRE3G

[0396] Nucleotide sequence bGH poly(A) sequence ctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaat aaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattg ggaagagaatagcaggcaggctgggga (SEQ ID NO: 46)

[0397] SV40 poly (A) sequence

[0398] Taagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgt aaccattataagctgcaataaacaagtt (SEQ ID NO: 47)

[0399] The activity of the suicide gene-based down-titration mechanism is qualified using an nLuc bioluminescent reporter (FIGs. 3A-3B). Activation of the self-silencing signal by delivery of an operator induces a permanent reduction by approximately 2x to 3x in the reporter transgene expression (FIGs. 3A-3B; “Suicide Gene - Strong Promoter” and “Suicide Gene - Weak Promoter”), and is a function of the promoter driving the suicide gene expression. When a strong promoter is used to drive the reporter transgene and the suicide gene post activation, the strong promoter produces a strong initial signal (FIG. 3A; “Suicide Gene - Strong Promoter”) and a more pronounced down-titration (FIG. 3B; “Suicide Gene - Strong Promoter”). Conversely, a low-strength promoter, produces a lower expression signal (FIG. 3A; “Suicide Gene - Weak Promoter”) and a lower level of down-titration following delivery of the operator (FIG. 3B; “Suicide Gene - Weak Promoter”).

[0400] Example 3

[0401] In the present example a secretable alpha-glucosidase is delivered under the regulation of a CMV promoter for the treatment of Pompe disease as an enzyme replacement therapy. To ensure that the enzyme level does not exceed the therapeutic limit, the genetic construct is delivered in cis with a toxin subunit is used to destabilize and inhibit elongation factor 2 (EF- 2) thereby preventing elongation and inhibiting protein synthesis. FIG. 4 shows a plasmid map for the genetic construct described in this example.

[0402] Full sequence (except nanoplasmid backbone)

[0403] 3NF1

[0404] Nucleotide sequence Amino acid sequence

[0405] P2A

[0406] Nucleotide sequence

[0407] Amino acid sequence

[0408] ATNFSLLKQAGDVEENPGP (SEQ ID NO: 29)

[0409] Tet-on 3G

[0410] Nucleotide sequence

[0411] Amino acid sequence

[0412] TRE3G promoter

[0413] Diptheria toxin subunit A (DTA):

[0414] Nucleotide sequence Amino acid sequence NO: 53)

[0415] KI 9 aptazyme Ggcgcgtcctggattcgtggtaaaacataccagatttcgatctggagaggtgaagaatacgaccacctactacatccagctgatgagt cccaaataggacgaaacgcgct (SEQ ID NO: 54)

[0416] Insulator sequence

[0417] Caaacaaacaaa (SEQ ID NO: 55) bGH poly(A) sequence

[0418] SV40 poly (A) sequence Taagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgt aaccattataagctgcaataaacaagtt (SEQ ID NO: 47)

[0419] Example 4

[0420] In the present example a GLP- 1 Receptor Antagonist Analog Exenatide is delivered under the regulation of the CBh promoter. In cis with the Exenatide sequence cDNA, which is coupled with a secretory peptide, the construct contains a KI 9 aptazyme and a destabilized Cre recombinase. Upon administration of Tetracycline the K19 aptazyme stabilizes the Cre transcript, which coupled with Trimethoprim activates recombinase activity, which targets a sequence within the promoter region of the construct to eliminate expression of the therapeutic transgene (Exanatide). FIG. 5 shows a plasmid map for the genetic construct described in this example. The construct components, actuators, effectors, and overall sequences are shown below:

[0421] Actuator: Tetracycline (for K19 aptazyme) and Trimethoprim CAS RN 738-70-5, molecular formula C14H18N4O3 (for destabilized Cre).

[0422] Full sequence (except nanoplasmid backbone)

[0423]

[0424] 3NF1

[0425] 3NF2 loxP site ataacttcgtatagcatacattatacgaagttat (SEQ ID NO: 57)

[0426] CBh promoter ggttgg (SEQ ID NO: 32) Exenatide-4

[0427] Nucleotide sequence a (SEQ ID NO: 58)

[0428] Amino acid sequence

[0429] MATDSRTSWLLTVSLLCLLWPQEASAFPAMPLSSLFSNAVLRARGKRHGEGTFTSDLS KQMEEEAVREFIEWEKNGGPSSGAPPPS (SEQ ID NO: 59)

[0430] EFS promoter

[0431] Destabilized Cre Recombinase (DD-CRE):

[0432] Nucleotide sequence

[0433] Amino acid sequence

[0434] KI 9 aptazyme

[0435] Insulator sequence

[0436] Caaacaaacaaa (SEQ ID NO: 55) bGH poly(A) sequence

[0437] SV40 poly (A) sequence The activity of the self-inactivating Cre-based down-titration mechanism is qualified using an nLuc bioluminescent reporter (FIG. 6). Activation of the self- silencing signal by delivery of an operator induces a permanent reduction by approximately 3x in the reporter transgene expression (FIG. 6; “A”).

[0438] Example 5

[0439] In the present example an enzyme replacement therapy is delivered under the regulation of a constitutive promoter. The down-titration is mediated by a Arbus precatorius L, plant-derived Ribosome Inhibiting Protein (RIP) Abrin-A under the regulation of a 3rdgeneration TetOn promoter / operator and a K19 aptazyme. The administration of tetracycline or doxycycline activates the TetOn operator / promoter, which drives expression of the RIP protein, which inhibits ribosomes and reduces the overall gene expression from the therapeutic construct. FIG. 7 shows a plasmid map for the genetic construct described in this example.

[0440] Full sequence (except nanoplasmid backbone)

[0441]

[0442] 3NF1

[0443] CBh promoter ctgagcaagaggtaagggtttaagggatggttggttggtggggtattaatgtttaattacctggagcacctgcctgaaatcactttttttca ggttgg (SEQ ID NO: 32) secGAA (sp7-delta8-hGAA)

[0444] Nucleotide sequence

[0445] P2A

[0446] Nucleotide sequence

[0447] Amino acid sequence

[0448] Tet-on 3G

[0449] Nucleotide sequence

[0450]

[0451] Amino acid sequence

[0452] TRE3G promoter

[0453] Arbin A-chain

[0454] Nucleotide sequence

[0455] Amino acid sequence

[0456] KI 9 aptazyme

[0457] Ggcgcgtcctggattcgtggtaaaacataccagatttcgatctggagaggtgaagaatacgaccacctactacatccagctgatgagt cccaaataggacgaaacgcgct (SEQ ID NO: 54)

[0458] Insulator sequence

[0459] Caaacaaacaaa (SEQ ID NO: 55) bGH poly(A) sequence

[0460] SV40 poly (A) sequence

[0461] Example 6

[0462] In the present example a Glucagon Like Peptide 1 Receptor Agonist (GLP-1RA) is delivered for the management of type 2 diabetes. The GLP-1RA peptide is delivered with a secretion signal under the regulation of a constitutive promoter. Dose-down titration is enabled by selfdirected, small-molecule induced epigenetic silencing of the promoter region of the construct. FIG. 8 shows a plasmid map for the genetic construct described in this example. Full sequence (except nanoplasmid backbone)

[0463] 3NF1

[0464] Ctggggactttccagcctggggactttccagctgggactttccagg (SEQ ID NO: 10)

[0465] 3NF2

[0466] Ctggggactttccagctggggactttccagctgggactttccaggag (SEQ ID NO: 11)

[0467] CBh promoter

[0468] Zink finger target site

[0469] TGGCGACAGACAGCCGGC (SEQ ID NO: 66)

[0470] GLP-1 RA

[0471] Nucleotide sequence

[0472] Amino acid sequence bGH poly(A) sequence ctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaat aaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattg ggaagagaatagcaggcaggctgggga (SEQ ID NO: 46)

[0473] SV40 poly (A) sequence

[0474] KI 9 aptazyme

[0475] Insulator sequence

[0476] Caaacaaacaaa (SEQ ID NO: 55)

[0477] Epigenetics effector (hD3A-mD3L-ZF-ZN627)

[0478] IASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNP

[0479]

[0480] The activity of the self- silencing epigenetic regulator is qualified using an nLuc bioluminescent reporter (FIG. 9). Activation of the self- silencing signal by delivery of an operator induces a reduction by approximately 2x in the reporter transgene expression.

[0481] Example 7

[0482] In the present example a Glucagon Like Peptide 1 and Gastric Inhibitor Peptide dual receptor agonist (GLP / GIP) is delivered for the treatment of Type 2 Diabetes or Obesity under the regulation of an EFS promoter. To enable dose down-titration should the therapeutic reach optimal therapeutic effect or to reduce the dose should the therapeutic exceed a tolerability limit, a small molecule actuator (Cumate) is used to activate an in-cis delivered epigenetic effector element to silence expression of the therapeutic transgene. FIG. 10 shows a plasmid map for the genetic construct described in this example. The specific elements of the constructs and the full construct design are shown below.

[0483] Actuator: water insoluble cumate, 4-Isopropylbenzoic acid (Sigma 268402) or USP-grade equivalent thereof.

[0484] Full sequence (except nanoplasmid backbone) ggcgggccatttaccgtaagttatgtaacgctggggactttccagctggggactttccagctgggactttccaggag (SEQ ID NO: 71)

[0485] Effector element: Our standard epigenetic silencer system hD3A-mD3L-ZF-ZN627

[0486] Nucleotide sequence

[0487] Amino acid sequence

[0488] Effector sequence: target DNA sequence of our standard epigenetic silencer system.

[0489] ZF target sequence (SEQ ID NO:66)

[0490] Actuator response element: Cumate inducible promoter, repressor, and promoter sequence

[0491] Cumate-inducible promoter (CMV5+CuO)

[0492]

[0493] CuO sequence

[0494] Atcaaacagaccagattgtctgtttgt (SEQ ID NO: 73)

[0495] CymR repressor

[0496] Nucleotide sequence (SEQ ID NO: 74)

[0497] Amino acid sequence

[0498] Therapeutic transgene for GIP / GLP dual receptor agonist: GIP / GLP-1 RA

[0499] Nucleotide sequence catct (SEQ ID NO: 76)

[0500] Amino acid sequence

[0501] APPPS (SEQ ID NO: 77)

[0502] 3NF1

[0503] Ctggggactttccagcctggggactttccagctgggactttccagg (SEQ ID NO: 10)

[0504] 3NF2

[0505] Ctggggactttccagctggggactttccagctgggactttccaggag (SEQ ID NO: 11)

[0506] EFS promoter

[0507] P2A

[0508] Nucleotide sequence

[0509] Amino acid sequence bGH poly(A) sequence SV40 poly (A) sequence Taagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgt aaccattataagctgcaataaacaagtt (SEQ ID NO: 47)

[0510] Example 8

[0511] In the present example, a system containing a dual actuator regulated effector element is intended to deliver biologically active recombinant acid alpha-glucosidase for the treatment of Glycogen storage disease type II (Pompe disease). The first actuator is a tetracycline or tetracycline derivative, which acts upon the TetOn inducible promoter and in conjunction with the TetOn operator activates expression of rapamycin inducible-Caspase9. The second actuator is Rapamycin or rapamycin analog or derivative, which activates inducible Caspase9 to promote the initiation of apoptosis in the cells carrying the therapeutic construct. FIG. 11 shows a plasmid map for the genetic construct described in this example.

[0512] 3NF1

[0513] 3NF2

[0514]

[0515] P2A

[0516] Nucleotide sequence

[0517] Amino acid sequence

[0518] ATNFSLLKQAGDVEENPGP (SEQ ID NO: 29) tTS

[0519] Nucleotide sequence

[0520] Amino acid sequence

[0521] EGRGSLLTCGD VEEN PGP (SEQ ID NO: 27) rtTA bGH poly(A) sequence

[0522] SV40 poly (A) sequence Taagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgt aaccattataagctgcaataaacaagtt (SEQ ID NO: 47)

[0523] The system is tested with a reporter gene with three concentrations of Rapamycin (ranging from InM to O.OlnM) and a constant (0.1 nM concentration of Tetracycline and facilitates reduction in expression of nLuc (as measured by the strength of the bioluminescence signal) (FIG. 12), Two promoters are used to drive initial transgene expression, a stronger CBh promoter and a weaker EFS promoter. Constructs with no downtitration genetic element (LNP-CBh-nLuc) are used as a reference control.

[0524] No change in expression relative to the “no treatment” control is observed for the LNP-CBh-nLuc constructs. On the other hand, the iCaspase9-containing constructs reduce gene expression in a Rapamycin dose-dependent manner following concurrent activation by tetracycline. During treatment, appropriate rapamycin and tetracycline concentrations can be obtained following administration of the actuator small molecules locally or systemically following treatment with the therapeutic construct.

[0525] Example 9

[0526] In the present example a single actuator-regulated effector element is intended to deliver biologically active recombinant insulin-like growth factor 2 for the treatment of Silver- Rus sell Syndrome. The actuator is tetracycline or a tetracycline derivative, which acts upon a 3rdgeneration TetOn inducible promoter and in conjunction with the TetOn operator activates expression of the N-terminal domain of gasdermin D. Gasdermin D oligomerizes to form pores in the cell membrane and initiates pyroptosis. FIG. 13 shows a plasmid map for the genetic construct described in this example. Full sequence (except nanoplasmid backbone)

[0527] NO: 81)

[0528] Transgene: insulin-like growth factor 2 (IGF2)

[0529] Nucleotide sequence

[0530] Amino acid sequence:

[0531] Actuator: Tetracycline or derivative

[0532] Actuator response element: TetRepressor / Gen 3 TetOn, third generation rtTA / TRE3G TRE3G Promoter

[0533] Effector element: N-terminal domain of gasdermin D Nucleotide sequence

[0534] Amino acid sequence 85)

[0535] Auxiliary sequences:

[0536] CBh promoter

[0537] Self-cleaving 2A peptide from porcine teschovirus-1

[0538] Nucleotide sequence

[0539] Amino acid sequence

[0540] Kozak

[0541] GCCACC bGH polyA

[0542] SV40 polyA

[0543] Example 10 In the present example a single actuator-regulated effector element is intended to deliver biologically active beta-glucocerebrosidase for the treatment of Gaucher’s disease. Beta-glucocerebrosidase is expressed under the CMV promoter. The actuator is tetracycline or a tetracycline derivative, which acts upon a 3rdgeneration TetOn inducible promoter. In conjunction with the TetOn operator, this activates expression of a viral DNA-binding protein (IE2 from human cytomegalovirus) that binds to the CMV promoter and inhibits expression of beta-glucocerebrosidase. Tetracycline and the TetOn operator additionally activate expression of a transcriptional transactivator (tTA) that continuously activates the TetOn operator after the withdrawal of tetracycline. FIG. 14 shows a plasmid map for the genetic construct described in this example.

[0544] Full sequence (except nanoplasmid backbone) CA AG T TC TC GT AG CT G A T GC CA A GC G CA CG AC AG T A C TT G T CT GA GA G A G GG

[0545] GTCTCGACTTCTTCACCCTGTTCTTCCTCGCTATCAGAGATCACGATACAGCCGGC

[0546] Actuator: Tetracycline or derivative, advanced tTA

[0547] Advanced tTA Nucleotide sequence

[0548] Amino acid sequence

[0549] Actuator response element: TetRepressor / Gen 3 TetOn, third generation rtTA / TRE3G

[0550] TRE3G TetOn3G

[0551] Nucleotide sequence

[0552] Amino acid sequence

[0553] Effector element: IE2 from human cytomegalovirus

[0554] Nucleotide sequence

[0555] Auxiliary sequences:

[0556] CMV promoter

[0557] Self-cleaving 2A peptide from porcine teschovirus-1

[0558] Nucleotide sequence

[0559] Amino acid sequence

[0560] Kozak

[0561] G bGH polyA A Example 11

[0562] In the present example a single actuator-regulated effector element is intended to deliver biologically active lamin A for the treatment of progeria. Lamin A is expressed under the constitutive CBh promoter, and the gene is flanked on either side by one of two serine- recombinase recognition sequences (attP and attB). The actuator is tetracycline or a tetracycline derivative, which acts upon a tetracycline-inducible promoter and, in conjuction with the tet operator, activates expression of the serine integrase derived from Bxbl bacteriophage. The integrase excises the genetic sequence of lamin A, turning gene expression off. FIG. 15 shows a plasmid map for the genetic construct described in this example.

[0563] Full sequence (except nanoplasmid backbone)

[0564] (SEQ ID NO: 96)

[0565] Actuator: Tetracycline or derivative

[0566] Actuator response element: TetRepressor / Gen 3 TetOn, third generation rtTA / TRE3G

[0567] TRE3G

[0568] TetOn3G

[0569] Nucleotide sequence

[0570] Effector element: Bxbl serine integrase, associated attB and attP sites attP site attB site

[0571] ID NO: 98)

[0572] Bxbl serine integrase

[0573] Nucleotide sequence CCGTGGCCCAGATGGAACTGGAAGCTATCAAGGAAAGAAATAGATCTGCCGCCCA TTTCAACATCAGAGCCGGCAAATACAGAGGATCTCTGCCCCCCTGGGGCTACCTG CCTACCAGAGTGGACGGCGAGTGGAGACTGGTGCCTGATCCTGTGCAGCGGGAG CGGATCCTGGAAGTGTACCACCGGGTCGTTGACAACCACGAGCCACTCCATCTGG

[0574] TGGCTCACGACTTGAACAGAAGGGGCGTGCTGTCTCCTAAGGACTACTTCGCCCA GCTGCAGGGAAGAGAGCCTCAGGGCAGAGAGTGGAGCGCCACAGCCCTGAAGC GCAGCATGATCAGCGAGGCCATGCTGGGATACGCCACCCTGAACGGCAAGACCGT GCGAGATGATGACGGCGCCCCTCTGGTGCGGGCCGAACCTATCCTGACAAGAGAG

[0575] CAGCTGGAGGCCCTGCGGGCCGAGCTGGTGAAGACCTCTAGAGCTAAACCTGCT GTGTCCACCCCAAGCCTGCTGCTGAGGGTCCTGTTCTGTGCCGTGTGCGGAGAAC CCGCCTACAAGTTCGCCGGCGGAGGCAGAAAGCACCCTAGATACCGGTGCAGATC CATGGGCTTCCCCAAACACTGCGGCAATGGCACCGTGGCCATGGCCGAGTGGGAT

[0576] GCCTTCTGCGAGGAACAAGTGCTGGATCTGCTCGGCGATGCTGAGAGACTCGAGA AGGTGTGGGTGGCCGGCAGCGACAGCGCCGTGGAACTGGCCGAGGTGAACGCCG AGCTGGTGGATCTGACCAGCCTGATCGGCAGCCCCGCCTATAGAGCCGGTTCTCC ACAGAGAGAAGCCCTGGACGCCAGAATCGCCGCTCTGGCTGCTAGGCAGGAGGA

[0577] ACTGGAGGGCCTGGAAGCCCGGCCTAGCGGATGGGAGTGGCGAGAAACCGGACA GCGGTTCGGCGACTGGTGGCGGGAACAGGACACAGCCGCCAAGAACACCTGGCT GCGGTCCATGAACGTGCGGTTGACATTCGATGTCCGGGGCGGCCTGACAAGAACC ATCGACTTCGGCGACCTACAGGAGTACGAGCAACACCTGAGACTGGGCAGCGTG

[0578] GTGGAAAGACTGCACACCGGCATGAGCTAG (SEQ ID NO: 99)

[0579] Amino acid sequence

[0580] MRALVVIRLSRVTDATTSPERQLESCQQLCAQRGWDVVGVAEDLDVSGAVDPFDRK RRPNLARWLAFEEQPFDVIVAYRVDRLTRSIRHLQQLVHWAEDHKKLVVSATEAHFD TTTPFAAVVIALMGTVAQMELEAIKERNRSAAHFNIRAGKYRGSLPPWGYLPTRVDG EWRLVPDPVQRERILEVYHRVVDNHEPLHLVAHDLNRRGVLSPKDYFAQLQGREPQ

[0581] GREWSATALKRSMISEAMLGYATLNGKTVRDDDGAPLVRAEPILTREQLEALRAELV KTSRAKPAVSTPSLLLRVLFCAVCGEPAYKFAGGGRKHPRYRCRSMGFPKHCGNGTV

[0582] AMAEWDAFCEEQVLDLLGDAERLEKVWVAGSDSAVELAEVNAELVDLTSLIGSPAY RAGSPQREALDARIAALAARQEELEGLEARPSGWEWRETGQRFGDWWREQDTAAK NTWLRSMNVRLTFDVRGGLTRTIDFGDLQEYEQHLRLGSVVERLHTGMS (SEQ ID NO: 100) Auxiliary sequences:

[0583] CBh promoter

[0584] Self-cleaving 2A peptide from porcine teschovirus-1

[0585] Nucleotide sequence

[0586] Amino acid sequence

[0587] GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 38)

[0588] Kozak bGH polyA

[0589] SV40 poly A

[0590] Example 12

[0591] In the present example a single actuator-regulated is intended to deliver a functional copy of cystic fibrosis transmembrane conductance regulator (CFTR) for treatment of cystic fibrosis. A CBh promoter controls expression of CFTR and inducible Caspase 9 (iCasp9), which are connected by a self-cleaving peptide (P2A). The actuator is AP20187, a ligand that causes dimerization of caspase 9 and induction of apoptosis. FIG. 16 shows a plasmid map for the genetic construct described in this example.

[0592] Full sequence (except nanoplasmid backbone)

[0593]

[0594] CTTCTAATGGTGATTATGGGAGAACTGGAGCCTTCAGAGGGTAAAATTAAGCACA

[0595]

[0596] Actuator: AP20187 or other dimerizer capable of dimerizing iCasp9

[0597] Effector element: iCasp9

[0598] Nucleotide sequence

[0599] Auxiliary sequences:

[0600] CBh promoter

[0601] Self-cleaving 2A peptide from porcine teschovirus-1

[0602] Nucleotide sequence

[0603] Amino acid sequence

[0604] Kozak bGH polyA

[0605] Example 13

[0606] In the present example a single actuator-regulated is intended to deliver a functional copy of hexosaminidase subunit alpha (HexA) for the treatment of Tay-Sachs Disease. A CBh promoter controls expression of HexA and inducible Caspase 8 (iCasp8), which are connected by a self-cleaving peptide (P2A). The actuator is AP20187, a ligand that causes dimerization of caspase 8 and induction of apoptosis. FIG. 17 shows a plasmid map for the genetic construct described in this example.

[0607] Full sequence (except nanoplasmid backbone)

[0608] Actuator: AP20187 or other dimerizer capable of dimerizing iCasp8 Effector element: iCasp8

[0609] Nucleotide sequence

[0610] Auxiliary sequences:

[0611] CBh promoter

[0612] Self-cleaving 2A peptide from porcine teschovirus-1

[0613] Nucleotide sequence

[0614] Amino acid sequence

[0615] Kozak bGH polyA

[0616] Example 14

[0617] In the present example, a system containing a toggle-regulated effector element is intended to deliver biologically active fibroblast growth factor 18 for the treatment of osteoarthritis. The system is constructed of a constitutive CMV promoter followed by several TetO sequences driving the expression of both fibroblast growth factor 18 and the lac repressor protein fused to KRAB. A second promoter followed by LacO or CuO sequences drives the expression of the Tet repressor protein fused to KRAB (tet transcriptional silencer / tTS). When tetracycline is added, fibroblast growth factor 18 and the lac repressor / cumate repressor protein are expressed, allowing the switch to reach a stable equilibrium in which the lac / cumate-responsive second promoter is continuously repressed, and fibroblast growth factor 18 is continuously produced. When IPTG or cumate is added, the lac / cumate repressor protein can no longer bind to the LacO / CuO sequences, allowing the TetR-KRAB fusion protein to be expressed. The switch reaches a new stable equilibrium in which the TetR-KRAB fusion protein is continuously expressed, repressing the expression of fibroblast growth factor 18. FIG. 18 shows a plasmid map for the genetic construct described in this example.

[0618] Full sequence (except nanoplasmid backbone)

[0619] Transgene: fibroblast growth factor 18

[0620] Nucleotide sequence

[0621] Amino acid sequence

[0622] Actuator: Tetracycline or derivative, IPTG or cumate

[0623] Actuator response elements: Tet Repressor- KRAB (tTS) / TRE3G, Lac Repressor- KRAB /

[0624] CMV-7x LacO or cumate repressor / CuOn promoter tTS

[0625] Nucleotide sequence

[0626] Amino acid sequence ID NO: 20)

[0627] 7x LacO Promoter NO: 113)

[0628] Lac repressor-KRAB

[0629] Nucleotide sequence

[0630] ID NO: 116)

[0631] Cumate repressor

[0632] Nucleotide sequence

[0633] Auxiliary sequences:

[0634] Self-cleaving 2A peptide from porcine teschovirus-1

[0635] Nucleotide sequence

[0636] Amino acid sequence

[0637] Kozak bGH polyA

[0638] EQUIVALENTS

[0639] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described in the present application. Such equivalents are intended to be encompassed by the following claims.

[0640] All references, including patent documents, are incorporated by reference in their entirety.

Claims

CLAIMSWhat is claimed is:

1. A system for permanently reducing gene expression from a therapeutic construct or a plethora of constructs within one or more cells comprised of at least:(a) a therapeutic transgene containing nucleic acid construct delivered in a formulation capable of entry into cells and enablement of stable therapeutic transgene expression for a period of at least 4 months;(b) an actuator comprised of one or more chemical or biological molecules;(c) an actuator response element and / or actuator response sequence that enables expression, activation, or stabilizes an effector element or an effector sequence;(d) an effector sequence delivered in cis with the therapeutic transgene; and(e) an effector element expressed from said effector sequence capable of permanently reducing expression of said therapeutic transgene in a manner that elicits minimal bystander effect and is therefore safe to surrounding tissues, organs, and organism in its entirety.

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

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 to 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 ophthalmic delivery.

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

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 the mucous membranes.

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

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 to be used in conjunction with therapeutic gene therapy or delivered in conjunction with a therapeutic transgene for the treatment of human, plant, or veterinary diseases or for the augmentation of human, veterinary, or plant function.

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

22. The system of any one of the preceding claims, wherein the therapeutic transgene encodes a protein or enzyme required for protein or enzyme replacement therapy.

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 aptazyme, aptamer, a ribozyme, or a genetic structure that is capable of enzymatic activity alone or in conjunction with a protein.

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

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

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

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

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

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

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

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

35. The system of any one of the preceding claims, wherein the system includes proteins delivered with the nucleic acid.

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

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 comprised at least in part from a tetracycline or tetracycline derivative.

39. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from a doxycycline or doxycycline derivative.

40. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from an antibiotic or antimicrobial peptide.

41. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from a rapamycin or derivative thereof.

42. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from a ganciclovir or derivative thereof.

43. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from a ganciclovir, valganciclovir, valaciclovir, or derivative thereof.

44. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from a tamoxifen or derivative thereof.

45. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from an estrogen or derivative thereof.

46. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from an acetaldehyde or derivative thereof.

47. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from an L-arginine or derivative thereof.

48. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from a Biotinyl- AMP or derivative thereof.

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

50. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from a 6-hydroxy nicotine or derivative thereof.

51. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from an erythromycin or derivative thereof.

52. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from a macrolide or derivative thereof.

53. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from a streptogramin or derivative thereof.

54. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from NADH or derivative thereof.

55. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from an element resulting in the formation of increased energy or heat.

56. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from SCB 1 or derivative thereof.

57. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from 3-oxo-C8-HSL or derivative thereof.

58. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from Phloretin or derivative thereof.

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

60. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from theophylline or derivative thereof.

61. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from IPTG or derivative thereof.

62. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from 5-fluorocytosine or derivative thereof.

63. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from propionate or derivative thereof.

64. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from acetate or derivative thereof.

65. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from cumate, cumate ester, or cumic acid, or derivative thereof.

66. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from gluconate or derivative thereof.

67. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from maltose or derivative thereof.

68. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from arabinose or derivative thereof.

69. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from mono- or di-saccharide.

70. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from a trimethoprim or derivative thereof.

71. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from an exogenously administered small molecule, peptide, protein, RNA, DNA, or pharmacological formulation.

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

73. The system of any one of the preceding claims, wherein the actuator is comprised at least in part from an endogenously expressed or presented peptide, hormone, protein, RNA, DNA, enzyme, mineral or chemical or biochemical substance and is capable of interacting with the actuator response element or actuator response sequence.

74. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from an inducible Caspase 9.

75. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from a natural Caspase 9.

76. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from a Zinc-Finger Nuclease.

77. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from a transcription activator- like effector nucleases (TALEN).

78. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from a meganuclease.

79. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from a deoxycytidine deamination-derived editor.

80. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from a deoxyadenosine deamination-derived base editor.

81. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from a DNA-binding peptide.

82. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from a viral or bacterial DNA-binding protein.

83. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from a natural Caspase 8.

84. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from a natural Caspase or a Caspase pathway protein.

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

86. The system of any one of the preceding claims, wherein the effector element is comprised in part from an FK506-binding protein 12 (with F36V mutation) fused with human caspase-9 protein deleted for 1-134 AA.

87. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from at least a part of a viral thymidine kinase.

88. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from at least a part of an Escherichia Coli cytosine deaminase.

89. The system of any one of the preceding claims, wherein the effector element is a protein with catalytic activity capable of chemically modifying a nucleotide, nucleoside, or nucleic acid.

90. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a diphtheria toxin subunit A.

91. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a 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 one of the preceding claims, wherein the effector element is comprised at least in part from, at least a part of the human or simian diphtheria toxin receptor.

94. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a bacterial, viral, or eukaryotic toxin.

95. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a protein capable of inhibiting transcription.

96. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a protein capable of inhibiting translation.

97. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a protein capable of inducing apoptosis.

98. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a protein capable of inducing senescence.

99. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a protein capable of reducing general gene expression.

100. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a nuclease.

101. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a nuclease capable of site specific mutagenesis.

102. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a nuclease and at least a part of a targeting element.

103. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a Cas nuclease and guide RNA targeting the nuclease to the sequence in any one of the preceding claims.

104. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a nuclease targeting the sequence in any one of the preceding claims.

105. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a nuclease targeting a promoter region of the sequence in any one of the preceding claims.

106. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a nuclease targeting a coding region of the sequence in any one of the preceding claims.

107. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a nuclease targeting a post translational regulation element region of the sequence in any one of the preceding claims.

108. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a recombinase.

109. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a Cre recombinase.

110. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a Kruppel-associated box domain from human gene ZNF10.

111. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a zinc finger protein.

112. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a cyclization recombinase with SV40 nuclear localization signal.

113. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a Cre recombinase with SV40 T-antigen intron inserted in the ORE114. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a Flpe recombinase.

115. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a Flpo recombinase.

116. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a Dre recombinase.

117. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of an SV40 T-antigen sequence.

118. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a piggyBac transposase.

119. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a transposase.

120. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a sleeping beauty transposase.

121. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of an argonaut protein.

122. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a DNA methyltransferase.

123. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a protein which makes up or interacts with chromatin remodeling complex.

124. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a protein that makes up or interacts with the DNA methylation pathway.

125. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a base editor.

126. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a prime editor.

127. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a nickase.

128. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a viral, bacterial, or eukaryotic gene silencing protein.

129. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a siRNA.

130. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a miRNA.

131. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of FRT.

132. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of an integrase.

133. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of an RNA capable of reducing gene expression.

134. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a protein enabling the effector degradation in the absence of an inducer.

135. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a dihydrofolate reductase.

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

137. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of transposon or retrotransposon.

138. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a Streptococcus pyogenes Cas9.

139. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a catalytically inactive Cas9.

140. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a codon optimized Cas9.

141. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of Cas9 with a D10A mutation.

142. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a dCas9 fused with KRAB.

143. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of dCas9 fused with a carboxy terminal bipartite repressor domain, KRAB-MeCP2 or derivative.

144. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a Campylobacter jujeni Cas9 ortholog.

145. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a CRISPR associated protein or an ortholog.

146. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a Casl2a or Casl2b protein.

147. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of Casl3.

148. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of Casl2f or Casl3a.

149. The system of any one of the preceding claims, wherein the effector element is comprised at least a part from at least a part of a DNA, DNA capable of expressing, RNA, RNA, protein, or combination thereof capable of effecting DNA sequences in a sequence specific manner to induce double stranded breaks, single stranded breaks, methylation, chromatin modification, or generally epigenetic silencing.

150. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from at least a part of a toxin, toxin derivative, modified toxin, or a homolog, ortholog, or paralog thereof.

151. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from at least a part of a bacterial, fungal, plant, or animal toxin.

152. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from at least a part of a vibrio cholerae toxin, shiga toxin, shigella toxin, ricin, saporin, staphylococcus aureus enterotoxin H, Clostridium perfringens enterotoxin, streptolysin 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 is comprised at least in part from at least a part of a viral thymidine kinase or derivative thereof.

154. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from at least a part of a bacterial cytosine deaminase or derivative thereof.

155. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from at least a part of a D-amino-acid oxidase or derivative thereof.

156. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from at least a part of a bacterial carboxypeptidase G2 or derivative thereof.

157. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from at least a part of a purine nucleoside phosphorylase or derivative thereof.

158. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from at least a part of a thymidine phosphorylase or derivative thereof.

159. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from at least a part of a xanthine-guanine phosphoribosyl transferase or derivative thereof.

160. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from at least a part of a nitroreductase or derivative thereof.

161. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from at least a part of a penicillin-G amidase or derivative thereof.

162. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from at least a part of a multi-drug activation enzyme or derivative thereof.

163. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from at least a part of a horseradish peroxidase or derivative thereof.

164. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from at least a part of a Beta-galactosidase or derivative thereof.

165. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from at least a part of a deoxyribonucleotide kinase or derivative thereof.

166. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from at least a part of a Beta-glucoronidase or derivative thereof.

167. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from at least a part of a carboxypeptidase A or derivative thereof.

168. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from at least a part of a cytochrome P450 or derivative thereof.

169. The system of any one of the preceding claims, wherein the effector element is comprised at least in part from at least a part of a deoxycytidine kinase or derivative thereof.

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

171. The system of any one of the preceding claims, wherein the effector sequence encodes at least a part of the consensus Lox sequence or a sequence capable of mediating the effect of a Lox sequence.

172. The system of any one of the preceding claims, wherein the effector sequence encodes at least a part of the consensus FRT sequence or a sequence capable of mediating the effect of an FRT sequence.

173. The system of any one of the preceding claims, wherein the effector sequence encodes at least a part of the consensus Rox sequence or a sequence capable of mediating the effect of a Rox sequence.

174. The system of any one of the preceding claims, wherein the effector sequence is comprised at least in part from GTGTAGTTCGACCATTCGTG (SEQ ID NO: 1).

175. The system of any one of the preceding claims, wherein the effector sequence is comprised at least in part from GTTCAGGATCACGTTACCGC (SEQ ID NO: 2).

176. The system of any one of the preceding claims, wherein the effector sequence is comprised at least in part from GGGCGAGGAGCTGTTCACCG (SEQ ID NO: 3).

177. The system of any one of the preceding claims, wherein the effector sequence is comprised at least in part from GAAGTTCGAGGGCGACACCC (SEQ ID NO: 4).

178. The system of any one of the preceding claims, wherein the effector sequence is comprised at least in part from GCAGATGTAGTGTTTCCACA (SEQ ID NO: 5).

179. The system of any one of the preceding claims, wherein the effector sequence is comprised at least in part from CCTAAGGTTAAGTCGCCCTCG (SEQ ID NO: 6).

180. The system of any one of the preceding claims, wherein the effector sequence is comprised at least in part from CAACAAGATGAAGAGCACCAA (SEQ ID NO: 7).

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

182. The system of any one of the preceding claims, wherein the effector sequence is comprised at least in part from genetic sequences, which can be targeted by integrases, recombinases, nucleases, nickases, chromatin complexes, or methylases or methyltransferases.

183. The system of any one of the preceding claims, wherein the effector sequence is comprised at least in part from genetic sequences, which can be targeted for silencing RNA or protein binding or can be itself silenced.

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

185. The system of any one of the preceding claims, wherein the effector sequence is a DNA sequence derived from viral or bacterial genomes.

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

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

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

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

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

191. The system of any one of the preceding claims, wherein the actuator response element is comprised at least in part from a tetracycline-controlled transcriptional silencer (tTS).

192. The system of any one of the preceding claims, wherein the actuator response element is comprised at least in part from a 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 composed at least in part of a reverse tetracycline-controlled transactivator.

194. The system of any one of the preceding claims, wherein the actuator response element is comprised at least in part of a viral protein such as VP 16 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 comprised of a tTS and rtTA driven by the same promoter.

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

197. The system of any one of the preceding claims, wherein the actuator response element is comprised at least in part from a transcriptional repressor.

198. The system of any one of the preceding claims, wherein the actuator response element is comprised at least in part from a transcriptional silencer.

199. The system of any one of the preceding claims, wherein the actuator response element is comprised at least in part from a DNA binding protein.

200. The system of any one of the preceding claims, wherein the actuator response element is comprised at least in part from a promoter binding protein.

201. The system of any one of the preceding claims, wherein the actuator response element is comprised at least in part from a transcription initiation complex binding protein.

202. The system of any one of the preceding claims, wherein the actuator response element is comprised at least in part of a protein responsible for DNA methylation.

203. The system of any one of the preceding claims, wherein the actuator response element is comprised at least in part of a protein responsible for histone modification.

204. The system of any one of the preceding claims, wherein the actuator response element is comprised at least in part of at least a part of a Tet-ON system.

205. The system of any one of the preceding claims, wherein the actuator response element is comprised at least in part of at least a part of a Tet-OFF system.

206. The system of any one of the preceding claims, wherein the actuator response element is comprised at least in part of at least a part of AlcR, ArgR, BirA, EthR, HdnoR, HucR, MphR(A), PIP, Rex, RheA, ScbR, TraR, TtgR or their analog, homolog, ortholog or protein fragment, protein, or fusion protein of similar functionality.

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

208. The system of any one of the preceding claims, wherein the actuator response element is comprised at least in part from at least a part of ABIl-Gal4DBD, ABA, PVL1-VP16, PHR- p65, CIBl-TetR, or their analog, homolog, ortholog or protein fragment, protein, or fusion protein of similar functionality.

209. The system of any one of the preceding claims, wherein the actuator response element is comprised at least in part from at least a part of a transcriptional activator, enhancer, derepressor, or protein, RNA, or DNA capable of increasing gene expression directly or indirectly in a targeted or generally cis-acting manner.

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