Adjustable gene therapy

Abstract

The present invention is applicable, at least in part, to therapeutic agents, and more specifically to gene therapy in humans or veterinary medicine.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 459,417, filed on April 14, 2023, entitled "ADJUSTABLE GENE THERAPY", the entire disclosure of which is incorporated herein by reference.

[0002] Reference to electronic sequence listings The contents of the electronic sequence listing (R087270003WO00 - SEQ - KVC.xml; size: 132,546 bytes; creation date: April 12, 2024) are incorporated herein by reference in their entirety.

[0003] Technical Field The present invention is applied, at least in part, to therapeutic agents, more specifically to gene therapy in human or veterinary medicine.

Background Art

[0004] Recent advances in gene therapy have demonstrated that this treatment modality can not only cure diseases such as single - gene disorders, reduce disease states, and prevent progression by replacing defective or malfunctioning genes, but also function as a means of gene augmentation when natural expression or protein function is insufficient. This enables the use of gene therapy in many applications that were previously considered second - best for gene - transfer - based treatments. However, improvements are needed.

Summary of the Invention

[0005] Aspects of the present disclosure relate to a modifiable therapeutic gene therapy system for delivery to any one of the tissues or cells provided herein, such as a subcutaneous space (e.g., one or more cell types within a subcutaneous space), the system comprising: a) a gene construct encoding at least one therapeutic gene, at least one promoter element, and optionally, under the control of one or more regulatory elements; b) a delivery carrier or vector encapsulating at least a portion of the gene construct, optionally allowing for the re-administration of a subsequent dose of the gene construct (e.g., with at least 10% efficiency compared to the initial dose of the gene construct); and c) means for increasing or decreasing the level of therapeutic transgene expression from the gene construct.

[0006] In some embodiments, means c) is a means for permanently reducing the level of therapeutic gene expression (for example, by at least 2%). In some embodiments, means of reducing the level of therapeutic gene expression involve the application of external stimuli.

[0007] In some embodiments, the gene construct encodes at least one functional portion of a human peptide or protein (e.g., a full-length human protein). In some embodiments, the gene construct encodes at least one functional portion of a human protein analog or antagonist. In some embodiments, the gene construct encodes at least one functional portion of a non-human peptide or protein (e.g., a full-length non-human protein). In some embodiments, the gene construct encodes at least one functional portion of a non-human protein analog or antagonist. In some embodiments, the gene construct encodes insulin or an insulin analog. In some embodiments, the insulin or insulin analog is modified furin-cleavable insulin. In some embodiments, the gene construct encodes GLP-1, a GLP-1 agonist, or a GLP-1 agonist analog. In some embodiments, the gene construct encodes a growth factor. In some embodiments, the gene construct encodes a cytokine. In some embodiments, the gene construct encodes an anti-inflammatory protein. In some embodiments, the gene construct encodes a complement protein. In some embodiments, the gene construct encodes a receptor agonist. In some embodiments, the gene construct encodes a receptor antagonist. In some embodiments, the gene construct encodes a fusion protein (e.g., composed of one or more functional elements of different proteins). In some embodiments, the subcutaneous space is the subcutaneous layer.

[0008] In some embodiments, the subcutaneous space is the layer between the superficial fascia and the deep fascia. In some embodiments, the subcutaneous space is the layer between the dermis and the superficial fascia. In some embodiments, the gene construct consists of one or more circular single-stranded DNA constructs. In some embodiments, the gene construct consists of one or more circular double-stranded DNA constructs. In some embodiments, the gene construct consists of one or more linear single-stranded DNA constructs. In some embodiments, the gene construct consists of one or more linear double-stranded DNA constructs. In some embodiments, the gene construct consists of at least a portion of DNA or RNA. In some embodiments, the gene construct consists of at least a portion of DNA and / or RNA. In some embodiments, the external stimulus consists of chemical DNA, RNA, or DNA and RNA derivatives.

[0009] In some embodiments, the promoter element includes a constitutive promoter. In some embodiments, the promoter element includes an inducible promoter. In some embodiments, the promoter element includes a tissue-specific promoter. In some embodiments, the promoter element includes a promoter containing a sequence from one or more of the CAG, EF1a, UBC, CBh, MSCV, hPGK, SFFV, and SV40 promoters. In some embodiments, the promoter element includes a tetOn inducible promoter construct. In some embodiments, the promoter element includes a promoter sequence comprising at least one of an enhancer, a regulator, an operator, and / or a repressor. In some embodiments, the promoter element includes an inducible promoter whose expression can be upregulated or downregulated in response to external or internal stimuli such as inflammation, heat, light, stress, steroids, tetracyclines, antibiotics, rapamycin, ganciclovir, or acyclovir administration, or is inducible by an upregulated or downregulated molecule (e.g., ROS, NOS, or cytokine release). In some embodiments, the promoter element includes a circadian rhythm or periodic promoter (for example, one that changes its activity level by at least 5% with some periodicity over a range of hours to months).

[0010] In some embodiments, the regulatory element includes post-translational regulatory elements. In some embodiments, the regulatory element includes at least some or all of the post-transcriptional regulatory elements (WPREs) of woodchuck hepatitis virus. In some embodiments, the regulatory element includes at least some or all of the optimized post-transcriptional regulatory elements (WPREs) of woodchuck hepatitis virus. In some embodiments, the regulatory element includes cis-acting elements that can increase cytoplasmic accumulation. In some embodiments, the regulatory element includes at least some or all of the HIV type 1 Rev-Rev responsive elements. In some embodiments, the regulatory element includes at least some or all of the post-translational regulatory elements of human hepatitis virus. In some embodiments, the regulatory element includes at least some or all of the one or more viral post-translational regulatory elements that can improve the expression of any other elements encoded by the gene or gene construct of interest.

[0011] In some embodiments, the gene construct comprises one or more functional gene sequences. In some embodiments, one or more functional gene sequences promote the transfer of the gene construct (e.g., at least 4% of the gene construct) to the nucleus of target cells. In some embodiments, one or more functional gene sequences comprises sequences encoding sequence-specific DNA-binding proteins bound to nuclear localization signal peptides. In some embodiments, one or more functional gene sequences comprises DNA nuclear target sequences recognized by one or more specific transcription factors. In some embodiments, one or more functional gene sequences comprises DNA nuclear target sequences (DTS) that are active in importin-mediated transport systems. In some embodiments, one or more functional gene sequences comprises at least some or all of the SV40 DTS. In some embodiments, one or more functional gene sequences comprises at least some or all of the glucocorticoid response element (GRE) DTS. In some embodiments, one or more functional gene sequences comprises a DTS sequence of Sox2 regulatory region 2. In some embodiments, one or more functional gene sequences include importin beta(1), importin 7, NF-kappa beta, or small molecule guanosine triphosphatase Ran interaction sequences. In some embodiments, one or more functional gene sequences (e.g., reducing expression from a gene construct) include an inducible suicide gene. In some embodiments, one or more functional gene sequences (e.g., reducing expression from a gene construct) include an inducible expression system encoding an RNA molecule that reduces the expression of at least a portion of a therapeutic gene construct. In some embodiments, one or more functional gene sequences (e.g., reducing expression from a gene construct) encode a protein capable of targeted epigenetic silencing. In some embodiments, one or more functional gene sequences (e.g., reducing expression from a gene construct) encode an HSV-TK suicide gene or system. In some embodiments, one or more functional gene sequences (e.g., reducing expression from a gene construct) encode a RapaCas9 suicide gene or system.In some embodiments, one or more functional gene sequences (e.g., reducing expression from a gene construct) encode one or more miRNAs, siRNAs, shRNAs, dsRNAs, ncRNAs, lncRNAs, piwi-interacting RNAs, PATs, eRNAs, and / or circRNAs. In some embodiments, one or more functional gene sequences (e.g., reducing expression from a gene construct) encode at least a guide RNA and a CRISPR-dCas system (e.g., bound to a protein that induces epigenetic silencing). In some embodiments, one or more functional gene sequences (e.g., reducing expression from a gene construct) encode at least a guide RNA and CRISPR-dCas9-KRAB. In some embodiments, one or more functional gene sequences (e.g., reducing expression from a gene construct) encode at least a guide RNA and CRISPR-dCas12b-KRAB. In some embodiments, one or more functional gene sequences (e.g., reducing expression from a gene construct) encode at least a guide RNA and CRISPR-dCas8c-KRAB. In some embodiments, one or more functional gene sequences (e.g., those that reduce expression from a gene construct) encode at least a guide RNA and CRISPR-dCas8a-KRAB. In some embodiments, one or more functional gene sequences (e.g., those that reduce expression from a gene construct) encode at least a guide RNA and CRISPR-dCas8b-KRAB. In some embodiments, one or more functional gene sequences (e.g., those that reduce expression from a gene construct) encode at least a TALEN, meganuclease, endonuclease, restriction enzyme, zinc finger protein, or other DNA or RNA-binding protein.

[0012] In some embodiments, the regulatory element alters the expression from the gene construct in response to temperature. In some embodiments, the regulatory element alters the expression from the gene construct in response to heat. In some embodiments, the regulatory element alters the expression from the gene construct in response to cold. In some embodiments, the regulatory element alters the expression from the gene construct in response to ultrasound. In some embodiments, the regulatory element alters the expression from the gene construct in response to electrical stimulation. In some embodiments, the regulatory element alters the expression from the gene construct in response to chemical stimulation. In some embodiments, the regulatory element alters the expression from the gene construct in response to changes in the physiological environment. In some embodiments, the regulatory element alters the expression from the gene construct in response to paracrine, endocrine, or autocrine factors. In some embodiments, the regulatory element alters the expression from the gene construct in response to the presence of inflammation. In some embodiments, the regulatory element includes a proximal promoter. In some embodiments, the regulatory element includes a distal promoter. In some embodiments, the regulatory element includes an insulator. In some embodiments, the regulatory element forms a secondary structure with the gene construct or other gene sequences.

[0013] In some embodiments, the delivery carrier or vector comprises lipid nanoparticles (e.g., functionalized lipid nanoparticles, solid lipid nanoparticles, lipid polymer hybrid nanoparticles). In some embodiments, the delivery carrier or vector comprises liposomes (e.g., functionalized liposomes, stealth liposomes) or micelles. In some embodiments, the delivery carrier or vector comprises one or more cells (e.g., functional cells), cellular components, or cell membranes. In some embodiments, the delivery carrier or vector comprises cubosomes, transfectosomes, endosomes, exosomes, or vesicle systems. In some embodiments, the delivery carrier or vector comprises polymer nanoparticles. In some embodiments, the delivery carrier or vector comprises non-immunogenic or low-immunogenic viral vectors. In some embodiments, the delivery carrier or vector comprises proteins or polypeptides. In some embodiments, the delivery carrier or vector is functionalized to reduce immunogenicity. In some embodiments, the delivery carrier or vector is functionalized to promote uptake (e.g., by one or more specific cell types). In some embodiments, the delivery carrier or vector is functionalized to promote endocytosis, pinocytosis, or enhance transport to the nucleus. In some embodiments, the delivery carrier or vector comprises an ionizable lipid. In some embodiments, the delivery carrier or vector comprises an ionizable element. In some embodiments, the delivery carrier or vector comprises an ionizable polymer. In some embodiments, the delivery carrier or vector comprises cholesterol. In some embodiments, the delivery carrier or vector comprises a cationic component. In some embodiments, the delivery carrier or vector comprises an amphiphilic polymer. In some embodiments, the delivery carrier or vector comprises polyethylene glycol. In some embodiments, the delivery carrier or vector comprises an antibody, nanobody, or antibody fragment. In some embodiments, the delivery carrier or vector comprises a peptide.In some embodiments, the delivery carrier or vector comprises nanoparticles chemically or physically conjugated with a peptide, protein, or a functional sequence derived from a peptide or protein. In some embodiments, the delivery carrier or vector comprises a nanoemulsion, a nanostructured lipid, or is composed of at least a portion of an amphiphilic polymer or oligomer. In some embodiments, the delivery carrier or vector comprises a phospholipid, a sphingolipid, a polyelectrolyte polymer, or a polyelectrolyte complex. In some embodiments, the delivery carrier or vector comprises metal or ceramic nanoparticles. In some embodiments, the delivery carrier or vector does not induce a humoral response or a cell-mediated immune response. In some embodiments, the delivery carrier or vector does not induce a memory immune response. In some embodiments, the delivery carrier or vector is minimally immunogenic (e.g., such that the memory immune response produced by the carrier or vector is not able to neutralize at least 10% of subsequent doses of the carrier or vector via the same route of administration in a test subject). In some embodiments, the delivery carrier or vector is minimally immunogenic (e.g., such that the memory immune response produced by the carrier or vector cannot neutralize at least 50% of subsequent doses of the carrier or vector via the same route of administration, such as in the test subject). In some embodiments, the delivery carrier or vector is minimally immunogenic (e.g., such that the memory immune response produced by the carrier or vector cannot neutralize at least 75% of subsequent doses of the carrier or vector via the same route of administration, such as in the test subject). In some embodiments, the delivery carrier or vector is minimally immunogenic (e.g., such that the memory immune response produced by the carrier or vector cannot neutralize at least 90% of subsequent doses of the carrier or vector via the same route of administration, such as in the test subject).In some embodiments, the delivery carrier or vector is minimally immunogenic (for example, such that the memory immune response induced by the carrier or vector is unable to neutralize at least 99% of subsequent doses of the carrier or vector via the same route of administration, such as in the test subject).

[0014] In some embodiments, the external stimulus induces cooling of the skin and subcutaneous tissue (for example, to induce adipocyte death). In some embodiments, the external stimulus induces cooling of the skin and subcutaneous tissue to induce adipocyte aging. In some embodiments, the external stimulus causes a temperature change in the subcutaneous tissue that is sufficient to alter the expression from gene constructs. In some embodiments, the external stimulus includes high-frequency ultrasound. In some embodiments, the external stimulus includes medium-frequency ultrasound. In some embodiments, the external stimulus includes low-frequency ultrasound. In some embodiments, the external stimulus includes high-frequency ultrasound-mediated cavitation. In some embodiments, the external stimulus includes medium-frequency ultrasound-mediated cavitation. In some embodiments, the external stimulus includes low-frequency ultrasound-mediated cavitation. In some embodiments, the external stimulus includes ultrasound at frequencies in the range of 1 to 10 kHz. In some embodiments, the external stimulus includes ultrasound at frequencies in the range of 10 to 28 kHz. In some embodiments, the external stimulus includes ultrasound at frequencies in the range of 28 to 40 kHz. In some embodiments, the external stimulus includes ultrasound at frequencies in the range of 40 to 60 kHz. In some embodiments, the external stimulus includes ultrasound in the frequency range of 1 to 320 kHz. In some embodiments, the external stimulus includes sound waves (e.g., capable of suppressing expression in adipocytes, inducing sentences in adipocytes, or inducing adipocyte death, such as at least in a portion of the subcutaneous space). In some embodiments, the external stimulus includes electromagnetic waves (e.g., capable of suppressing expression in adipocytes, inducing sentences in adipocytes, or inducing adipocyte death, such as at least in a portion of the subcutaneous space). In some embodiments, the external stimulus includes electrical signals (e.g., capable of suppressing expression in adipocytes, inducing sentences in adipocytes, or inducing adipocyte death, such as at least in a portion of the subcutaneous space). In some embodiments, the external stimulus includes administration of a formulation containing rapamycin, rapalog, or a derivative of rapamycin. In some embodiments, the external stimulus includes administration of a formulation containing tetracycline or a derivative thereof.In some embodiments, the external stimulus includes the administration of rapamycin or rapalog, or a derivative of rapamycin, in combination with a tetracycline or tetracycline derivative. In some embodiments, the external stimulus includes the administration of ganciclovir or a derivative thereof. In some embodiments, the external stimulus includes the administration of doxycycline or a derivative thereof. In some embodiments, the external stimulus includes the administration of one or more pharmacologically active small molecules. In some embodiments, the external stimulus includes the administration of one or more peptides, with or without conjugation. In some embodiments, the external stimulus includes the administration of one or more hormones or analogs thereof. In some embodiments, the external stimulus includes the administration of tamoxifen or a derivative thereof. In some embodiments, the external stimulus includes the administration of one or more agents that induce an inflammatory response. In some embodiments, the external stimulus includes the administration of one or more steroids. In some embodiments, the external stimulus includes the administration of one or more antibodies, with or without functionalization.

[0015] In some embodiments, the external stimulus includes the administration of one or more receptor ligands. In some embodiments, the external stimulus includes the administration of one or more antibody fragments. In some embodiments, the external stimulus includes the administration of one or more proteins. In some embodiments, the external stimulus includes the administration of one or more fusion proteins. In some embodiments, the external stimulus includes the administration of one or more RNA molecules. In some embodiments, the external stimulus includes the administration of one or more lipids. In some embodiments, the external stimulus includes the administration of one or more metabolites. In some embodiments, the external stimulus includes the administration of agents that induce an anti-inflammatory or immunomodulatory response. In some embodiments, the external stimulus includes the administration of one or more neurotransmitters. In some embodiments, the external stimulus includes the administration of one or more proteins that can directly or indirectly interact with a promoter, RNA, or DNA sequence. In some embodiments, the external stimulus includes the administration of one or more RNA or DNA molecules that can directly or indirectly interact with a promoter, RNA, or DNA sequence.

[0016] Aspects of this disclosure relate to the therapeutic gene expression method described herein, which includes administering a controllable therapeutic gene therapy system at least once. Aspects of this disclosure relate to methods of therapeutic transgene expression, comprising administering a gene construct encoding at least one therapeutic gene to any one of the tissues or cells provided herein, such as a subcutaneous space (e.g., one or more cell types within a subcutaneous space), under the control of at least one promoter element, and optionally one or more regulatory elements, and optionally under the control of a delivery carrier or vector encapsulating at least a portion of the gene construct.

[0017] In some embodiments, the gene construct is any one of the gene constructs described herein. In some embodiments, one or more tuning elements are any one of the tuning elements described herein. In some embodiments, the delivery carrier or vector is one of the carriers or vectors described herein.

[0018] In some embodiments, the method further comprises killing, reducing, removing, or aging cells in the subcutaneous space to which the gene construct has been administered. In some embodiments, the method further comprises reducing the expression of transgenes from the gene construct. In some embodiments, the method further comprises increasing the expression of transgenes from the gene construct.

[0019] In some embodiments, the external stimulus is applied to cells in the subcutaneous space to which the gene construct has been administered. In some embodiments, the external stimulus is any one of the external stimuli described herein. In some embodiments, the external stimulus is heat, cold, electromagnetic radiation, ultrasound, sound waves, pressure, electrical stimulation, or chemical or physical means that increase or decrease the expression of the therapeutic gene, or elements that can increase or decrease the expression of the therapeutic gene. In some embodiments, a tunable gene therapy system and / or gene construct includes means for up- or down-regulating transgene expression, wherein the means are any one of the means provided herein for such purposes.

[0020] In some embodiments, the method further comprises administering to a subcutaneous space (e.g., one or more cell types within the subcutaneous space) (1) a modifiable gene therapy system, or (2) a gene construct encoding at least one therapeutic gene, under the control of at least one promoter element, and optionally one or more regulatory elements, and optionally under the control of a delivery carrier or vector encapsulating at least a portion of the gene construct.

[0021] In some embodiments, transgene expression is permanently reduced (for example, compared to baseline levels of expression of the administered transgene). In some embodiments, the administration is for treating a disease in a subject (e.g., a human subject). In some embodiments, the administration is for treating a disease in a non-human subject. In some embodiments, the administration is for enhancing or improving the health, physical condition, mental state, or physical or mental capacity of a human or animal. In some embodiments, the administration is for increasing the lifespan of a human or animal and / or increasing the healthy lifespan of a human or animal.

[0022] In some embodiments, the target cells are adipocytes. In some embodiments, the target cells are preadipocytes. In some embodiments, the target cells are progenitor cells capable of differentiating into adipocytes. In some embodiments, the target cells are stem cells capable of differentiating into adipocytes. In some embodiments, the target cells are resident cells of the subcutaneous tissue. In some embodiments, the target cells are transient cells or stem cells, but can be resident cells of the subcutaneous tissue.

[0023] In some embodiments, the adjustable therapeutic system or method is for the treatment of monogenic disorders. In some embodiments, the adjustable therapeutic system or method is for the treatment of enzyme deficiency disorders. In some embodiments, the adjustable therapeutic system or method is for protein replacement therapy. In some embodiments, the adjustable therapeutic system or method is for the treatment of metabolic disorders. In some embodiments, the adjustable therapeutic system or method is for the treatment of autoimmune disorders. In some embodiments, the adjustable therapeutic system or method is for the treatment of oncology. In some embodiments, the adjustable therapeutic system or method is for the treatment of neurological disorders. In some embodiments, the adjustable therapeutic system or method is for the treatment of cardiovascular conditions. In some embodiments, the adjustable therapeutic system or method is for the treatment of musculoskeletal disorders. In some embodiments, the adjustable therapeutic system or method is for the treatment of hematological disorders. In some embodiments, the adjustable therapeutic system or method is for the treatment of dermatological disorders. In some embodiments, the adjustable therapeutic system or method is for the treatment of immune system disorders. In some embodiments, the adjustable therapeutic system or method is for the treatment of pulmonary system disorders. In some embodiments, the adjustable therapeutic system or method is for the treatment of kidney or bladder disorders. In some embodiments, the adjustable therapeutic system or method is for the delivery of therapeutic antibodies via expression from a gene construct. In some embodiments, the adjustable therapeutic system or method is for the delivery of therapeutic proteins via expression from a gene construct. In some embodiments, the adjustable therapeutic system or method is for the delivery of fusion proteins via expression from a gene construct. In some embodiments, the adjustable therapeutic system or method is for the delivery of peptides via expression from a gene construct. In some embodiments, the adjustable therapeutic system or method is for the delivery of immunogens via expression from a gene construct.In some embodiments, an adjustable treatment system or method is for the delivery of a vaccine via expression from a genetic construct.

[0024] In some embodiments, an adjustable treatment system or method further comprises, or the administration thereof comprises, one or more markers for subsequent localization, visualization, analysis, enabling upregulation, enabling downregulation, subsequent dosing selection, or general visualization, or means therefor. In some embodiments, an adjustable treatment system or method further comprises, or the administration thereof comprises, a surface or internal marker for subsequent localization, visualization, analysis, enabling upregulation, enabling downregulation, subsequent dosing selection, or general visualization at the site of delivery, or means therefor.

[0025] In some embodiments, one or more markers comprise any one of the markers as described herein (e.g., one or more metals, ceramics, polymers, or composites, or a gene code encoding a reporter protein, optionally directly or that can be visualized after application of a stimulus that enables or enhances visualization).

[0026] In some embodiments, the stimulus is any one of the stimuli as described herein (e.g., one or more energies, cold, heat, administration of one or more chemical substances, therapeutic molecules or atoms that change the physical, chemical or physiological state of the system and enable the reporting, visualization, reading or interaction of the marker, thereby providing information regarding the treatment). In some embodiments, the administration occurs more than once, such as multiple times, during the treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] [Figure 1A]Figures 1A and 1B show plasmid maps for npRB2.6v3 (CBh-FC-hINS-CMV-rapaCasp9-K19) (Figure 1A) and npRB2.6v7 (CBh-FC-FLuc-CMV-rapaCasp9-K19) (Figure 1B). [Figure 1B] Figures 1A and 1B show plasmid maps for npRB2.6v3 (CBh-FC-hINS-CMV-rapaCasp9-K19) (Figure 1A) and npRB2.6v7 (CBh-FC-FLuc-CMV-rapaCasp9-K19) (Figure 1B). [Figure 2] Figure 2 shows the results of a cell apoptosis assay quantified by FLuc expression at 76 hpi. [Figure 3] Figure 3 shows RapaCasp9 inducing apoptosis in HdAD at 24 hpi. Results are shown for 0 mM, 0.05 mM, and 0.1 mM Tet. [Figure 4] Figure 4 is a plasmid map for gcCBh-fLuc-SV40IpA. [Figure 5A-5C] Figures 5A–5C show the results of animal monitoring after subcutaneous injection of the initial construct dose. Figures 5A–5B show that IVIS confirmed the durability of the initial signal (transgene expression) throughout up to day 22. Figures 5A and 5C demonstrate that the initial dose can be upregulated in a predictable, dose-dependent manner. [Figure 6] Figure 6 shows the total flow from the region of interest as a function of time (days). [Figure 7] Figure 7 shows the total flow from the upward adjustment of ROI one week later (left) and one month later (right). [Figure 8] Figure 8 shows the mean flow before and after downward adjustment in albino mice. [Figure 9] Figure 9 shows the plasmid map of npRB2.10v1(EFS-NLuc+EFS-Cre-ERT2). [Figure 10] Figure 10 is the plasmid map of npRB2.6v8(CBh-NLuc). [Modes for carrying out the invention]

[0028] In its current form, gene therapy is a single-dose treatment that delivers the initial dose without the ability to adjust the dose up or down after the initial administration. In addition, gene therapy treatments delivered by viral vectors cannot be re-administered because the viral vector often elicits a memory immune response that neutralizes subsequent doses in a way that varies from patient to patient and from organism to organism. This can depend on numerous factors, including the initial administration route, the delivered dose, the patient's immune status, pre-existing immunity to the vector, and the immune system status at the time of potential re-administration. Moreover, multiple in vivo studies have shown variable responses to re-administration of viral gene therapy using the same vector within the same organism, with some subsequent doses showing expression levels similar to or equivalent to the initially delivered dose, while others showed no expression from the therapeutic construct. This variability is hypothesized to be caused by the aforementioned immune and vector-related factors, resulting in a lack of predictability for re-administration, up-adjustment, or dose increases. Target particles are used for drug delivery, and virus-like particles are attempted as a means of narrowing the targeting of gene therapy procedures, with the targeted portion in such particles also preventing re-administration of gene therapy and thus up-adjustment after the initial administration. In addition, while attempting to target tissues, the majority of gene therapies are administered intravenously or directly to the organs requiring gene replacement. Gene therapies administered intravenously, regardless of whether viral or non-viral vectors are used, and regardless of whether targeted or non-targeted particles are applied, are primarily taken up by the liver. Gene therapy delivered to target organs can be effective in transducing target tissue, but it sometimes comes with increased complications due to immunogenic reactions within that organ, difficulties in accessing target tissues (such as the brain or retina), and overall procedural complexity. Conversely, the vast majority of protein replacement therapy or protein-based therapeutic interventions are administered subcutaneously, which is a route of administration to tissues that offer simple access, excellent bioavailability, and are generally tolerant of stimulation without significant impact on overall biological function.

[0029] Furthermore, while gene therapies using non-immunogen carriers have been attempted, these procedures lack the ability to adjust the initial dose downward in a controllable and predictable manner. The inability to adjust gene therapy procedures after initial delivery hinders their use in numerous applications where upward and downward adjustments of treatment are required after the initial assessment of safety and efficacy in a given patient. At the same time, many treatments require upward and downward adjustments for effective action. For example, GLP-1 receptor agonists (or GLP-1 analogs) and basal insulin are initially administered at low doses to assess patient-specific tolerability, and after the initial assessment, which may take weeks to months, the therapeutic dose is scrutinized for adjustment to optimize safety, tolerability, or efficacy. Other procedures require downward adjustment of the initial dose to achieve efficacy and reduce safety-related risks. For example, anti-VEGF therapy for age-related macular degeneration (wet) is initially administered at high frequency or high mean in situ concentrations and will be reduced (in dose or frequency) to minimize potential treatment-related side effects. Therefore, in order to extend the applicability of gene therapy treatments to therapies that require dose adjustment after initial administration or that may benefit from dose adjustment, dose-adjustable or adjustable gene therapy treatments have been developed that allow for at least one, but in some embodiments, multiple, arbitrary upward and / or downward adjustments after initial delivery. Ultimately, the route of administration of the gene therapy treatment is preferably optimized in some embodiments to allow for upward and downward adjustment of the effective profile, the safety profile of the treatment, and / or pharmacokinetics suitable for a wide range of therapeutic applications. Accordingly, provided herein are means and related compositions and methods for enabling and using adjustable gene therapy treatments that can be delivered at an initial dose and subsequently adjusted upward and downward after initial administration.

[0030] The therapeutic gene therapy formulations provided herein can be used for the treatment of diseases or the management of disease symptoms. More specifically, the therapeutic formulations disclosed herein can be used for human or veterinary purposes and comprise at least one gene construct or gene construct encoding at least one therapeutic gene or gene sequence capable of beneficial effects in human or veterinary purposes. Provided herein are means for delivering a number of functional elements in cis or trans configuration along with the therapeutic gene or gene construct, which allows for or optimizes the up and / or down adjustment of the therapeutic formulation or, in particular, the therapeutic gene or gene construct after initial administration. In one embodiment, an adjustable dose of gene therapy elements (single / plural) that can be adjusted up or down at least once after initial administration is provided. The formulations provided herein can be used to optimize the safety, efficacy and / or tolerability of the initially delivered dose. In one embodiment, the provided means are for delivering a gene therapy treatment to a subcutaneous space that is generally well-vascularized and capable of providing optimal pharmacokinetics for a bioagent delivered subcutaneously, as well as means of downmodulation, such as by applying an agent or means to reduce the expression level of the gene therapy in the target cells, or optionally reduce the viability of the target cells, or optionally induce senescence in the target cells or senescence of the target cells. Such agents or means may be one or more of ultrasound, focused ultrasound, radiofrequency ultrasound, intermediate or medium-frequency ultrasound, low-frequency ultrasound, ultrasound-mediated cavitation, ultracavitation, radiofrequency, cold, temperature flux, cooling effect, temperature reduction, electromagnetic radiation, and focused energy.

[0031] Generally, what is provided herein are therapeutic gene therapy formulations for the treatment of diseases or pathological conditions in humans or animals, and the dose delivered after the initial administration may also be adjustable up and / or down. The formulation may include at least one therapeutic gene construct of interest encoding at least one peptide, protein, or non-coding RNA and a promoter sequence that modulates the expression of the therapeutic gene of interest. Any one of the formulations may be delivered cis with post-translational regulatory elements. Any one of the formulations may be delivered cis or trans with a gene sequence that can promote the transfer of at least a portion of the therapeutic gene construct to the nucleus of target cells. Any one of the formulations may include at least one gene sequence that can reduce the expression of the gene construct from one or more elements by acting on cells containing the gene construct, the gene construct itself, or one or more elements within cells containing the gene construct. Any one of the gene constructs may be at least partially encapsulated by a carrier or vector that does not induce a memory immune response and / or can neutralize at least a portion of the therapeutic dose of the same or substantially similar composition subsequently re-administered via the same route of administration in general. In a therapeutic gene therapy system described herein that does not include regulatory sequences that allow for the stepwise suppression of therapeutic gene expression through silencing, removal of the gene construct, induction of apoptosis, or a general reduction of gene expression in cells containing the construct, the reduction of therapeutic gene expression can be achieved by applying an external stimulus that can eliminate cells containing the therapeutic construct, reduce cell viability, general gene expression, general transcription, translation, or protein secretion, or induce apoptosis or senescence in cells containing the construct. In some embodiments, the cells can be removed by surgical means or other means such as liposuction.

[0032] Facilitating nuclear transfer may include the transfer of a sufficient amount of the therapeutic construct to the nucleus to enable the efficacy of the construct without compromising the safety or tolerability of the treatment, ideally at least 4% of the construct, and most ideally at least 20% of the construct, on a molar basis. Similarly, in some embodiments, the occurrence of an adaptive immune response includes the production of neutralizing antibodies or reactive T cells that can exclude or neutralize subsequent doses in a manner that limits efficacy, ideally with neutralization of less than 50% of the dose, most ideally with neutralization of less than 10%, and preferably without significant construct or carrier-specific neutralization.

[0033] In some embodiments, the downregulation is complete, while in other embodiments, the downregulation is incremental and may include any amount between 2% to 15%, 90%, or 2% to 100% of the total expression. Incremental downregulation may be performed all at once, or as a series of stimuli to reduce expression in multiples of the initial downregulation or as needed to obtain optimal transgene expression. The route of administration for delivery of the formulation may generally be systemic or topical, and may consist of one or more clinically established routes of administration for therapeutic agents. More specifically, the route of administration may be limited to one or more topical tissues or organs, or it may be systemic, including intestinal or parenteral routes of administration. In some embodiments, the route of administration may include one or more intravenous, subcutaneous, subdermal, intra-articular, intraventricular, intra-intramuscular, subarachnoid, vaginal, rectal, inhalation, intravitreous, oral, oral cavity, sublingual, ocular, transdermal, pulmonary, or intraperitoneal. In some embodiments, one preferred method of administering gene therapy is to subcutaneous or subcutaneous adipose tissue. This may be the safest means of down-adjusting gene therapy, while still providing optimal pharmacokinetics for treatment, allowing for multiple up-adjustments, and, if necessary, administration of numerous gene therapy constructs.

[0034] Provided herein are therapeutic formulations containing a genetically encoded therapeutic gene, with or without functional elements or genetic components involved in regulation. More specifically, a therapeutic formulation may encode at least one functional portion of a human peptide or protein, at least one functional portion of a human peptide or protein analog or antagonist, at least one functional portion of a non-human peptide or protein, or at least one functional portion of a non-human peptide or protein analog or antagonist. In some embodiments, a therapeutic formulation may encompass at least a portion of a gene, or more specifically, at least a coding region of a gene. In yet another embodiment, the therapeutic gene of interest may encode modified furin-cleavable insulin, insulin or insulin analog, GLP-1 peptide, GLP-1 agonist, or GLP-1 agonist analog. In yet another embodiment, the therapeutic formulation may encode at least one growth factor, cytokine, anti-inflammatory protein, complement protein, or at least a portion of proteins of the immune system or immune system regulators, as well as at least a receptor agonist or antagonist, a secreted protein or a portion thereof, or a receptor or a functional element thereof. In yet another embodiment, the therapeutic formulation may encode at least a portion of a fusion protein composed of one or more functional elements of at least two different proteins or peptides. Alternatively, the therapeutic formulation may encode non-protein-coding elements of the human or animal genome, generally encompassing structural or regulatory elements, such as transfer RNA (tRNA), ribosomal RNA (rRNA), long non-coding RNA (lncRNA), microRNA (miRNA), silencing RNA (siRNA), inducible RNA (iRNA), endogenous silencing-inducing RNA, piwi-interacting RNA (piRNA), or other types of RNA or DNA that can perform intracellular or extracellular functions, whether or not they can be involved in protein coding.

[0035] A therapeutic gene construct may include at least one promoter that drives or modulates the expression of a therapeutic gene or sequence of interest, and optionally one or more additional promoters that can drive or modulate the expression of dose-regulating elements, nuclear localization signals, or accessory elements that are necessary for or provide an auxiliary function of the therapeutic gene therapy.

[0036] In some embodiments, the promoter sequence intended to drive the expression of a therapeutic sequence may be one or more mammalian promoters, constitutive promoters, inducible promoters, or tissue-specific promoters. In other embodiments, the promoter sequence may be derived in at least part from one or more viral promoters, bacterial promoters, archaeal promoters, or promoters or regulatory sequences of human or animal genomes, and may optionally contain introns, untranslated regions, enhancers, or other regulatory elements necessary for gene expression or regulation. The promoter sequence may contain at least one element of the following: chicken β-actin promoter and rabbit β-globin splice receptor site (CAG) promoter, EF1α (EF1a) promoter, polyubiquitin C gene (UBC) promoter, CBh promoter (composed of a modified version of CAG, consisting of a cytomegalovirus (CMV) early enhancer element, promoter region, the first intron and first exon of the chicken β-actin gene, and the splice receptor of the rabbit β-globin gene), mouse stem cell virus (MSCV) promoter, phosphoglycerate kinase (PGK) promoter, spleen fociform virus (SFFV) promoter, or monkey virus 40 (SV40) promoter. The promoter sequence may be a tetracycline-on (tetOn) inducible promoter construct or a tetracycline-off (tetOff) inducible promoter construct. Regulation may be additionally provided by one or more enhancers, regulators, operators, or repressor sequences, which may be delivered cis or trans with the construct. In some embodiments, the promoter sequence may consist at least in part of an inducible promoter whose expression can be upregulated or downregulated in response to external or internal stimuli such as inflammation, heat, light, stress, steroids, tetracyclines, antibiotics, rapamycin, ganciclovir, or acyclovir administration, or may be generally inducible by upregulated or downregulated reactive oxygen species (ROS), reactive nitrogen species (NOS), or cytokine release.Furthermore, in yet another embodiment, the promoter sequence may change its expression level in response to internal or external stimuli by altering in accordance with natural biological cycles. In some embodiments, the promoter sequence may consist at least in part of a circadian rhythm or periodic promoter that changes its activity level by at least 5%, or preferably at least 50%, with some periodicity ranging from several hours to several months, preferably from several days to several weeks.

[0037] In other embodiments, promoter sequences intended to drive the expression of dose-regulating sequences or co-components such as peptides that promote nuclear localization, transport, or stability, or peptides that regulate expression, may be one or more mammalian promoters, constitutive promoters, inducible promoters, or tissue-specific promoters. In other embodiments, promoter sequences may be derived in at least part from one or more viral promoters, bacterial promoters, or promoters or regulatory sequences of human or animal genomes, and may optionally contain introns, untranslated regions, enhancers, or other regulatory elements necessary for expression or regulation. In some embodiments, promoter sequences may contain one or more elements of the CAG promoter, EF1α (EF1a) promoter, UBC promoter, CBh promoter, MSCV promoter, hPGK promoter, SFFV promoter, or SV40 promoter. Promoter sequences may be tetOn-inducible promoter constructs or tetOff-inducible promoter constructs. Regulation may be additionally provided by one or more enhancers, regulators, operators, or repressor sequences, which may be delivered cis or trans with the construct. In some embodiments, the promoter sequence may consist at least in part of an inducible promoter whose expression can be upregulated or downregulated in response to external or internal stimuli such as inflammation, heat, light, stress, steroids, tetracyclines, antibiotics, rapamycin, ganciclovir, or acyclovir administration, or may be generally induced by upregulated or downregulated ROS, NOS, or cytokine release. Furthermore, in yet another embodiment, the promoter sequence may change its expression level in response to internal or external stimuli by changing in accordance with natural biological cycles. In some embodiments, the promoter sequence may consist at least in part of a circadian rhythm or periodic promoter that changes its activity level by at least 5%, preferably at least 50%, with some periodicity ranging from several hours to several months, preferably from several days to several weeks.

[0038] In some embodiments, to enable additional functionality and / or optimal expression of therapeutic genes and any relevant regulatory elements or sequences, the construct may contain one or more posttranslational regulatory elements that may include elements that regulate expression, elements that conditionally regulate expression, elements that stabilize transcribed RNA, or elements that form a secondary or tertiary structure with one or more elements of the therapeutic construct, genomic DNA, or other constructs delivered in cis or trans. Some embodiments may contain one or more polyA signals, or signals that encode polyA, signals that regulate the length of the polyA tail, promote or enhance capping, promote or enhance ribozyme assembly, or generally promote or enhance assembly or transcription or translation mechanisms (after the regulatory elements have been transcribed). In some embodiments, the post-translational regulators incorporated into the therapeutic construct include at least a portion of woodchuck hepatitis virus post-translational regulators (WPREs), at least a portion of optimized woodchuck hepatitis virus post-translational regulators, gene sequences capable of increasing the accumulation or stability of nuclear or cytoplasmic mRNA, at least a portion of human immunodeficiency virus type 1 Rev-Rev responsive elements, at least a portion of human hepatitis virus post-translational regulators, at least a portion of one or more viral post-translational regulators capable of improving the expression of a gene of interest, or elements encoded by any other construct. In some embodiments, one or more post-transcriptional regulators may be incorporated in series, in tandem, or delivered trans with the construct. In yet another embodiment, the regulators may be upstream or downstream of a sequence encoding a therapeutic gene or RNA and may optionally regulate the transcription, translation, or expression of the auxiliary elements of the therapeutic construct.

[0039] In some embodiments, a gene sequence, its carrier element, or general formulation may contain one or more elements that facilitate the transfer of the sequence to the nucleus or other cellular compartments or organelles in order to enable additional or optional expression, localization of the construct, durability of the construct within cells, and / or prevent congenital or adaptive immune responses. The elements may consist of at least part DNA, RNA, protein, peptide, lipid, polymer, cholesterol, or other molecules that can induce the aforementioned effects of expression optimization, construct localization, stability, durability, and immune response optimization. In some embodiments, the therapeutic construct contains a gene sequence delivered in cis or trans that facilitates the transfer of at least a portion of the gene construct, ideally at least 4% on a molar basis, and at least 20% on a molar basis, of the therapeutic construct to the nucleus of target cells. In some embodiments, the gene sequence consists of at least part a protein-coding sequence encoding a protein that transports the therapeutic gene construct to the nucleus, accompanied by a dedicated promoter. In another embodiment, the gene sequence comprises at least part a sequence encoding a sequence-specific DNA-binding protein bound to a nuclear localization signal peptide, or at least part a DNA nuclear target sequence recognized by one or more specific transcription factors, or at least part a DNA nuclear target sequence (DTS) active in the importin-mediated transport system. In yet another embodiment, the sequence that promotes the transfer of the therapeutic construct to the nuclease comprises at least part an SV40 DTS, 3NF DTS, Sox2 regulatory region 2 DTS sequence, or an importin β(1), importin 7, NF-κβ, or small guanosine triphosphatase Ran interaction sequence. In yet another embodiment, the gene sequence that can induce the transfer of the gene construct or other elements delivered trans-to the nucleus encodes DNA that can bind to an intracellular transport protein, or RNA that can act as an intermediary between the intracellular transport protein and the therapeutic construct or elements delivered trans-to the nucleus.

[0040] In some embodiments, to facilitate downregulation after the initial administration of gene therapy, the therapeutic construct contains one or more elements that can reduce the expression level of a therapeutic gene, protein, or RNA of interest. The therapeutic construct may contain gene sequences that can reduce the expression of a therapeutic input gene by altering its expression level upon activation by an external stimulus, transiently or permanently alter the tertiary structure of the therapeutic construct, exclude the therapeutic construct from cells, exclude cells that hold the therapeutic construct, induce cellular senescence, generally reduce transcription within cells containing the therapeutic construct, induce apoptosis in cells containing the therapeutic construct, or induce epigenetic changes in the therapeutic construct, elements of the therapeutic construct, or genomic DNA, thereby altering the transcription, translation, or generally expression of a therapeutic gene or RNA coding element contained within the therapeutic construct. In some embodiments, sequences that can reduce expression from the therapeutic construct are suicide genes, or induceable suicide genes, or suicide genes under an induceable promoter. In other embodiments, gene sequences capable of reducing expression from a therapeutic construct are inducible systems that, under the control of an inducible promoter encoding a protein capable of targeted epigenetic silencing of at least some of the therapeutic gene construct, encode RNA molecules capable of reducing the expression of at least some of the therapeutic gene construct or protein-coding genes. In some embodiments, gene sequences capable of reducing the expression of a therapeutic construct are delivered cis or trans with the therapeutic construct and encode one or more elements of a herpes simplex virus thymidine kinase or HSV-TK suicide gene system, a rapamycin caspase 9 or RapaCas9 suicide gene system, or other suicide gene systems capable of inducing programmed cell death or apoptosis in cells containing the construct. In yet another embodiment, gene sequences capable of reducing the expression of a gene construct encode one or more miRNAs, siRNAs, shRNAs, dsRNAs, ncRNAs, lncRNAs, piwi-interacting RNAs, PATs, eRNAs, or circRNAs.In yet another embodiment, the sequence encodes a DNA-binding protein with targeting capability that can induce sequence-specific epigenetic changes through its domain or binding domain, thereby reducing expression from therapeutic constructs. In yet another embodiment, the sequence encodes a CRISPR-dCas system bound to at least a guide RNA and a protein capable of inducing epigenetic silencing, or at least a guide RNA and CRISPR-dCas9-KRAB, or at least a guide RNA and CRISPR-dCas12b-KRAB (a functional fragment of a protein analog or homolog of KRAB = Kruppel-associated box protein), or at least a guide RNA and CRISPR-dCas8c-KRAB, or at least a guide RNA and CRISPR-dCas8a-KRAB, or at least a guide RNA and CRISPR-dCas8b-KRAB. In another embodiment, gene sequences that can reduce the expression of a therapeutic construct and enable dose-downadjustment encode at least a transcriptional activator-like effector nuclease or TALEN, meganuclease, endonuclease, restriction enzyme, zinc finger protein, or other DNA or RNA-binding protein.

[0041] In another embodiment, means for downregulating a therapeutic construct are contained within the genome, nucleus, mitochondria, or cytoplasm of the cell to which the therapeutic construct is delivered, and in yet another embodiment, means for downregulating are applied to at least one cell and / or adjacent tissue of the cell carrying the therapeutic construct via external stimuli such as ultrasound, heat, cold, heat flux, electromagnetic radiation, non-electromagnetic radiation, shock, pressure, or direct or indirect aspiration to the cell or to tissue adjacent to the tissue containing the cell or at least some of the cells holding the therapeutic construct. Such stimuli may induce cell death, necrosis, apoptosis, inflammation, a reduction in the level of intracellular transcription or translation, intracellular senescence, or a combination thereof.

[0042] The therapeutic constructs described herein may be delivered without a carrier, encapsulated in a carrier, partially encapsulated in a carrier, or interact with a carrier in some physical or chemical manner. Multiple carriers or carrier systems may be used to deliver the therapeutic constructs and may include one or more of liposomes, lipid nanoparticles, polymer nanoparticles, exosomes, microsomes, nanosomes, or exosome-like structures, ionizable lipid-containing particles, lipid and polymer-containing nanoparticles, targeted ligand-containing nanoparticles, peptides, proteins, or nanoparticles chemically or physically conjugated to functional sequences derived from peptides or proteins, lipid nanoemulsions, solid lipid nanoparticles, and nanostructured lipid carriers. In some embodiments, the carrier may contain one or more of amphiphilic oligomers, phospholipids, sphingolipids, ionizable lipids, or cholesterol. In yet another embodiment, the carrier may consist at least in part of a polymer electrolyte complex, cationic liposomes, stealth liposomes, cubosomes, lipoplexes, nanocells, or lipid bilayer sheets. A carrier system or carrier element can bind to one or more targeted ligands, structures or molecules that increase carrier stability, binding ligands, structures or molecules that promote cellular uptake, increase the half-life of the carrier or therapeutic construct, facilitate internalization, facilitate escape from endosomes, control, regulate, or promote intracellular transport, and increase transport to the nucleus or other organelles.

[0043] Some specific examples of concentration ranges for formulations and reagents that can be used to deliver gene cargo are listed below. [Table 1] [Table 2] [Table 3] [Table 4] Possible variants of the helper lipid include: [Table 5-1] [Table 5-2] Possible variants of pegylated lipids include: [Table 6]

[0044] The injection volume may range from 10 μL to 50 mL, depending on the disease, the animal or human being treated, body weight, or other physiological factors. For a reference formulation containing 5 μg of DNA, 141.6 μg of lipid is used as a carrier in a 100 μL formulation.

[0045] The following table lists reference gene constructs that can be used in formulations, as reporter genes, or in combination with reporter genes or therapeutic genes as regulatory sequences for assessing gene expression: [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5]

[0046] One embodiment comprises a formulation containing a modifiable therapeutic gene therapy system intended for delivery to the subcutaneous space, particularly to one or more cell types within the subcutaneous space, the system comprising a gene construct encoding at least one therapeutic gene under the control of at least one promoter element and optionally one or more regulatory elements, a delivery carrier or vector encapsulating at least a portion of the gene construct, and means for increasing the level of therapeutic transgene expression and / or for reducing the level of therapeutic transgene expression (e.g., permanently) (e.g., via the application of external stimuli). The delivery carrier or vector encapsulating at least a portion of the gene construct may be low immunogenic, minimally immunogenic, or non-immunogenic, thereby allowing the same formulation or elements of the same formulation to be repeatedly administered to the same organism without inducing a neutralizing immune response that reduces the efficiency of the re-administered dose by more than approximately 10%. In another embodiment, the delivery carrier or vector is non-immunogenic, allowing the efficiency of the re-administered dose to remain essentially or substantially the same as the initially delivered dose. In another embodiment, this is regardless of the time frame for subsequent dose administration. In some embodiments, the construct holds at least one coding or non-coding therapeutic gene of interest that codes for at least one functional portion of a human peptide or protein, or at least one functional portion of a human peptide or protein analog or antagonist, or at least one functional portion of a non-human peptide or protein, or at least one functional portion of a non-human peptide or protein analog or antagonist. In some embodiments, the therapeutic gene coded by the gene therapy system is insulin, or furin-cleavable insulin, or a modified insulin analog, or an insulin receptor agonist, or a glucagon-like peptide-1 (GLP-1) peptide, a GLP-1 receptor agonist, or a GLP-1 agonist analog. In another embodiment, the therapeutic gene codes for gastric suppressor peptide (GIP) or an analog, a GLP-1 / GIP dual receptor agonist, a coagonist, or an analog thereof.In another embodiment, therapeutic genes of interest include growth factors, cytokines, anti-inflammatory proteins, pro-inflammatory proteins, complement proteins, receptor agonists or antagonists, hormones, peptides, fusion proteins composed of one or more functional elements of different proteins, immunogens, vaccines, or one or more subunits or combinations thereof.

[0047] In some embodiments, therapeutic gene therapy is intended for delivery to the subcutaneous layer, or generally the subcutaneous space, or the tissue layer between the superficial and deep fascia, or any tissue containing adipocytes. Preferably, gene therapy can be adjusted upward or downward in dosage by means other than systemic or local administration of peptides, hormones, small molecule compounds, proteins, or other chemicals. In some embodiments, gene therapy is delivered to the subcutaneous fat body.

[0048] In some embodiments, therapeutic gene therapy is formulated to deliver one or more circular single-strand DNA constructs, circular double-strand DNA constructs, linear single-strand DNA constructs, or linear double-strand DNA constructs. In other embodiments, the gene cargo may generally consist of one or more DNA, RNA, or DNA-RNA hybrids, or chemical derivatives thereof. The gene sequence of the construct may contain at least one promoter, which may consist of a constitutive promoter, an inductive promoter, a tissue-specific promoter, or more specifically, one or more CAG, EF1a, UBC, CBh, MSCV, hPGK, SFFV, or SV40 promoters, or combinations thereof. In other embodiments, the promoter may be a tetOn inductive promoter construct or a tetOff promoter construct, and may optionally contain one or more enhancers, regulators, operators, or repressors. In other embodiments, the promoter element may include an inducible promoter whose expression can be upregulated or downregulated in response to external or internal stimuli such as inflammation, heat, light, stress, steroids, tetracyclines, antibiotics, rapamycin, ganciclovir, or acyclovir, or may be generally induced by upregulated or downregulated ROS, NOS, or cytokine release. In other embodiments, the promoter may respond to a circadian rhythm that changes its activity level by at least 5% with some periodicity ranging from several hours to several months, or may be a periodic promoter. The gene therapy construct may contain a woodchuck hepatitis virus posttranslational regulator (WPRE), or an optimized woodchuck hepatitis virus posttranslational regulator, or a cis-acting element capable of increasing the accumulation of WPRE or cytoplasmic mRNA, or at least a portion of a human immunodeficiency virus type 1 Rev-Rev responsive element, or at least a portion of a human hepatitis virus posttranslational regulator, or at least a portion of one or more viral posttranslational regulators capable of improving the expression of a gene of interest, or one or more posttranslational regulators comprising elements encoded by any other construct.The gene therapy construct may contain one or more functional gene sequences capable of promoting the transfer of at least 4% of the gene construct to the nucleus of target cells containing the therapeutic construct. In some embodiments, the therapeutic construct may contain a protein coding sequence with a dedicated promoter that codes for a protein transporting the therapeutic gene construct to the nucleus or a functional gene sequence that promotes the transfer of at least 4% of the gene construct to the nucleus, and optionally codes for a sequence-specific DNA-binding protein bound to a nuclear localization signal peptide. Other means of transferring the gene therapy construct to the nucleus include coding for a DNA nuclear target sequence (DTS) recognized by one or more specific transcription factors, or a DNA nuclear target sequence (DTS) that is active in an importin-mediated transport system, or at least a portion of an SV40 DTS, or at least a portion of a glucocorticoid response element (GRE), or at least a portion of a Sox2 regulatory region 2 DTS sequence, or at least a portion of importin β(1), importin 7, NF-κβ, or small signaling factors. (GRE)DTS, or at least a portion of the Sox2 regulatory region 2DTS sequence, or at least a portion of the import in beta(1), import in 7, NF-κB, or small guanosine triphosphate Ran interaction sequence.

[0049] Gene therapies as provided herein may be downregulated by physical, chemical, or physiological stimuli. In some embodiments, the downregulation is facilitated by a functional gene sequence or gene element within the gene therapy construct, which may be induceable by one or more chemical, physical, or physiological stimuli, and may be an inducible suicide gene, an inducible expression system encoding an RNA molecule capable of reducing the expression of at least a portion of the therapeutic gene construct, a protein-coding gene under the regulation of an inducible promoter encoding a protein capable of targeted epigenetic silencing of at least a portion of the therapeutic gene, or more specifically, one or more of the HSV-TK suicide gene system, the RapaCas9 suicide gene system, or one or more of miRNAs, siRNAs, shRNAs, dsRNAs, ncRNAs, lncRNAs, piwi-interfering RNAs, PATs, eRNAs, or circRNAs. In other embodiments, the therapeutic gene construct may encode a guide RNA and CRISPR-dCas system conjugated to a protein capable of inducing epigenetic silencing, or guide RNA and CRISPR-dCas9-KRAB, or guide RNA and CRISPR-dCas12b-KRAB, or guide RNA and CRISPR-dCas8c-KRAB, or guide RNA and CRISPR-dCas8a-KRAB, or guide RNA and CRISPR-dCas8b-KRAB, or a TALEN, meganuclease, endonuclease, restriction enzyme, zinc finger protein, or other DNA or RNA-binding protein, or a combination thereof. Factors that modulate expression may include factors that can alter the expression of one or more therapeutic transgenes in response to temperature, or generally heat, cold, heat flux, ultrasound, focused energy, electromagnetic radiation, shock, mechanical pressure, depressurization, vacuum, suction, vibration, agitation, electrical stimulation, chemical stimulation, changes in the physiological environment, changes in the level, concentration, gradient, or activity of paracrine factors, endocrine factors, or autocrine factors, changes in the level of local inflammation, or the presence or absence of inflammatory cells or molecules, or a combination thereof, whether for upregulation or downregulation, or for permanent or transient regulation.In some embodiments, gene therapy can be downregulated by cell removal via liposuction, microliposuction, or microsurgery procedures, or by irradiation with ionizing or non-ionizing radiation. Regulatory elements may include gene sequences, aptamers, aptazymes (e.g., K19 aptazyme), protein-binding sequences (e.g., histone-binding sequences), ribosomes, RNA, proteins, or sequences expressing RNA-protein complexes, or combinations thereof, that alter their structure in response to internal or external stimuli. The aforementioned regulatory elements may include at least a segment of a proximal promoter, distal promoter, insulator, or combination thereof, and may be positioned to regulate a therapeutic gene, dose regulation mechanism, both, or other segments of the expression system. The aforementioned regulatory elements may themselves be capable of forming secondary structures with other genomic sequences or other sequences within the therapeutic construct. These secondary structures may function as means of regulating expression, stability, transport of the construct to the nucleus, or a combination thereof.

[0050] In some embodiments, the gene construct may be encapsulated in a delivery carrier or vector, which may be a vehicle, capsule, envelope, capsid, structural element, or a combination of one or more structures or structural elements. The delivery carrier or vector may partially or completely envelope one or more gene constructs and may be functionalized on the outside, inside, or on multiple surfaces. The delivery carrier or vector may consist at least in part of lipid nanoparticles, liposomes, micelles, cubosomes, functionalized lipid nanoparticles, functionalized liposomes, stealth liposomes, solid lipid nanoparticles, lipid-polymer hybrid nanoparticles, transfectosomes, endosomes, exosomes, cell membranes, vesicle systems, polymers, polymer nanoparticles, non-immunogenic or low-immunogenic viral vectors, proteins or polypeptides, or combinations thereof. One or more elements of the delivery carrier or vector may be functionalized with one or more functional elements to reduce immunogenicity, promote uptake by one or more specific cell types, promote uptake, endocytosis, pinocytosis, enhance transport to the nucleus, or a combination thereof, or to optimize overall physical, physiological, chemical activity or stability. In some embodiments, gene therapy can be delivered intracellularly, or into organelles, or into at least a portion of a cell. In other embodiments, the gene cargo can be delivered intracellularly or as part of cell therapy, and cell therapy can be administered subcutaneously and have the potential for re-administration and / or up or down-modulation. The delivery carrier or vector may contain one or more ionizable lipids, ionizable elements, ionizable polymers, or ionizable oligomers or monomers, and may optionally contain cationic or anionic elements, cholesterol, one or more amphiphilic polymers, polyethylene glycol or derivatives thereof, one or more antibodies, nanobodies, or antibody fragments, peptides or derivatives thereof, DNA or derivatives thereof, RNA or derivatives thereof, or other chemical or physical elements or combinations thereof.Some embodiments include a delivery carrier or vector comprising at least a portion of a delivery carrier or vector containing a peptide, a protein, or a functional sequence derived from a peptide or protein, and nanoparticles or nanostructures chemically or physically conjugated with such a functional sequence. In other embodiments, the delivery vector may be a nanoemulsion, a nanostructured lipid, or comprise at least a portion of an amphiphilic polymer or oligomer. In other embodiments, the delivery carrier or vector may comprise at least a portion of a phospholipid, sphingolipid, ionizable lipid, polyelectrolyte polymer, polyelectrolyte complex, or cholesterol. In yet another embodiment, the delivery carrier or vector may comprise at least a portion of a metal or ceramic nanoparticle. Any embodiment of the compositions or methods provided herein may involve the use of one or more delivery carriers or vectors to deliver one or more gene constructs for the purpose of delivering one or more gene constructs without inducing a humoral or cell-mediated immune response, without inducing a memory immune response, or with the aim of inducing a minimal immune response that allows for re-administration of a therapeutic agent. The re-administration efficiency after the initial dose may be at least 10% at the initial dose level, and an immune response that does not result in severe adverse reactions may be induced. The delivery carrier or vector intended for the proposed gene therapy may be designed to be optimally or minimally immunogenic such that the efficiency of subsequent doses is at least 10%, ideally 50%, most ideally 75%, or 99%, or some value within the aforementioned efficiencies, so that any memory immune response produced by the carrier or vector or gene therapy cannot neutralize the formulation and reduce the delivery efficiency by at least 90%, ideally 50%, and most ideally 25%, or 1%. Therefore, some embodiments provide a delivery carrier or vector that is minimally immunogenic such that a memory immune response produced by the carrier or vector cannot be neutralized by at least 50%, at least 75%, at least 90%, or at least 99% by a subsequently re-administered therapeutic formulation or a substantially similar therapeutic formulation and / or substantially the same route of administration.In some embodiments, therapeutic gene therapy constructs can be downregulated using external stimuli, which optionally induce one or more of the following: cooling of the skin and subcutaneous tissue to induce adipocyte death; cooling of the skin and subcutaneous tissue to induce adipocyte aging; a subcutaneous temperature change sufficient to alter the expression of the therapeutic gene; or a change in the physical environment of adipocytes such that the change induces a reduction in gene expression from the therapeutic gene. The external stimuli (one or more) may consist of at least a portion of high-frequency ultrasound, medium-frequency ultrasound, low-frequency ultrasound, or sound waves capable of suppressing expression, inducing sentences in adipocytes, or inducing adipocyte death in at least a portion of the subcutaneous space; or electromagnetic waves capable of suppressing expression, inducing sentences in adipocytes, or inducing adipocyte death in at least a portion of the subcutaneous space; or electrical signals capable of suppressing expression, inducing sentences in adipocytes, or inducing adipocyte death in at least a portion of the subcutaneous space. The stimulus may be chemically active in combination with or on its own, and may include, for example, rapamycin or its derivatives, tetracycline or its derivatives, a combination of rapamycin and tetracycline or their derivatives, ganciclovir or its derivatives, doxycycline or its derivatives, or generally the administration of a formulation containing one or more pharmacologically active small molecule compounds. Alternatively, the downregulation may be applied indirectly by delivery of a formulation containing one or more peptides, conjugated or unconjugated, or one or more hormones or their analogues, or more specifically, tamoxifen or its derivatives, which may act directly on DNA or proteins expressed from the therapeutic formulation, act on receptors on cells containing the gene construct, or act via other secondary means.Alternatively, or in combination with other external stimuli, downregulation may be achieved through the administration of one or more agents that induce inflammatory responses, preparations containing one or more steroids, preparations containing one or more antibodies with or without functionalization, preparations containing one or more receptor ligands, preparations containing one or more antibody fragments, preparations containing one or more proteins, preparations containing one or more fusion proteins, preparations containing one or more RNA molecules, preparations containing one or more lipids, preparations containing one or more metabolites, the administration of agents that induce anti-inflammatory or immunomodulatory responses, the administration of preparations containing one or more neurotransmitters, or a combination thereof. In some embodiments, the external stimulus may be performed or constituted by administering a formulation containing one or more proteins capable of directly or indirectly interacting with a promoter, RNA, or DNA sequence. In other embodiments, the external stimulus may be performed or constituted by administering a formulation containing one or more RNA or DNA molecules capable of directly or indirectly interacting with a promoter, RNA, or DNA sequence.

[0051] The gene therapy constructs disclosed herein may have the ability to increase the dose or up-adjust the treatment after the initial administration. This up-adjustment may be permanent or transient and may result from an increase in transgene expression from the original dose or an additional dose of the gene therapy, providing an absolute or relative increase from the original dose. In some embodiments, the dose increase or up-adjustment is achieved via the administration of a second dose of the adjustable gene therapy, the second dose being at least 1% of the original dose, or at least 2% of the original dose, or at least 5%, 10%, 15%, or 25% of the original dose. In other embodiments, the increase in therapeutic transgene expression is achieved via the administration of an additional dose(s) of the gene therapy system. In other embodiments, means of increasing therapeutic transgene expression or up-adjustment may be achieved by the administration of a formulation containing at least DNA, RNA, protein, or a combination thereof, which can transiently or permanently increase therapeutic transgene expression. Subsequent administration of therapeutic gene therapy may be carried out in part or in whole to achieve upregulation, and therefore means of increasing transgene expression may consist of administering at least 10% of at least one of the components of the gene therapy system provided herein. In some embodiments, upregulation or increase of therapeutic transgene expression may be achieved by administering a small molecule that can permanently or transiently increase gene expression, or by administering external stimuli such as heat, cold, electromagnetic radiation, ultrasound, sound waves, pressure, electrical stimulation, or other chemical or physical means that can increase or suppress the expression of elements that can increase the expression of therapeutic transgenes or suppress the expression of therapeutic transgenes.

[0052] Increases or decreases in gene expression from therapeutic constructs may be transient or permanent, and in the case of decreases, they may include less than 25%, 40%, 60%, 80%, or 90% of the total expression of the administered treatment; and in the case of increases, they may include more than 10%, 20%, 50%, or 100% of the total expression. In some embodiments, permanent reductions may be complete or near-complete and greater than 90% of the total expression of the administered therapeutic gene therapy; in other cases, the reduction may be localized, localized, or affect only a portion of the delivered gene, isolated by construct type, response element, anatomical area, expressed transgen, or any other element that distinguishes the portion.

[0053] An additional optional element is the use of markers to identify the site of the procedure. These markers can generally be classified into superficial (defined as observable from the surface with minimal or no intervention) and / or internal (defined as observable from the surface after intervention). Markers may be used to identify one or more of the type, site, dose, formulation, date, or other relevant information or aspects of the treatment. While markers can have many functions, they can generally be used for locating the treatment for subsequent examination, selecting a site for up-adjustment, down-adjustment of the initial or other procedure, selecting an injection site for a drug, administering an inducer, or administering a stimulus that affects or prevents changes in the chemical, physical, or physiological environment of the treatment. Markers can also be used to store, preserve, transmit, or interact with information that is somehow related to the procedure.

[0054] The marker (e.g., a surface marker) may consist of one or more of the following: a skin tattoo, a skin mark, a skin tag, or a means of marking the skin with information, and may range from a simple dot to a complex barcode, a two-dimensional data matrix, or a QR code. The mark may be permanent or semi-permanent, lasting for days, weeks, months, or years. The mark may be visible directly in daylight, or visible under ultraviolet light or any other spectral range, may be fluorescent, may require initial excitation before visibility, or may become visible by initial surface administration of mechanical energy such as electromagnetic spectrum, ultrasound, or, for example, palpation.

[0055] The marker (e.g., an internal marker) may be delivered with the formulation, encoded in a gene construct within the formulation or as part of the formulation, or administered after injection of the formulation. The aforementioned marker may consist of one or more of tantalum, gold, platinum, barium sulfate, or other metals or metal salts, and may consist at least in part of silicone, polyvinyl alcohol, or other biostable or biodegradable polymers, with or without a carrier. In another embodiment, the marker may consist at least in part of a ceramic or composite material. The marker may be passive, like a radiopaque material, or may actively emit a signal, or may exist in an off or on state that can switch to the opposite or another state after the application of a stimulus. The marker may be radiopaque, radiopaque, or semi-radiopaque to ultrasound or other detection or measurement techniques typically used for the visualization of the body or body structures. The marker may be atomic, molecular, or nanostructured and may be part of the formulation. In another embodiment, the marker may be encoded in a gene construct delivered as part of the formulation. For example, a marker may encode a fluorescent protein, which is expressed by cells along with the therapeutic introduction gene, and this expression may be activated by permanent, periodic, or external stimuli such as the administration of heat, cold, radiation, light, energy, or molecular compounds, such as tetracycline or its derivatives, rapamycin or its derivatives, ganciclovir or its derivatives, steroids or their derivatives, tamoxifen or its derivatives, or other therapeutic molecular compounds or formulations, or combinations thereof, to the whole body or around the original treatment site. In another embodiment, a marker may be a bioluminescent protein, such as nanoluciferase or firefly luciferase, or any other bioluminescent protein, which may be constitutively luminescent in the body, or may be luminescent after the administration of a substrate, such as flimazine or its derivatives, or any other small molecule, metal, polymer, or ceramic substrate for the encoded marker enzyme.The reporter gene may be expressed constitutively, or after physical or chemical stimuli such as heat or tetracycline, or generally after the administration of energy within the electromagnetic spectrum, ultrasound, heat, cold, or pharmacological or chemically active substances.

[0056] The gene therapy is intended to treat a disease in a human or animal and may be categorized in some embodiments as a treatment, and in other embodiments as an enhancement or improvement of the health, physical condition, mental state, or physical or mental function of a human or animal. In other embodiments, the gene therapy can be used to increase or alter the lifespan, healthy lifespan, or lifespan or healthy lifespan of a particular tissue, organ, cell, or combination thereof in a human or animal.

[0057] In some embodiments, the target cell type is one or more adipocytes, preadipocytes, progenitor cells capable of differentiating into adipocytes, stem cells capable of differentiating into adipocytes, resident cell types of subcutaneous tissue, or transient cells or stem cells that can become resident cells of subcutaneous tissue. Gene therapy is intended to treat or prevent any human or animal disease, but in some specific embodiments, it may be used to treat monogenic disorders, enzyme deficiencies, protein deficiencies (as protein replacement therapy), metabolic disorders, autoimmune diseases, oncology, neurological disorders, cardiovascular disorders, musculoskeletal disorders, hematological disorders, infectious diseases, dermatological disorders, immune system disorders, respiratory diseases, kidney or bladder diseases, or diseases of other organs, tissues, systems, or combinations thereof. In one embodiment, a therapeutic gene therapy system is intended to deliver therapeutic or prophylactic antibodies via expression from gene constructs, therapeutic, prophylactic, augmentative, or prophylactic proteins, fusion proteins, peptides, immunogens, antigens, enzymes, ribozymes, mRNA, non-coding RNA, miRNA, shRNA, or other RNAs, or molecules that can function as vaccines or adjuvants via expression from gene constructs.

[0058] The disclosed gene therapy treatment may be delivered in vials for subsequent administration, or in pre-filled syringes or cartridges. Alternatively, the treatment may be administered via auto-injectors or patch injectors, via ultrasound guidance or other guidance techniques to ensure delivery to the correct location. Alternatively, and optionally, the treatment may also be delivered by a set of needles or via a set of injections to more evenly distribute the construct at the treatment site. Gene therapy treatments may be delivered via patches, microneedles, sets of microneedles, jet injections, or carriers, or by other means, to alter the overall structure of the tissue through which the therapeutic agent passes or is injected.

[0059] Within the scope of the compositions or methods provided herein, it should be understood that formulations, materials, gene constructs, sequences, biological and chemical compositions, markers, or methods of use may be modified by those skilled in the art, provided that the structures described herein perform the desired function and remain within the scope of the compositions or methods described herein. Various parts, components, or features can be used in combination, with or without modification by those skilled in the art, to achieve the desired functionality of the aforementioned formulations.

[0060] Furthermore, all individual features and methods of use described herein, and every combination of two or more such features and methods of use, are included within the scope of the compositions or methods provided herein, provided that the features and methods of use in such combinations are not inconsistent with each other. It is understood that certain parts or combinations of such parts can be modified by those skilled in the art while still achieving the objective(s) of the compositions or methods provided herein.

[0061] Ultimately, it is understood that the specific scope provided herein is not limited, but rather intended, for example. [Examples]

[0062] Example 1 This example outlines a minimal plasmid construct delivered within a lipid nanoparticle formulation containing an inducible promoter, post-translational regulators, a gene of interest (GLP-1 receptor agonist), nuclear targeting elements, and gene regulators that enable reduction of gene expression of interest after ganciclovir administration.

[0063] Gene construct map (excluding plasmid backbone): Full-length construct sequence (excluding plasmid backbone): AAV2 ITR: ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct (SEQ ID NO: 15) 3NF1: ctggggactttccagcctggggactttccagctgggactttccagg (SEQ ID NO: 7) 3NF2: Ctggggactttccagctggggactttccagctgggactttccaggag (SEQ ID NO: 8) TRE promoter: Ggtaccgagctcgactttcacttttctctatcactgatagggagtggtaaactcgactttcacttttctctatcactgatagggagtggtaaactcgactttcact tttctctatcactgatagggagtggtaaactcgactttcacttttctctatcactgatagggagtggtaaactcgactttcacttttctctatcactgatagggag tggtaaactcgactttcacttttctctatcactgatagggagtggtaaactcgactttcacttttctctatcactgatagggagtggtaaactcgacctatataag cagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagagacaccgggaccgatccagcctccgcggccccgaattg (Sequence number 16) Kozak array: gccaccatgg (SEQ ID NO: 17) GLP-1 receptor agonist (RA) nucleotide sequence: atgcacggcgagggcacattcaccagcgacctgagcaagcagatggaagaggaagccgtgagactgttcatcgagtggctgaagaacggcggaccctccagcggcgcccctccttctaagaaaaagaaaaagaagtaa (SEQ ID NO: 18) GLP-1 RA amino acid sequence: MHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK (Sequence ID 19) SV40 late pA: Cagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccatta taagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggtttttaaagcaagtaaaacctctacaaatgtggta (Sequence number 20) CBh promoter: cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattgtgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgcgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtattaatgtttaattacctggagcacctgcctgaaatcactttttttcaggttgg (SEQ ID NO: 2) tTS nucleotide sequence: atgtctagattagataaaagtaaagtgattaacagcgcattagagctgcttaatgaggtcggaatcgaaggtttaacaacccgtaaactcgcccagaagctaggtgtagagcagcctacattgtattggcacgtgcgcaacaagcagactcttatgaacatgctttcagaggcaatactggcgaagcatcacacccgttcagcaccgttaccgactgagagttggcagcagtttctccaggaaaatgctctgagtttccgtaaagcattactggtccatcgtgatggagcccgattgcatatagggacctctcctacgcccccccagtttgaacaagcagaggcgcaactacgctgtctatgcgatgcagggttttcggtcgaggaggctcttttcattctgcaatctatcagccattttacgttgggtgcagtattagaggagcaagcaacaaaccagatagaaaataatcatgtgatagacgctgcaccaccattattacaagaggcatttaatattcaggcgagaacctctgctgaaatggccttccatttcgggctgaaatcattaatatttggattttctgcacagttagatgaaaaaaagcatacacccattgaggatggtaataaaccaaaaaagaagagaaagctagcagtgtcagtgacatttgaagatgtggctgtgctctttactcgggacgagtggaagaagctggatctgtctcagagaagcctgtaccgtgaggtgatgctggagaattacagcaacctggcctccatggcaggattcctgtttaccaaaccaaaggtgatctccctgttgcagcaaggagaggatccctgg (SEQ ID NO: 21) tTS amino acid sequence: MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVRNKQTLMNMLSEAILAKHHTRSAPLPTESWQQFLQENALSFRKALLVHRDGARLHIGTSPTPPQFEQAEAQLRCLCDAGFSVEEALFILQSISHFTLGAVLEEQATNQIENNHVIDAAPPLLQEAFNIQARTSAEMAFHFGLKSLIFGFSAQLDEKKHTPIEDGNKPKKKRKLAVSVTFEDVAVLFTRDEWKKLDLSQRSLYREVMLENYSNLASMAGFLFTKPKVISLLQQGEDPW (Sequence ID 22) T2A linker nucleotide sequence: ggctccgga T2A linker amino acid sequence: GSG T2A nucleotide sequence: gagggcaggggaagtcttctaacatgcggggacgtggaggaaaatcccggcccc (Sequence ID 23) T2A amino acid sequence: EGRGSLLTCGDVEENPGP (Sequence ID 24) rtTA nucleotide sequence: atgtctagactggacaagagcaaagtcataaacggcgctctggaattactcaatggagtcggtatcgaaggcctgacgacaaggaaactcgctcaaaagctgggagttgagcagcctaccctgtactggcacgtgaagaacaagcgggccctgctcgatgccctgccaatcgagatgctggacaggcatcatacccacttctgccccctggaaggcgagtcatggcaagactttctgcggaacaacgccaagtcattccgctgtgctctcctctcacatcgcgacggggctaaagtgcatctcggcacccgcccaacagagaaacagtacgaaaccctggaaaatcagctcgcgttcctgtgtcagcaaggcttctccctggagaacgcactgtacgctctgtccgccgtgggccactttacactgggctgcgtattggaggaacaggagcatcaagtagcaaaagaggaaagagagacacctaccaccgattctatgcccccacttctgagacaagcaattgagctgttcgaccggcagggagccgaacctgccttccttttcggcctggaactaatcatatgtggcctggagaaacagctaaagtgcgaaagcggcgggccggccgacgcccttgacgattttgacttagacatgctcccagccgatgcccttgacgactttgaccttgatatgctgcctgctgacgctcttgacgattttgaccttgacatgctccccgggtaa (SEQ ID NO: 25) Amino acid sequence of rtTA: MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG (Sequence ID 26) BGH pA: ctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatc gcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgg (Sequence number 27) EF1a promoter: HSV-TK sequence: atgcctacactgctgcgggtttacatcgacggccctcacggcatgggcaagaccaccacaacacagctgctcgtggccctgggctccagagacgacattgtgtacgtgcctgagcccatgacctactggcgggtcctgggagcctctgaaaccatcgccaatatctacaccactcagcacagactggatcagggcgagatcagcgctggcgacgccgccgtggtgatgaccagcgcccagatcaccatgggcatgccctacgccgtcaccgacgccgtgctggctcctcatattggcggcgaagccggtagcagccacgcccctcctccagctctgacccttatcttcgaccggcaccccatcgccgctctgctgtgctaccccgctgctagatacctgatgggctccatgacacctcaggccgtgctggccttcgtggccctgatcccccccaccctgcctggcacaaacatcgtgctgggcgccctgccagaagatagacacatcgatagactggccaagcggcagcggccaggcgagagactggatctggccatgctggctgctatccgcagagtgtatggcctgctggccaataccgtgagatacctgcagggcggcggaagctggagagaggactggggccagctgagcgggaccgccgtgcccccccagggcgccgagcctcaaagcaacgccggccctagaccccacatcggcgatacgctgtttaccctgttcagagcccctgagctgctggcccctaacggcgacctgtacaacgtgtttgcctgggccctcgacgtgctggctaaaagactgaggcctatgcacgtgttcatcctggactacgaccagtctcctgccggatgtagagatgcactgctgcaactgacaagcggcatgatccagacccacgtgaccacacctggatctatccctaccatctgcgacctggccagaacattcgcccgggaaatgggagaggccaactaa(Sequence ID 29) HSV-TK amino acid sequence: MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRVLGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPE DRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAVPPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDALLQLTSGMIQTHVTTPGSIPTICDLARTFAREMGEAN (Sequence number 30) Insulator: caaacaaacaaa (Sequence code 31) K19 Aptazyme: ggcgcgtcctggattcgtggtaaaacataccagatttcgatctggagaggtgaagaatacgaccacctactacatccagctgatgagtcccaaataggacgaaacgcgct (SEQ ID NO: 32) SV40 pA: Taagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagtt (SEQ ID NO: 33)

[0064] Example 2 This example outlines a hairpin DNA construct delivered within a polymeric lipoplex containing a constitutive promoter, post-translational regulators, a gene of interest (furin-cleavable insulin), nuclear targeting elements, and gene regulators that enable reduction of gene expression of interest after steroid and second-inducible agent administration.

[0065] Genetic construct map: Full-length construct sequence (excluding plasmid backbone): SV40 DTS: ggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaacca (SEQ ID NO: 6) CAG Promoter (CAGp): Furin-cleavable human insulin (FC-hINS): atggccctgtggatgagactgctgcctctgctggccctcctggccctgtggggccccgaccccgccgccgcttttgtgaaccagcacctgtgcggaagcgacctggtcgaggccctgtacctggtgtgcggcgagcggggcttcttcttacacccctagaaccaagc gggaagccgaggatctgcaggtgggccaggtggagctgggcggcggaccaggcgccggctccctgcaacctctggctctggaaggatctagacagaagagaggcatcgtggagcagtgttgtacaagcatctgcagcctgtatcagctggaaaactactgcaattaa (Sequence number 36) FC-hINS amino acid sequence: MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSDLVEALYLVCGERGFFYTPRTKREAEDLQVGQVELGGGPGAGSLQPLALEGSRQKRGIVEQCCTSICSLYQLENYCN (Sequence ID 37) Optimized WPRE (oPRE): gagcatcttaccgccatttatacccatatttgttctgtttttcttgatttgggtatacatttaaatgttaataaaacaaaatggtggggcaatcatttacatttttagggatatgtaattactagttcaggtgtattgccacaagacaaacatgttaagaaactttcccgttatttacgctctgttcctgttaatcaacctctggattacaaaatttgtgaaagattgactgatattcttaactatgttgctccttttacgctgtgtggatatgctgctttatagcctctgtatctagctattgcttcccgtacggctttcgttttctcctccttgtataaatcctggttgctgtctcttttagaggagttgtggcccgttgtccgtcaacgtggcgtggtgtgctctgtgtttgctgacgcaacccccactggctggggcattgccaccacctgtcaactcctttctgggactttcgctttccccctcccgatcgccacggcagaactcatcgccgcctgccttgcccgctgctggacaggggctaggttgctgggcactgataattccgtggtgttgtc (SEQ ID NO: 4) CMV promoter (CMVp): tagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatc (SEQ ID NO: 1) Synthetic intron IVS8: gtaagtgtcttcctcctgtttccttcccctgctattctgctcaaccttcctatcagaaactgcagtatctgtatttttgctagcagtaatactaacggttctttttttctcttcacag (SEQ ID NO: 38) SWITCH nucleic acid sequence: SWITCH amino acid sequence: MDSQQPDLKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLEFPGVDQKKFNKVRVVRALDAVALPQPVGVPNESQALSQRFTFSPGQDIQLIPPLINLLMSIE PDVIYAGHDNTKPDTSSSLLTSLNQLGERQLLSVVKWSKSLPGFRNLHIDDQITLIQYSWMSLMVFGLGWRSYKHVSGQMLYFAPDLILNEQRMKESSFYSLCLTMWQIPQEFVKLQVSQEEFLCMKVLLLLNTIPLEGLRSQTQFEEMRSSYIRELIKAIGLR QKGVVSSSQRFYQLTKLLDNLHDLVKQLHLYCLNTFIQSRALSVEFPEMMSEVIAGSTPMEFQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISS (Sequence ID 40) hGH pA: gggtggcatccctgtgaccctccccagtgcctctcctggccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaagttgcatcattttgtctgactaggtgtc cttctataatattatggggtggaggggggtggtatggagcaaggggcaagttgggaagacaacctgtagggcctgcggggtctattgggaaccaagctggagtgcagtggcacaatctt ggctcactgcaatctccgcctcctgggttcaagcgattctcctgcctcagcctcccgagttgttgggattccaggcatgcatgaccaggctcagctaatttttgttttttggtagaga cggggtttcaccatattggccaggctggtctccaactcctaatctcaggtgatctacccaccttggcctcccaaattgctgggattacaggcgtgaaccactgctcccttccctgtcctt (Sequence number 41) GAL4 UAS: Cggagtactgtcctccgagcggagtactgtcctccgactcgagcggagtactgtcctccgatcggagtactgtcctccgcgaattccggagtactgtcctccg (SEQ ID NO: 42) Minimum Ad promoter (ADp): Ggggggctataaaagggggtgggggcgttcgtcctcactct (Sequence ID 43)

[0066] Example 3 This example outlines a minimal DNA minicircle construct delivered within a lipid nanoparticle formulation containing an inflammatory response promoter, post-translational regulators, a gene of interest (an interleukin-1 receptor antagonist), a nuclear targeting element, and a gene regulator that enables reduction of gene expression of interest after rapamycin administration.

[0067] Gene construct map (excluding plasmid backbone): Full-length construct sequence (excluding plasmid backbone): 5xNFkB DTS: tggggactttccgctggggactttccgctggggactttccgctggggactttccgctggggactttccgc (SEQ ID NO: 45) NFKB response element (RE): gggaatttccggggactttccgggaatttccggggactttccgggaatttcc (UC46) Minimum promoter (minP): Tagagggtatataatggaagctcgacttccag (Sequence ID 47) MVM Intron: gtaagggtttaagggatggttggttggtggggtattaatgtttaattacctggagcacctgcctgaaatcactttttttcag (Sequence ID 48) Interleukin-1 receptor antagonist (IL-1 RA) sequence: atggaaatctgtagaggcctgaggtctcatctgattaccctgctgctgctgttcctgttccac agcgaaaccatctgcagacctagcggcagaaagagcagcaagatgcaagctttcagaatc tgggatgtgaaccagaagaccttctacctgcggaacaaccagctggtggccggctacctg cagggccctaatgtgaatctggaagagaaaatcgacgtcgtgcccatcgagcctcacgcc ctgtttctgggcatccacggcggcaagatgtgcctgagctgcgtgaagtctggagatgag acaagactgcagctcgaggccgtgaacatcacagacctgtccgagaacagaaaacaggac aagcggttcgccttcatccggagcgacagcggaccaaccaccagcttcgagtctgccgct tgtcccggctggttcctgtgcacagccatggaagccgaccagcctgtgtccctgaccaac atgcctgatgaaggcgtgatggtgaccaagttctactttcaggaggacgagtaa (Sequence code 49) Interleukin-1 receptor antagonist (IL-1 RA) amino acid sequence: MEICRGLRSHLITLLLFLFHSETICRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO: 50) rapaCas9 sequence: rapaCas9 amino acid sequence: MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKLEYSGGGSLEGVQVETISPGDGRTFPKRGQTC VVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGGSGGGGSGGGGSGVDGFGDVGALESLRGNADLAYILSMEP CGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS (Sequence ID 13)

[0068] Example 4 This example outlines a double-stranded DNA construct delivered within a ligand-targeted polymeric particle containing a constitutive promoter, post-translational regulators, a gene of interest (hBDNF), nuclear targeting elements, and gene regulators that enable reduction of gene of interest expression after doxycycline administration.

[0069] Genetic construct map: Full-length construct sequence (excluding plasmid backbone): WPRE: Cgataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctcctttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttccccgtatggctttcatttctctcccttgt ataaatcctggtgctgtctctttatgaggagttgtggccccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccaccctgtcagctcctttccgggactttcgctttcccc tccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttcgggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgcggcctctgcggctctgcggctcttccgcgtcttcgccttcgccctcagacgagtcggatctccccttgggccgcctccccgcatcgg (sequence number 5) Mature BDNF (mBDNF) sequence: Atgaccatcctgttcctgaccatggtgatctcctactttggctgcatgaaagcccactctgatcctgccagaaggggcgagctgagcgtgtg cgacagcatcagcgagtgggtcacagccgctgataagaaaaccgccgtggacatgagcggcggaacagtgaccgtgctggaaaaggtgcctgtgtccaagggccagct gaagcagtacttctacgagacaaaatgcaaccccatgggctacacaaaggaaggctgtagaggcatcgacaagcgccactggaacagccagtgcagaaccacccaatc ttatgtgcgggccctgacaatggacagcaagaagcggatcggctggcggttcatcagaatcgacaccagctgcgtgtgtaccctgaccattaagagaggaagataatag (Sequence number 53) mBDNF amino acid sequence: MTILFLTMVISYFGCMKAHSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGR (Sequence ID 54) GRE DTS: Ggtacattttgttctagaacaaaatgtaccggtacattttgttctggtacattttgttct (Sequence ID 55)

[0070] Example 5 Therapeutic gene therapy formulations containing candidate genes for human protein substitution were produced by combining the following lipid components per 1 ml of the mixture. [Table 8] The formulation was combined with the gene construct at an N / P ratio of 4:10 using impact jet mixing, and the encapsulation efficiency was evaluated. [Table 9] [Table 10] The biophysical properties of LNP particles were characterized using dynamic light scattering instruments, as illustrated below. [Table 11]

[0071] The gene constructs shown in Figure 1A-1B were encapsulated in formulations with an N / P ratio of 4 to 10. The formulations were then used to treat primary human adipocytes and preadipocytes at doses of 50–500 ng / well. Both constructs contained a suicide gene downregulatory element consisting of the rapaCasp9 gene fused to a K19 aptazyme, which requires the presence of two regulators, tetracycline and rapamycin, for activation.

[0072] The firefly luciferase reporter gene expressed from primary human adipocytes transduced with LNP102-npRB2.6v7 was used to quantify the response of a single regulator, rapamycin, and the rapamycin-induced suicide gene downregulatory element, while the effective dose of the other regulator, tetracycline, was fixed at 0.1 mM. Specifically, a false-negative control was used to assess the absence of luminescence (no cells expressing fLuc). A 0 nM rapamycin-positive control was used to assess baseline expression of fLuc without downregulatory adjustment. The rapamycin concentration was increased from 0 nM to 0.1 nM, and a clear dose-dependent reduction in transgene expression was observed using quantitative bioluminescence spectroscopy. Thus, within 76 hours of inducer activation, induction of the downregulatory suicide gene element dose-dependently reduced reporter gene expression (Figure 2).

[0073] In parallel, an Annexin NanoBiT, 500-fold bioluminescence assay was used to measure the functionality and downregulatory / downmodulation of the construct in primary human adipocyte cultures transduced with LNP102-npRB2.6v3. Rapamycin was administered to the cultured cells in the range of 0.01 to 1 nM, while a second regulator, tetracycline, was added at concentrations of 0 mM, 0.05 mM, or 0.1 mM. No induction of apoptosis was observed in the absence of tetracycline, demonstrating the functionality of the aptazyme, while clear dose-dependent cellular apoptosis was observed after administration of tetracycline at concentrations of 0.05 mM and 0.1 mM for all rapamycin concentrations. Post-apoptosis, expression from the therapeutic transgene was effectively eliminated, enabling downregulation of gene therapy (Figure 3).

[0074] Example 6 Lipid-based carrier formulations were prepared and mixed in an N / P ratio of 7 for subsequent administration to C57BL / 6 mice or C6 albino background mice. Total formulations of 2–10 μg were injected into mice as "initial dose" or "upward-adjusted dose," which were administered 2–4 weeks after the initial dose to allow for the formation of any potential immune response. The size of the administered constructs varied between 6203 and 3144 bp, encompassing the constructs shown in Figures 1B and 4.

[0075] Following subcutaneous injection of the initial dose, animals were monitored for serious adverse events, general safety, and signal (reporter transgene expression) durability using in vivo bioluminescence imaging (IVIS). IVIS confirmed the durability of the initial signal (transgene expression) throughout up to day 22 (Figures 5A and 5B) and the ability to upregulate the initial dose in a predictable, dose-dependent manner, enabling not only re-administration but also control of the upregulation of the initial treatment, as shown in Figures 5A and 5C. The combination therapy was then continued to evaluate the long-term durability of reporter transgene expression. A total of six IVIS measurements were performed over a 90-day period. Signals were consistently localized at the original injection site, clearly observed at levels statistically exceeding background luminescence, demonstrating the high stability and excellent long-term durability potential of the gene therapy (Figures 6-7).

[0076] This example illustrates the minimum observable downward adjustment, ranging from approximately 2% to 15%, which may subsequently increase after additional stimulation.

[0077] Example 7 In this example, 3 μg of LNP preparation N / P7, prepared in a 4 mL batch of 144 μL of 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid, 1-octyrnonyl ester, 128 μL of 1,2-distearoylphosphatidylcholine, 1184 μL of cholesterol, 1512 μL of 1,2-dimyristyl-rac-glycero-3-methoxy-polyethylene glycol (2,000), and 1032 μL of ethanol, containing a 5496 nt gene construct encoding the firefly luciferase reporter gene under the regulation of a CBh promoter (hybrid cytomegalovirus enhancer / chicken β-actin), was subcutaneously delivered to C57BL / 6 albino mice. After 3 months of imaging to confirm signal stability, animals were subjected to cryolipolysis-mediated signal downmodulation using a 1.5 cm² diameter probe delivering a 3 W / cm² heat flux at 0°C (a heat flux previously confirmed to allow subcutaneous tissue cooling to approximately 6°C) (Figure 8). Three series of treatments were administered twice daily, with a total cooling time of 10 minutes after equilibration of the probe and skin. Glycerol was applied to the tip of the probe to prevent crystal formation and potential damage to the dermis. The reduction in adipocytes can be extrapolated to changes in therapeutic induction gene expression after treatment. If necessary, additional treatments may be applied sequentially to further reduce subcutaneous adipocyte counts and completely block gene expression.

[0078] The reporter gene can be replaced with any therapeutically relevant transgene, which is either currently undergoing clinical evaluation to assess therapeutic efficacy via subcutaneous delivery as a protein or mRNA, or is already qualified. Dosage, formulation, concentration, number of injections, promoter, or other regulatory sequences can be adjusted to optimize the therapeutic efficacy of the transgene. When administered to non-rodent animal tissues, such as companion animals, humans, or horses, to achieve cryolipolysis, the flux, probe area, time, and cooling / heating profile, as well as cooling aids, can be adjusted.

[0079] Example 8 This example describes a method for downward adjustment using the Lox-Cre system: 3NF1 DTS: Ctggggactttccagcctggggactttccagctgggactttccagg (SEQ ID NO: 7) 3NF2 DTS: ctggggactttccagctggggactttccagctgggactttccaggag (Sequence ID 8) EFS promoter: GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGATCCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGG (Sequence number 3) loxP site: ATAACTTCGTATAgcatacatTATACGAAGTTAT (Sequence ID 57) NLuc coding array: ATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATC CAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGAT GATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTA ACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGTAA (Sequence number 58) NLuc amino acid sequence: MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA (Sequence ID 59) BGH polyA signal: Ctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaa attgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagagaatagcaggcaggctgggga (Sequence number 60) Cre-ERT2 coding sequence: Cre-ERT2 amino acid sequence: MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLG IAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDS ETGAMVRLLEDGDLEPSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEGFPATA (Sequence ID 11) Insulator: caaacaaacaaa (Sequence ID 31) K19 Aptazyme: ggcgcgtcctggattcgtggtaaaacataccagatttcgatctggagaggtgaagaatacgaccacctactacatccagctgatgagtcccaaataggacgaaacgcgct (SEQ ID NO: 32) SV40 pA: Taagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagtt (SEQ ID NO: 33)

[0080] Example 9 This example describes therapeutic gene therapy for downregulation of gene expression or viability in adipocytes or preadipocytes expressing gene therapy, via the application of temperature reduction devices, ultrasound, or electromagnetic radiation. Gene construct sequence (excluding plasmid backbone): 3NF1 DTS: Ctggggactttccagcctggggactttccagctgggactttccagg (SEQ ID NO: 7) 3NF2 DTS: ctggggactttccagctggggactttccagctgggactttccaggag (Sequence ID 8) CBh Promoter: Cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaatgataacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatgg cccgcctggcattgtgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccaccccaattttgtatttatttttttttaatttttgtgcagcgatgggggggggggg ggggggggggcgcgcgccaggcggggcggggcgaggggcggggcggggcgaggcggggcgaggcgggaggcgggaggaggtgcggcggcagccaatcagagcggcgcctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcggcgggagtcgctgcgcgctgccttcgccccgtgccccgctccgccgccgccgccgccgccgccgctctgactgaccggttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggttggttttggttattaatgtttaattacctggagcacctgcctgaaatcacttttttcaggttgg (sequence number 2) NLuc coding sequence: Atggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaattttaaggtggtgtaccctgtggat gatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgta acagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaacccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcgtaa (Sequence number 58) NLuc amino acid sequence: MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA (Sequence ID 59) BGH polyA signal: ctgtgccttctagttgccagccatctgttgtttgccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaa attgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagagaatagcaggcaggctgggga (Sequence number 60)

[0081] Example 10 This example describes means of down-preparing or down-regulating gene expression from therapeutic constructs via the implantation, expression, and / or activation of a targeted epigenetic silencer that can be induced by the administration of one or more exogenous stimuli. Specifically, exemplary sequences of DNA methyltransferases (or other epigenetic silencers) from humans or other target species (e.g., mice): Human DNMT3A (CD): NHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACV (Sequence ID 63) or Mouse DNMT3L (CD): GPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPL (Sequence ID 64) It is combined with a targeting element such as a zinc finger motif or protein, and has the following sequence as an example: ZN627_Hu: DSVAFEDVAVNFTLEEWALLDPSQKNLYRDVMRETFRNLASVGKQWEDQNIEDPFKIPRRNISHIPERLCESKEGGQGEE (Sequence ID: 65) By optionally combining NLS sequences such as MAPKKKRKV (SEQ ID NO: 66)) and flexible spacer or linker sequences such as SGGGGSGGGGS (SEQ ID NO: 67), the following fusion proteins can be produced: hD3A(CD)-mD3L(CD)-ZF(NLucTSS34)-ZN627: MAPKKKRKVNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTV NDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVS SGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPL GSSCDRCPGWYMFQFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRS RSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLSGGGGGSGGGGSLEPGEKPYKCPECGKSFSDPGHLVRHQRTHTGEKPYKCPECGKSFSDCRDLARHQRTHT GEEKPY (Sequence ID 12) It is designed to target the following nucleotide sequence: Target site as part of the construct sequence (TSS downstream pos 34): TGGCGACAGACAGCCGGC (SEQ ID NO: 68): ZF target region (total 400 nt, 200 nt upstream and 200 nt downstream of TSS): Ccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtattaatgtttaattacctggagcacctgcctgaaatcactttttttcaggttgggttggacgcgtcgctagcgccaccatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtct (SEQ ID NO: 69) ZF: LEPGEKPYKCPECGKSFSDPGHLVRHQRTHTGEKPYKCPECGKSFSDCRDLARHQRTHTGEKPYKCPECGKSFSSPADLTRHQRTHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSQSGHLTEHQRTHTGEKPYKCPECGKSFSRSDHLTTHQRTHTGKKTS (SEQ ID NO: 70) or alternative example for DNMT3A-3L-ZF-KRAB fusion: MAPKKKRKVMNHDOEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIOVDRYIASEVCEDSITVGMVRHOGKI MYVGDVRSVTOKHIOEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRA RYFWGNLPGMNRPLASTVNDKLELOECLEHGRIAKFSKVRTITTRSNSIKOGKDOHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARORLLGRSWSVPVIRHL FAPLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKROPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVY GSTOPLGSSCDRCPGWYMFQFHRILOYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLOTEAVTLODVRGRDYNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRS KLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLSGGGGGSGGGGSVGIHGVPSRPGERPFQCRICMRNFSHKSSLTRHTRTHTGEKPFQCRICMRNFSRTEHLARHLRTHTG SQKPFQCRICMRNFSQSAHLKRHTRTHTGEKPFQCRICMRNFSRTEHLARHLRTHTGGGGSQKPFQCRICMRNFSHKSSLTRHTRTHTGEKPFQCRICMRNFSRPESLAPHLRTHLRGSGGGSMDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKSSV (Sequence ID 72) DNMT3A:MNHDOEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIOVDRYIASEVCEDSITVGMVRHOGKIMYVGDVRSVTOKHIOEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELOECLEHGRIAKFSKVRTITTRSNSIKOGKDOHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARORLLGRSWSVPVIRHLFAPLKEYFACV (Sequence ID 73) DNMT3L:SSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKROPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTOPLGSSCDRCPGWYMFQFHRILOYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLOTEAVTLODVRGRDYNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLSGGGGSGGGGSVGIHGVP (Sequence ID 74) Linker (GGGS (SEQ ID NO: 75)), SV40 NLS (PKKKRKV (SEQ ID NO: 76)), ZF (SRPGERPFQCRICMRNFSHKSSLTRHTRTHTGEKPFQCRICMRNFSRTEHLARHLRTHTGSQKPFQCRICMRNFSQSAHLKRHTRTHTGEKPFQCRICMRNFSRTEHLARHLRTHTGGGGSQKPFQCRICMRNFSHKSSLTRHTRTHTGEKPFQCRICMRNFSRPESLAPHLRTHLRGS (SEQ ID NO: 77))

Claims

1. A modifiable therapeutic gene therapy system for delivery to any one of the tissues or cells provided herein, such as the subcutaneous space (for example, one or more cell types within the subcutaneous space): a. A gene construct encoding at least one therapeutic gene, regulated by at least one promoter element and optionally one or more regulatory elements. b. Optionally, a delivery carrier or vector that encapsulates at least a portion of the gene construct, enabling the re-administration of a subsequent dose of the gene construct (for example, with at least one 0% efficiency compared to the initial dose of the gene construct), c. The system comprising means for increasing or decreasing the level of therapeutic gene expression from a gene construct.

2. The adjustable therapeutic gene therapy system according to claim 1, wherein means c) is a means for permanently reducing the level of therapeutic gene expression (for example, by at least 2%).

3. A tunable therapeutic gene therapy system according to claim 1 or 2, wherein the means for reducing the level of therapeutic gene expression is via the application of an external stimulus.

4. A tunable therapeutic gene therapy system according to any one of the preceding claims, wherein the gene construct encodes at least one functional portion of a human peptide or protein (e.g., a full-length human protein).

5. A tunable therapeutic gene therapy system according to any one of the preceding claims, wherein the gene construct encodes at least one functional portion of a human protein analog or antagonist.

6. A tunable therapeutic gene therapy system according to any one of the preceding claims, wherein the gene construct encodes at least one functional portion of a non-human peptide or protein (e.g., a full-length non-human protein).

7. A tunable therapeutic gene therapy system according to any one of the preceding claims, wherein the gene construct encodes at least one functional portion of a non-human protein analog or antagonist.

8. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the gene construct encodes insulin or an insulin analog.

9. The adjustable therapeutic gene therapy system according to claim 8, wherein insulin or an insulin analog is modified furin-cleavable insulin.

10. A modifiable therapeutic gene therapy system according to any one of claims 1 to 7, wherein the gene construct encodes GLP-1, a GLP-1 agonist, or a GLP-1 agonist analog.

11. A configurable therapeutic gene therapy system according to any one of claims 1 to 7, wherein the gene construct encodes a growth factor.

12. A modifiable therapeutic gene therapy system according to any one of claims 1 to 7, wherein the gene construct encodes a cytokine.

13. A modifiable therapeutic gene therapy system according to any one of claims 1 to 7, wherein the gene construct encodes an anti-inflammatory protein.

14. A modifiable therapeutic gene therapy system according to any one of claims 1 to 7, wherein the gene construct encodes a complement protein.

15. A modifiable therapeutic gene therapy system according to any one of claims 1 to 7, wherein the gene construct encodes a receptor agonist.

16. A modifiable therapeutic gene therapy system according to any one of claims 1 to 7, wherein the gene construct encodes a receptor antagonist.

17. A tunable therapeutic gene therapy system according to any one of claims 1 to 7, wherein the gene construct encodes a fusion protein (for example, composed of one or more functional elements of different proteins).

18. A controllable therapeutic gene therapy system according to any one of the preceding claims, wherein the subcutaneous space is a subcutaneous tissue layer.

19. A tunable therapeutic gene therapy system according to any one of the preceding claims, wherein the subcutaneous space is the layer between the superficial fascia and the deep fascia.

20. A tunable therapeutic gene therapy system according to any one of the preceding claims, wherein the subcutaneous space is the layer between the dermis and the superficial fascia.

21. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the gene construct comprises one or more circular single-stranded DNA constructs.

22. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the gene construct comprises one or more circular double-stranded DNA constructs.

23. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the gene construct comprises one or more linear single-stranded DNA constructs.

24. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the gene construct comprises one or more linear double-stranded DNA constructs.

25. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the gene construct is composed of at least a portion of DNA or RNA.

26. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the gene construct is composed of at least a portion of DNA and / or RNA.

27. A tunable therapeutic gene therapy system according to any one of the preceding claims, wherein the external stimulus comprises chemical DNA, RNA, or a DNA and RNA derivative.

28. A configurable therapeutic gene therapy system according to any one of claims 1 to 27, wherein the promoter element includes a constitutive promoter.

29. A tunable therapeutic gene therapy system according to any one of claims 1 to 27, wherein the promoter element includes an inducible promoter.

30. A modifiable therapeutic gene therapy system according to any one of claims 1 to 27, wherein the promoter element comprises a tissue-specific promoter.

31. A modifiable therapeutic gene therapy system according to any one of claims 1 to 27, wherein the promoter element comprises a promoter containing a sequence from one or more of the CAG, EF1a, UBC, CBh, MSCV, hPGK, SFFV, and SV40 promoters.

32. A tunable therapeutic gene therapy system according to any one of claims 1 to 27, wherein the promoter element comprises a tetOn-inducible promoter construct.

33. A tunable therapeutic gene therapy system according to any one of the preceding claims, wherein the promoter element comprises a promoter sequence comprising at least one of an enhancer, a regulator, an operator, and / or a repressor.

34. A tunable therapeutic gene therapy system according to any one of claims 1 to 27, wherein the promoter element includes an inducible promoter whose expression can be upregulated or downregulated in response to an external or internal stimulus such as inflammation, heat, light, stress, steroids, tetracycline, antibiotics, rapamycin, ganciclovir, or acyclovir administration, or is inducible by an upregulated or downregulated molecule (e.g., ROS, NOS, or cytokine release).

35. A tunable therapeutic gene therapy system according to any one of claims 1 to 27, wherein the promoter element includes a circadian rhythm or periodic promoter (for example, one that changes its activity level by at least 5% with some periodicity over a range of hours to months).

36. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the regulatory element includes a post-translational regulatory element.

37. The adjustable therapeutic gene therapy system according to claim 36, wherein the regulatory element comprises at least some or all of the post-transcriptional regulatory elements (WPREs) of woodchuck hepatitis virus.

38. The adjustable therapeutic gene therapy system according to claim 36, wherein the regulatory element comprises at least some or all of the optimized post-transcriptional regulatory elements (WPREs) of woodchuck hepatitis virus.

39. The adjustable therapeutic gene therapy system according to claim 36, wherein the regulatory element includes a cis-acting element that can increase cytoplasmic accumulation.

40. The adjustable therapeutic gene therapy system according to claim 36, wherein the regulatory elements include at least some or all of the HIV type 1 Rev-Rev responsive elements.

41. The adjustable therapeutic gene therapy system according to claim 36, wherein the regulatory element comprises at least some or all of the post-translational regulatory elements of human hepatitis virus.

42. The adjustable therapeutic gene therapy system according to claim 36, wherein the regulatory elements comprise at least some or all of one or more viral posttranslational regulatory elements that can improve the expression of any other element encoded by the gene or gene construct of interest.

43. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the gene construct comprises one or more functional gene sequences.

44. A tunable therapeutic gene therapy system according to claim 43, wherein one or more functional gene sequences promote the transfer of a gene construct (e.g., at least 4% of the gene construct) to the nucleus of a target cell.

45. The adjustable therapeutic gene therapy system according to claim 43, comprising one or more functional gene sequences that encode a sequence-specific DNA-binding protein bound to a nuclear localization signal peptide.

46. The adjustable therapeutic gene therapy system according to claim 43, comprising one or more functional gene sequences, each comprising a DNA nuclear target sequence recognized by one or more specific transcription factors.

47. The adjustable therapeutic gene therapy system according to claim 43, wherein one or more functional gene sequences include DNA nuclear target sequences (DTS) that are active in importin-mediated transport systems.

48. A customizable therapeutic gene therapy system according to claim 43, wherein one or more functional gene sequences comprise at least some or all of the SV40 DTS.

49. A modifiable therapeutic gene therapy system according to claim 43, wherein one or more functional gene sequences comprise at least some or all of the glucocorticoid-responsive element (GRE)DTS.

50. A tunable therapeutic gene therapy system according to claim 43, comprising one or more functional gene sequences including a DTS sequence of Sox2 regulatory region 2.

51. A tunable therapeutic gene therapy system according to claim 43, wherein one or more functional gene sequences include importin beta(1), importin 7, NF-kappa beta, or a small molecule guanosine triphosphatase Ran interaction sequence.

52. The adjustable therapeutic gene therapy system according to claim 43, wherein one or more functional gene sequences (e.g., those that reduce expression from a gene construct) include an inducible suicide gene.

53. The adjustable therapeutic gene therapy system according to claim 43, comprising an inducible expression system in which one or more functional gene sequences (e.g., reducing expression from a gene construct) encode an RNA molecule that reduces the expression of at least a portion of a therapeutic gene construct.

54. A tunable therapeutic gene therapy system according to claim 43, wherein one or more functional gene sequences (e.g., reducing expression from a gene construct) encode a protein capable of targeted epigenetic silencing.

55. A tunable therapeutic gene therapy system according to claim 43, wherein one or more functional gene sequences (e.g., reducing expression from a gene construct) encode an HSV-TK suicide gene or system.

56. A tunable therapeutic gene therapy system according to claim 43, wherein one or more functional gene sequences (e.g., those that reduce expression from a gene construct) encode the RapaCas9 suicide gene or system.

57. A tunable therapeutic gene therapy system according to claim 43, wherein one or more functional gene sequences (for example, reducing expression from a gene construct) encode one or more miRNAs, siRNAs, shRNAs, dsRNAs, ncRNAs, lncRNAs, piwi-interacting RNAs, PATs, eRNAs, and / or circRNAs.

58. A tunable therapeutic gene therapy system according to claim 43, wherein one or more functional gene sequences (e.g., those that reduce expression from a gene construct) encode at least a guide RNA and a CRISPR-dCas system (e.g., bound to a protein that induces epigenetic silencing).

59. A tunable therapeutic gene therapy system according to claim 43 or 58, wherein one or more functional gene sequences (e.g., reducing expression from a gene construct) encode at least a guide RNA and CRISPR-dCas9-KRAB.

60. A tunable therapeutic gene therapy system according to claim 43 or 58, wherein one or more functional gene sequences (e.g., reducing expression from a gene construct) encode at least a guide RNA and CRISPR-dCas12b-KRAB.

61. A tunable therapeutic gene therapy system according to claim 43 or 58, wherein one or more functional gene sequences (e.g., reducing expression from a gene construct) encode at least a guide RNA and CRISPR-dCas8c-KRAB.

62. A tunable therapeutic gene therapy system according to claim 43 or 58, wherein one or more functional gene sequences (e.g., reducing expression from a gene construct) encode at least a guide RNA and CRISPR-dCas8a-KRAB.

63. A tunable therapeutic gene therapy system according to claim 43 or 58, wherein one or more functional gene sequences (e.g., reducing expression from a gene construct) encode at least a guide RNA and CRISPR-dCas8b-KRAB.

64. A tunable therapeutic gene therapy system according to claim 43, wherein one or more functional gene sequences (for example, reducing expression from a gene construct) encode at least a TALEN, meganuclease, endonuclease, restriction enzyme, zinc finger protein, or other DNA or RNA-binding protein.

65. A controllable therapeutic gene therapy system according to any one of the preceding claims, wherein a regulatory element alters the expression from a gene construct in response to temperature.

66. A controllable therapeutic gene therapy system according to any one of claims 1 to 64, wherein a regulatory element alters the expression from a gene construct in response to heat.

67. A controllable therapeutic gene therapy system according to any one of claims 1 to 64, wherein a regulatory element alters the expression from a gene construct in response to cold.

68. A controllable therapeutic gene therapy system according to any one of claims 1 to 64, wherein a regulatory element alters the expression from a gene construct in response to ultrasound.

69. A controllable therapeutic gene therapy system according to any one of claims 1 to 64, wherein a regulatory element alters the expression from a gene construct in response to electrical stimulation.

70. A controllable therapeutic gene therapy system according to any one of claims 1 to 64, wherein a regulatory element alters the expression from a gene construct in response to a chemical stimulus.

71. A controllable therapeutic gene therapy system according to any one of claims 1 to 64, wherein a regulatory element alters the expression from a gene construct in response to changes in the physiological environment.

72. A tunable therapeutic gene therapy system according to any one of claims 1 to 64, wherein a regulatory element alters the expression from a gene construct in response to a paracrine, endocrine, or autocrine factor.

73. A controllable therapeutic gene therapy system according to any one of claims 1 to 64, wherein a regulatory element alters the expression from a gene construct in response to the presence of inflammation.

74. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the regulatory element includes a proximal promoter.

75. A controllable therapeutic gene therapy system according to any one of claims 1 to 73, wherein the regulatory element includes a distal promoter.

76. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the regulatory element includes an insulator.

77. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the regulatory element forms a secondary structure with a gene construct or other gene sequence.

78. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector comprises lipid nanoparticles (e.g., functionalized lipid nanoparticles, solid lipid nanoparticles, lipid polymer hybrid nanoparticles).

79. A customizable therapeutic gene therapy system according to any one of claims 1 to 78, wherein the delivery carrier or vector comprises liposomes (e.g., functionalized liposomes, stealth liposomes) or micelles.

80. A customizable therapeutic gene therapy system according to any one of claims 1 to 78, wherein the delivery carrier or vector comprises one or more cells (e.g., functional cells), cellular components, or a cell membrane.

81. A customizable therapeutic gene therapy system according to any one of claims 1 to 78, wherein the delivery carrier or vector comprises a cubosome, transfectosome, endosome, exosome, or vesicle system.

82. A modifiable therapeutic gene therapy system according to any one of claims 1 to 78, wherein the delivery carrier or vector comprises polymer nanoparticles.

83. A customizable therapeutic gene therapy system according to any one of claims 1 to 78, wherein the delivery carrier or vector comprises a non-immunogenic or low-immunogenic viral vector.

84. A customizable therapeutic gene therapy system according to any one of claims 1 to 78, wherein the delivery carrier or vector comprises a protein or polypeptide.

85. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector is functionalized to reduce immunogenicity.

86. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector is functionalized (e.g., by one or more specific cell types) to facilitate uptake.

87. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector is functionalized to promote endocytosis, pinocytosis, or enhance transport to the nucleus.

88. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector comprises an ionizable lipid.

89. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector comprises an ionizable element.

90. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector comprises an ionizable polymer.

91. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector comprises cholesterol.

92. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector comprises a cationic component.

93. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector comprises an amphiphilic polymer.

94. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector comprises polyethylene glycol.

95. A customizable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector comprises an antibody, a nanobody, or an antibody fragment.

96. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector comprises a peptide.

97. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector comprises nanoparticles chemically or physically conjugated with a peptide, protein, or a functional sequence derived from a peptide or protein.

98. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector comprises a nanoemulsion, a nanostructured lipid, or at least a portion of an amphiphilic polymer or oligomer.

99. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector comprises a phospholipid, a sphingolipid, a polyelectrolyte polymer, or a polyelectrolyte complex.

100. A tunable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector comprises metal or ceramic nanoparticles.

101. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector does not induce a humoral response or a cell-mediated immune response.

102. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector does not induce a memory immune response.

103. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector is minimally immunogenic (for example, such that the memory immune response produced by the carrier or vector is unable to neutralize at least 10% of subsequent doses of the carrier or vector via the same route of administration in the test subject).

104. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector is minimally immunogenic (for example, such that the memory immune response produced by the carrier or vector is unable to neutralize at least 50% of subsequent doses of the carrier or vector via the same route of administration in the test subject).

105. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector is minimally immunogenic (for example, such that the memory immune response produced by the carrier or vector is unable to neutralize at least 75% of subsequent doses of the carrier or vector via the same route of administration in the test subject).

106. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector is minimally immunogenic (for example, such that the memory immune response produced by the carrier or vector is unable to neutralize at least 90% of subsequent doses of the carrier or vector via the same route of administration in the test subject).

107. A modifiable therapeutic gene therapy system according to any one of the preceding claims, wherein the delivery carrier or vector is minimally immunogenic (for example, such that the memory immune response produced by the carrier or vector is unable to neutralize at least 99% of subsequent doses of the carrier or vector via the same route of administration, such as in a test subject).

108. A tunable therapeutic gene therapy system according to any one of the preceding claims, wherein an external stimulus induces cooling of the skin and subcutaneous tissue (for example, to induce adipocyte death).

109. A controllable therapeutic gene therapy system according to any one of the preceding claims, wherein an external stimulus induces cooling of the skin and subcutaneous tissue to cause aging of adipocytes.

110. A tunable therapeutic gene therapy system according to any one of the preceding claims, wherein an external stimulus causes a temperature change in subcutaneous tissue that is sufficient to alter the expression from a gene construct.

111. An adjustable therapeutic gene therapy system according to any one of the preceding claims, wherein the external stimulus includes high-frequency ultrasound.

112. A controllable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus includes medium-frequency ultrasound.

113. An adjustable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus includes low-frequency ultrasound.

114. A controllable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus includes high-frequency ultrasound-mediated cavitation.

115. A tunable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus includes medium-frequency ultrasound-mediated cavitation.

116. A tunable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus includes low-frequency ultrasound-mediated cavitation.

117. A tunable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus includes ultrasound in the frequency range of 1 to 10 kHz.

118. A tunable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus includes ultrasound in the frequency range of 10 to 28 kHz.

119. A tunable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus includes ultrasound in the frequency range of 28 to 40 kHz.

120. A tunable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus includes ultrasound in the frequency range of 40 to 60 kHz.

121. A tunable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus includes ultrasound in the frequency range of 1 to 320 kHz.

122. A tunable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus includes sound waves (for example, capable of suppressing expression in adipocytes, inducing sentences in adipocytes, or inducing adipocyte death, such as in a portion of the subcutaneous space).

123. A tunable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus includes electromagnetic waves (for example, capable of suppressing expression in adipocytes, inducing sentences in adipocytes, or inducing adipocyte death, such as in a portion of the subcutaneous space).

124. A tunable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus includes an electrical signal (for example, capable of suppressing expression in adipocytes, inducing sentences in adipocytes, or inducing adipocyte death, such as in a portion of the subcutaneous space).

125. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus comprises the administration of a formulation comprising rapamycin, rapalog, or a derivative of rapamycin.

126. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus comprises the administration of a formulation containing tetracycline or a derivative thereof.

127. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus includes administration of rapamycin or rapalog, or a derivative of rapamycin, in combination with a tetracycline or tetracycline derivative.

128. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus comprises the administration of ganciclovir or a derivative of ganciclovir.

129. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus comprises the administration of doxycycline or a derivative of doxycycline.

130. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus comprises the administration of one or more pharmacologically active small molecules.

131. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus comprises the administration of one or more peptides, whether or not they are conjugated.

132. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus comprises the administration of one or more hormones or analogues.

133. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus comprises the administration of tamoxifen or a derivative of tamoxifen.

134. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein an external stimulus includes the administration of one or more agents that induce an inflammatory response.

135. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus comprises the administration of one or more steroids.

136. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus comprises the administration of one or more antibodies, whether or not they are functionalized.

137. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus comprises the administration of one or more receptor ligands.

138. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus comprises the administration of one or more antibody fragments.

139. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus comprises the administration of one or more proteins.

140. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus comprises the administration of one or more fusion proteins.

141. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus comprises the administration of one or more RNA molecules.

142. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus comprises the administration of one or more lipids.

143. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus comprises the administration of one or more metabolites.

144. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein an external stimulus includes the administration of a drug that induces an anti-inflammatory or immunomodulatory response.

145. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus comprises the administration of one or more neurotransmitters.

146. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus comprises the administration of one or more proteins capable of directly or indirectly interacting with a promoter, RNA, or DNA sequence.

147. A modifiable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulus comprises the administration of one or more RNA or DNA molecules capable of directly or indirectly interacting with a promoter, RNA, or DNA sequence.

148. A method for expressing a therapeutic gene, comprising administering at least once the adjustable therapeutic gene therapy system described in any one of the preceding claims.

149. A method for therapeutic gene expression, comprising administering a gene construct encoding at least one therapeutic gene to any one of the tissues or cells provided herein, such as a subcutaneous space (e.g., one or more cell types within a subcutaneous space), under the control of at least one promoter element, and optionally under the control of one or more regulatory elements, and optionally under the control of a delivery carrier or vector encapsulating at least a portion of the gene construct.

150. The method according to claim 149, wherein the gene construct is one of the gene constructs defined in any of the preceding claims or described herein.

151. The method according to claim 149 or 150, wherein one or more adjustment elements are any one of the adjustment elements defined in any of the preceding claims or described herein.

152. The method according to any one of claims 149 to 151, wherein the delivery carrier or vector is one of the delivery carriers or vectors as defined in any one of the preceding claims or as described herein.

153. The method according to any one of claims 149 to 152, wherein the method further comprises killing, reducing, removing, or aging cells in a subcutaneous space to which a gene construct has been administered.

154. The method according to any one of claims 149 to 152, further comprising reducing the expression of a transgene from a gene construct.

155. The method according to any one of claims 149 to 152, further comprising increasing the expression of a transgene from a gene construct.

156. The method according to any one of claims 148 to 155, wherein an external stimulus is applied to cells in a subcutaneous space to which a gene construct has been administered.

157. The method according to claim 156, wherein the external stimulus is one of the external stimuli defined in any one of the preceding claims or described herein.

158. The method of claim 156, wherein the external stimulus is heat, cold, electromagnetic radiation, ultrasound, sound waves, pressure, electrical stimulation, or chemical or physical means that increase or decrease the expression of a therapeutic gene, or an element that can increase or decrease the expression of a therapeutic gene.

159. The method according to any one of claims 148 to 158, wherein the adjustable gene therapy system and / or gene construct includes means for upregulating or downregulating transgene expression, and optionally, the means is one of the means for such purposes provided herein.

160. The method according to any one of claims 148 to 159, further comprising administering a second dose of (1) a modifiable gene therapy system, or (2) a gene construct encoding at least one therapeutic gene, into a subcutaneous space (e.g., one or more cell types in a subcutaneous space) under the control of at least one promoter element, and optionally under the control of one or more regulatory elements, and optionally under the control of a delivery carrier or vector encapsulating at least a portion of the gene construct.

161. The method according to any one of claims 148 to 160, wherein the transgene expression is permanently reduced (for example, compared to a baseline level of expression of the administered transgene).

162. The method according to any one of claims 148 to 161, wherein the administration is for the purpose of treating a disease in a subject (e.g., a human subject).

163. The method according to any one of claims 148 to 161, wherein the administration is for treating a disease in a non-human subject.

164. The method according to any one of claims 148 to 163, wherein the administration is for the purpose of enhancing or improving the health, physical condition, mental state, or physical or mental capacity of a human or animal.

165. The method according to any one of claims 148 to 163, wherein the administration is for the purpose of increasing the lifespan of a human or animal and / or increasing the healthy lifespan of a human or animal.

166. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims, wherein the target cells are adipocytes.

167. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims, wherein the target cells are preadipocytes.

168. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims, wherein the target cells are progenitor cells capable of differentiating into adipocytes.

169. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims, wherein the target cells are stem cells capable of differentiating into adipocytes.

170. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims, wherein the target cells are resident cells of the subcutaneous tissue.

171. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims, wherein the target cells are transient cells or stem cells, but can become resident cells of the subcutaneous tissue.

172. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims for the treatment of a single gene disorder.

173. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims for the treatment of enzyme deficiency.

174. A modifiable therapeutic gene therapy system or method for protein replacement therapy according to any one of the prior claims.

175. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims for the treatment of metabolic disorders.

176. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims for the treatment of an autoimmune disorder.

177. A modifiable therapeutic gene therapy system or method according to any one of the prior claims for the treatment of oncology.

178. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims for the treatment of neurological disorders.

179. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims for the treatment of a cardiovascular disease.

180. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims for the treatment of musculoskeletal disorders.

181. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims for the treatment of hematological disorders.

182. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims for the treatment of dermatological disorders.

183. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims for the treatment of an immune system disorder.

184. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims for the treatment of lung system disorders.

185. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims for the treatment of a kidney or bladder disorder.

186. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims for the delivery of a therapeutic antibody via expression from a gene construct.

187. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims for delivery of a therapeutic protein via expression from a gene construct.

188. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims for delivery of a fusion protein via expression from a gene construct.

189. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims for the delivery of peptides via expression from a gene construct.

190. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims for delivery of an immunogen via expression from a gene construct.

191. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims for the delivery of a vaccine via expression from a gene construct.

192. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims, further comprising one or more markers for subsequent localization, visualization, analysis, enabling upmodulation, enabling downmodulation, selection of subsequent drug administration, or general visualization, or means for such purposes, or administration thereof.

193. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims, further comprising, or administering, a surface or internal marker for subsequent localization, visualization, analysis, upmodulation, downmodulation, selection of subsequent drug delivery, or general visualization at the delivery site.

194. A tunable therapeutic gene therapy system or method according to claim 192 or 193, wherein one or more markers include any one of the markers described herein (for example, one or more metals, ceramics, polymers, or complexes, or a gene code encoding a reporter protein, which can optionally be visualized directly or after the application of a stimulus that enables or enhances visualization).

195. A tunable therapeutic gene therapy system or method according to claim 194, wherein the stimulus is any one of the stimuli described herein (including, for example, the administration of one or more energies, cold, heat, one or more chemicals, therapeutic molecules or atoms, which alter the physical, chemical or physiological state of the system and enable the reporting, visualization, reading or interaction of markers, thereby providing information about the treatment).

196. A modifiable therapeutic gene therapy system or method according to any one of the preceding claims, wherein administration occurs more than once, such as multiple times, during the procedure.

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