Modulatable gene therapy

Abstract

The invention is applied at least in part to therapeutic agents, and more particularly to gene therapy for human or veterinary medicine.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 459,417 entitled “ADJUSTABLE GENE THERAPY”, filed on April 14, 2023, under 35 USC § 119(e), the disclosure of which is incorporated herein by reference in its entirety.

[0003] Reference to electronic sequence listing

[0004] The contents of the electronic serial number (R087270003WO00-SEQ-KVC.xml; size: 132,546 bytes; and creation date: April 12, 2024) are incorporated herein by reference in their entirety. Technical Field

[0005] This invention applies at least in part to therapeutic agents, and more specifically, to gene therapy in humans or veterinary medicine. Background Technology

[0006] Recent advances in gene therapy have demonstrated that this approach can not only cure, alleviate pathological conditions, or prevent the progression of diseases (such as monogenic diseases) by replacing defective or dysfunctional genes, but also serve as a means of gene enhancement when naturally expressed or when proteins are under-expressed. This allows gene therapy to be used in many applications previously considered suboptimal compared to treatments delivered via gene delivery. However, improvements are needed. Summary of the Invention

[0007] Some aspects of this disclosure relate to a modulated therapeutic gene therapy system for delivery to any of the tissues or cells provided herein, such as delivery to a subcutaneous space (e.g., delivery to one or more cell types in a subcutaneous space), the system comprising: a) a genetic construct encoding at least one therapeutic gene, said therapeutic gene being regulated by at least one promoter element and optionally one or more regulatory elements; b) a delivery vector or vector encapsulating at least a portion of said genetic construct, optionally allowing reapplication of subsequent doses of said genetic construct (e.g., with at least 10% efficiency compared to the initial dose of said genetic construct); and c) means for increasing or decreasing the expression level of a therapeutic transgene from the genetic construct.

[0008] In some implementations, c) means are means for permanently reducing the level of therapeutic transgene expression (e.g., reducing it by at least 2%).

[0009] In some implementations, the means of reducing therapeutic transgene expression levels is by applying external stimuli.

[0010] In some embodiments, the genetic construct encodes at least one functional portion of a human peptide or protein (e.g., a full-length human protein). In some embodiments, the genetic construct encodes at least one functional portion of a human protein analog or antagonist. In some embodiments, the genetic 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 genetic construct encodes at least one functional portion of a non-human protein analog or antagonist. In some embodiments, the genetic construct encodes insulin or an insulin analog. In some embodiments, the insulin or insulin analog is modified, furin-cleavable insulin. In some embodiments, the genetic construct encodes GLP-1, a GLP-1 agonist, or a GLP-1 agonist analog. In some embodiments, the genetic construct encodes a growth factor. In some embodiments, the genetic construct encodes a cytokine. In some embodiments, the genetic construct encodes an anti-inflammatory protein. In some embodiments, the genetic construct encodes a complement protein. In some embodiments, the genetic construct encodes a receptor agonist. In some embodiments, the genetic construct encodes a receptor antagonist. In some embodiments, the genetic 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.

[0011] 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 genetic construct consists of one or more circular single-stranded DNA constructs. In some embodiments, the genetic construct consists of one or more circular double-stranded DNA constructs. In some embodiments, the genetic construct consists of one or more linear single-stranded DNA constructs. In some embodiments, the genetic construct consists of one or more linear double-stranded DNA constructs. In some embodiments, the genetic construct is at least partially composed of DNA or RNA. In some embodiments, the genetic construct consists of DNA and / or RNA. In some embodiments, the external stimulus consists of a chemical DNA derivative, an RNA derivative, or a combination of DNA and RNA derivatives.

[0012] In some embodiments, the promoter element comprises a constitutive promoter. In some embodiments, the promoter element comprises an inducible promoter. In some embodiments, the promoter element comprises a tissue-specific promoter. In some embodiments, the promoter element comprises a component containing... A promoter element comprising one or more sequences of a promoter. In some embodiments, the promoter element comprises a tetOn inducible promoter construct. In some embodiments, the promoter element comprises at least one promoter sequence having one or more enhancers, regulators, operators, and / or repressors. In some embodiments, the promoter element comprises an inducible promoter that can be upregulated or downregulated in response to external or internal stimuli (e.g., inflammation, heat, light, stress, administration of steroids, tetracyclines, antibiotics, rapamycin, ganciclovir, acyclovir), or can be induced by upregulated or downregulated molecules (e.g., ROS, NOS, or cytokine release). In some embodiments, the promoter element comprises a circadian or cyclic promoter (e.g., which alters its activity level by at least 5% over several cycles ranging from hours to months).

[0013] In some embodiments, the regulatory element comprises a post-translational regulatory element. In some embodiments, the regulatory element comprises at least some or all of a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). In some embodiments, the regulatory element comprises at least some or all of an optimized Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). In some embodiments, the regulatory element comprises a cis-acting element capable of increasing cytoplasmic mRNA accumulation. In some embodiments, the regulatory element comprises at least some or all of an HIV type 1 Rev-Rev response element. In some embodiments, the regulatory element comprises at least some or all of a human hepatitis virus post-translational regulatory element. In some embodiments, the regulatory element comprises at least some or all of one or more viral post-translational regulatory elements capable of increasing the expression of a target gene or any other element encoded by a genetic construct.

[0014] In some embodiments, the genetic construct comprises one or more functional genetic sequences. In some embodiments, one or more functional genetic sequences facilitate translocation of the genetic construct (e.g., at least 4% of the genetic construct) to the nucleus of a target cell. In some embodiments, one or more functional genetic sequences comprise sequences encoding sequence-specific DNA-binding proteins coupled to nuclear localization signal peptides. In some embodiments, one or more functional genetic sequences comprise DNA nuclear target sequences recognized by one or more specific transcription factors. In some embodiments, one or more functional genetic sequences comprise DNA nuclear target sequences (DTS) active in input protein-mediated transport systems. In some embodiments, one or more functional genetic sequences comprise at least a portion or all of the SV40 DTS. In some embodiments, one or more functional genetic sequences comprise at least a portion or all of the glucocorticoid response element (GRE) DTS. In some embodiments, one or more functional genetic sequences comprise the Sox2 regulatory region 2 DTS sequence. In some embodiments, one or more functional genetic sequences comprise an input protein β(1), an input protein 7, NF-κβ, or a guanosine triphosphatase Ran interaction sequence. In some embodiments, one or more functional genetic sequences (e.g., reducing expression from a genetic construct) comprise an inducible suicide gene. In some embodiments, one or more functional genetic sequences (e.g., reducing expression from a genetic construct) comprise an inducible expression system encoding an RNA molecule that reduces the expression of at least a portion of a therapeutic genetic construct. In some embodiments, one or more functional genetic sequences (e.g., reducing expression from a genetic construct) encode a protein capable of targeted epigenetic silencing. In some embodiments, one or more functional genetic sequences (e.g., reducing expression from a genetic construct) encode an HSV-TK suicide gene or system. In some embodiments, one or more functional genetic sequences (e.g., reducing expression from a genetic construct) encode a RapaCas9 suicide gene or system. In some embodiments, one or more functional genetic sequences (e.g., those reducing expression from a genetic construct) encode one or more of miRNA, siRNA, shRNA, dsRNA, ncRNA, lncRNA, piwi-interacting RNA, PAT, eRNA, and / or circRNA. In some embodiments, one or more functional genetic sequences (e.g., those reducing expression from a genetic construct) encode at least a guide RNA and a CRISPR-dCas system (e.g., coupled to a protein that induces epigenetic silencing).In some embodiments, one or more functional genetic sequences (e.g., reducing expression from a genetic construct) encode at least a guide RNA and CRISPR-dCas9-KRAB. In some embodiments, one or more functional genetic sequences (e.g., reducing expression from a genetic construct) encode at least a guide RNA and CRISPR-dCas12b-KRAB. In some embodiments, one or more functional genetic sequences (e.g., reducing expression from a genetic construct) encode at least a guide RNA and CRISPR-dCas8c-KRAB. In some embodiments, one or more functional genetic sequences (e.g., reducing expression from a genetic construct) encode at least a guide RNA and CRISPR-dCas8a-KRAB. In some embodiments, one or more functional genetic sequences (e.g., reducing expression from a genetic construct) encode at least a guide RNA and CRISPR-dCas8b-KRAB. In some implementations, one or more functional genetic sequences (e.g., those that reduce expression from a genetic construct) encode at least TALEN, megnuclease, endonuclease, restriction enzyme, zinc finger protein, or other DNA-binding or RNA-binding protein.

[0015] In some embodiments, the regulatory element alters the expression of a genetic construct in response to temperature. In some embodiments, the regulatory element alters the expression of a genetic construct in response to heat. In some embodiments, the regulatory element alters the expression of a genetic construct in response to cold. In some embodiments, the regulatory element alters the expression of a genetic construct in response to ultrasound. In some embodiments, the regulatory element alters the expression of a genetic construct in response to electrical stimulation. In some embodiments, the regulatory element alters the expression of a genetic construct in response to chemical stimulation. In some embodiments, the regulatory element alters the expression of a genetic construct in response to changes in the physiological environment. In some embodiments, the regulatory element alters the expression of a genetic construct in response to paracrine, endocrine, or autocrine factors. In some embodiments, the regulatory element alters the expression of a genetic construct in response to the presence of inflammation. In some embodiments, the regulatory element comprises a proximal promoter. In some embodiments, the regulatory element comprises a distal promoter. In some embodiments, the regulatory element comprises an insulator. In some embodiments, the regulatory element forms a secondary structure with a genetic construct or other genetic sequence.

[0016] In some embodiments, the delivery vector or carrier comprises lipid nanoparticles (e.g., functionalized lipid nanoparticles, solid lipid nanoparticles, lipid-polymer hybrid nanoparticles). In some embodiments, the delivery vector or carrier comprises liposomes (e.g., functionalized liposomes, stealth liposomes) or micelles. In some embodiments, the delivery vector or carrier comprises one or more types of cells (e.g., functional cells), cellular components, or cell membranes. In some embodiments, the delivery vector or carrier comprises a cubosome, transfection body, endosome, efflux body, or vesicle system. In some embodiments, the delivery vector or carrier comprises polymer nanoparticles. In some embodiments, the delivery vector or carrier comprises a non-immunogenic or low-immunogenic viral vector. In some embodiments, the delivery vector or carrier comprises a protein or peptide. In some embodiments, the delivery vector or carrier is functionalized to reduce immunogenicity. In some embodiments, the delivery vector or carrier is functionalized to promote uptake (e.g., uptake by one or more specific cell types). In some embodiments, the delivery vector or carrier is functionalized to promote endocytosis, pinocytosis, or enhance transport to the cell nucleus. In some embodiments, the delivery carrier or support comprises ionizable lipids. In some embodiments, the delivery carrier or support comprises ionizable elements. In some embodiments, the delivery carrier or support comprises ionizable polymers. In some embodiments, the delivery carrier or support comprises cholesterol. In some embodiments, the delivery carrier or support comprises a cationic component. In some embodiments, the delivery carrier or support comprises amphiphilic polymers. In some embodiments, the delivery carrier or support comprises polyethylene glycol. In some embodiments, the delivery carrier or support comprises antibodies, nanobodies, or antibody fragments. In some embodiments, the delivery carrier or support comprises peptides. In some embodiments, the delivery carrier or support comprises nanoparticles chemically or physically conjugated to peptides, proteins, or functional sequences derived from peptides or proteins. In some embodiments, the delivery carrier or support comprises nanoemulsions, nanostructured lipids, or is at least partially composed of amphiphilic polymers or oligomers. In some embodiments, the delivery carrier or support comprises phospholipids, sphingolipids, polyelectrolyte polymers, or polyelectrolyte complexes. In some embodiments, the delivery carrier or support comprises metal or ceramic nanoparticles. In some embodiments, delivery of the vector or carrier does not elicit a humoral or cell-mediated immune response. In some embodiments, delivery of the vector or carrier does not elicit a memory immune response. In some embodiments, delivery of the vector or carrier is minimally immunogenic (e.g., such that the memory immune response generated by the vector or carrier cannot neutralize at least 10% of any subsequent administration of the vector or carrier via the same route of administration, e.g., in the test subject).In some embodiments, the delivery vector or carrier is of minimal immunogenicity (e.g., such that the memory immune response generated by the vector or carrier cannot neutralize at least 50% of any subsequent administration of the vector or carrier via the same route of administration, for example, in the test subject). In some embodiments, the delivery vector or carrier is of minimal immunogenicity (e.g., such that the memory immune response generated by the vector or carrier cannot neutralize at least 75% of any subsequent administration of the vector or carrier via the same route of administration, for example, in the test subject). In some embodiments, the delivery vector or carrier is of minimal immunogenicity (e.g., such that the memory immune response generated by the vector or carrier cannot neutralize at least 90% of any subsequent administration of the vector or carrier via the same route of administration, for example, in the test subject). In some embodiments, the delivery vector or carrier is of minimal immunogenicity (e.g., such that the memory immune response generated by the vector or carrier cannot neutralize at least 99% of any subsequent administration of the vector or carrier via the same route of administration, for example, in the test subject).

[0017] In some embodiments, the external stimulus causes cooling of the skin and subcutaneous tissue (e.g., to induce adipocyte death). In some embodiments, the external stimulus causes cooling of the skin and subcutaneous tissue to induce adipocyte senescence. In some embodiments, the external stimulus causes a temperature change in the subcutaneous tissue sufficient to alter the expression of a genetic construct. In some embodiments, the external stimulus includes high-frequency ultrasound. In some embodiments, the external stimulus includes mid-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 mid-frequency ultrasound-mediated cavitation. In some embodiments, the external stimulus includes low-frequency ultrasound-mediated cavitation. In some embodiments, the external stimulus includes ultrasound with a frequency in the range of 1 to 10 kHz. In some embodiments, the external stimulus includes ultrasound with a frequency in the range of 10 to 28 kHz. In some embodiments, the external stimulus includes ultrasound with a frequency in the range of 28 to 40 kHz. In some embodiments, the external stimulus includes ultrasound with a frequency in the range of 40 to 60 kHz. In some embodiments, the external stimulus includes ultrasound with a frequency range of 1 to 320 kHz. In some embodiments, the external stimulus includes sound waves (e.g., capable of inhibiting expression in adipocytes, inducing senescence in adipocytes, or inducing adipocyte death, for example, in at least a portion of the subcutaneous space). In some embodiments, the external stimulus includes electromagnetic waves (e.g., capable of inhibiting expression in adipocytes, inducing senescence in adipocytes, or inducing adipocyte death, for example, in at least a portion of the subcutaneous space). In some embodiments, the external stimulus includes electrical signals (e.g., capable of inhibiting expression in adipocytes, inducing senescence in adipocytes, or inducing adipocyte death, for example, in at least a portion of the subcutaneous space). In some embodiments, the external stimulus includes administration of a formulation comprising rapamycin or a rapamycin analog or a rapamycin derivative. In some embodiments, the external stimulus includes administration of a formulation comprising tetracycline or a derivative thereof. In some embodiments, the external stimulus includes administration of rapamycin or a rapamycin analog or a rapamycin derivative in combination with tetracycline or a tetracycline derivative. In some embodiments, the external stimulus includes administration of ganciclovir or a ganciclovir derivative. In some embodiments, the external stimulus includes administration of doxycycline or a doxycycline derivative. 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 conjugations. In some embodiments, the external stimulus includes the administration of one or more hormones or their analogues. In some embodiments, the external stimulus includes the administration of tamoxifen or a tamoxifen derivative.In some embodiments, the external stimulus includes the administration of one or more agents that elicit 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 functionalized or unfunctionalized antibodies. 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 elicit 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 capable of directly or indirectly interacting with a promoter, RNA, or DNA sequence. In some embodiments, the external stimulus includes the administration of one or more RNA or DNA molecules capable of directly or indirectly interacting with a promoter, RNA, or DNA sequence.

[0018] Some aspects of this disclosure relate to methods of therapeutic transgenic expression, which include at least one administration of the adjustable therapeutic gene therapy system described herein.

[0019] Some aspects of this disclosure relate to methods of therapeutic transgenic expression, which include administering to any of the tissues or cells provided herein, such as to a subcutaneous space (e.g., to one or more cell types in a subcutaneous space): a genetic construct encoding at least one therapeutic gene, said therapeutic gene being regulated by at least one promoter element and optionally one or more regulatory elements; and optionally, a delivery vector or vector encapsulating at least a portion of said genetic construct.

[0020] In some implementations, the genetic construct is any of the genetic constructs described herein.

[0021] In some implementations, one or more control elements are any of the control elements described herein.

[0022] In some implementations, the delivery carrier or vehicle is any of the carriers or vehicles described herein.

[0023] In some embodiments, the method further includes killing, reducing, removing cells, or inducing cellular senescence in the subcutaneous space where the genetic construct has been applied. In some embodiments, the method further includes reducing transgene expression from the genetic construct. In some embodiments, the method further includes increasing transgene expression from the genetic construct.

[0024] In some embodiments, an external stimulus is applied to cells in the subcutaneous space where the genetic construct has been applied. In some embodiments, the external stimulus is any of the external stimuli described herein. In some embodiments, the external stimulus is heat, cold, electromagnetic radiation, ultrasound, sound waves, pressure, electrical stimulation, or other chemical or physical means to increase or decrease the expression of the therapeutic transgene or element that can increase or decrease the expression of the therapeutic transgene.

[0025] In some implementations, the modifiable gene therapy system and / or genetic construct includes means for upregulating or downregulating transgene expression, optionally wherein such means are any of the means provided herein for such purposes.

[0026] In some embodiments, the method further includes administering a second dose to a subcutaneous space (e.g., to one or more cell types in the subcutaneous space) of: i) a modulated gene therapy system or ii) a genetic construct encoding at least one therapeutic gene, said therapeutic gene being regulated by at least one promoter element and optionally one or more regulatory elements, and optionally, a delivery vector or vector encapsulating at least a portion of said genetic construct.

[0027] In some implementations, transgene expression is permanently reduced (e.g., relative to the baseline expression level of the applied transgene).

[0028] In some embodiments, the application is used to treat a disease in a subject (e.g., a human subject). In some embodiments, the application is used to treat a disease in a non-human subject. In some embodiments, the application is used to enhance or improve the health, physical condition, mental state, or physical or mental capacity of a person or animal. In some embodiments, the application is used to increase the lifespan of a person or animal and / or increase the healthy lifespan of a person or animal.

[0029] 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 subcutaneous tissue. In some embodiments, the target cells are transient cells or stem cells, but capable of becoming resident cells of subcutaneous tissue.

[0030] In some embodiments, the modifiable therapeutic system or method is used to treat monogenic diseases. In some embodiments, the modifiable therapeutic system or method is used to treat enzyme deficiency diseases. In some embodiments, the modifiable therapeutic system or method is used for protein replacement therapy. In some embodiments, the modifiable therapeutic system or method is used to treat metabolic diseases. In some embodiments, the modifiable therapeutic system or method is used to treat autoimmune diseases. In some embodiments, the modifiable therapeutic system or method is used to treat tumors. In some embodiments, the modifiable therapeutic system or method is used to treat neurological diseases. In some embodiments, the modifiable therapeutic system or method is used to treat cardiovascular pathologies. In some embodiments, the modifiable therapeutic system or method is used to treat musculoskeletal diseases. In some embodiments, the modifiable therapeutic system or method is used to treat hematological diseases. In some embodiments, the modifiable therapeutic system or method is used to treat skin diseases. In some embodiments, the modifiable therapeutic system or method is used to treat immune system diseases. In some embodiments, the modifiable therapeutic system or method is used to treat pulmonary diseases. In some embodiments, the modifiable therapeutic system or method is used to treat kidney or bladder diseases. In some embodiments, a modulotherapy system or method is used to deliver a therapeutic antibody via expression from a genetic construct. In some embodiments, a modulotherapy system or method is used to deliver a therapeutic protein via expression from a genetic construct. In some embodiments, a modulotherapy system or method is used to deliver a fusion protein via expression from a genetic construct. In some embodiments, a modulotherapy system or method is used to deliver a peptide via expression from a genetic construct. In some embodiments, a modulotherapy system or method is used to deliver an immunogen via expression from a genetic construct. In some embodiments, a modulotherapy system or method is used to deliver a vaccine via expression from a genetic construct.

[0031] In some embodiments, the adjustable therapeutic system or method further includes, or applies thereof, one or more biomarkers for subsequent positioning, visualization, analysis, achievement of increased titration, achievement of decreased titration, selection of subsequent dose, or overall visualization, or the means thereof. In some embodiments, the adjustable therapeutic system or method further includes, or applies thereof, surface or internal biomarkers at the delivery site for subsequent positioning, visualization, analysis, achievement of increased titration, achievement of decreased titration, selection of subsequent dose, or overall visualization, or the methods thereof.

[0032] In some implementations, one or more markers include any of the markers described herein (e.g., one or more of metals, ceramics, polymers or composites, or the genetic code encoding a reporter protein, optionally which can be visualized directly or after stimulation is applied to allow or enhance visualization).

[0033] In some implementations, the stimulus is any of the stimuli described herein (e.g., including one or more of the following: energy, cold, heat, application of one or more chemicals, treatments, molecules or atoms, to alter the physical, chemical or physiological state of the system, thereby enabling biomarker reporting, visualization, readout or interaction to provide information about treatment).

[0034] In some implementations, the administration occurs more than once during treatment, for example, multiple times. Attached Figure Description

[0035] Figures 1A to 1B It is for npRB2.6v3 (CBh-FC-hINS-CMV-rapaCasp9-K19) Figure 1A ) and npRB2.6v7 (CBh-FC-FLuc-CMV-rapaCasp9-K19) Figure 1B The plasmid map of ).

[0036] Figure 2 The results of the apoptosis assay are shown, which were quantified by FLuc expression at 76 hpi.

[0037] Figure 3 Apoptosis induced by RapaCasp9 in HdAD at 24 hpi is shown. Results for 0 mM, 0.05 mM, and 0.1 mM Tet are shown.

[0038] Figure 4 This is a plasmid map for gcCBh-fLuc-SV40IpA.

[0039] Figures 5A to 5C Animal monitoring results are shown after subcutaneous injection of the initial construct dose. Figures 5A to 5B The results showed that IVIS confirmed the durability of the initial signal (transgenic expression) lasting up to day 22. Figure 5A and 5C This demonstrates the ability to titrate the initial dose in a predictable dose-dependent manner.

[0040] Figure 6 The total throughput from the destination region is shown as a function of time (days).

[0041] Figure 7The total throughput of titrations from the ROI is shown after one week (left) and one month (right).

[0042] Figure 8 The average flux in albino mice before and after the reduction titration is shown.

[0043] Figure 9 This is a plasmid map of npRB2.10v1 (EFS-NLuc+EFS-Cre-ERT2).

[0044] Figure 10 This is a plasmid map of npRB2.6v8 (CBh-NLuc). Detailed Implementation

[0045] In its current form, gene therapy is a single-dose treatment that delivers an initial dose, which cannot be titrated up or down after the initial administration. Furthermore, gene therapy treatments delivered by viral vectors cannot be re-administered because viral vectors typically elicit a memory immune response to neutralize subsequent doses in a variable manner between patient-to-patient and organism-to-organism interactions, and this response can depend on many factors, such as the initial route of administration, the dose delivered, the patient's immune status, pre-existing immunity to the vector, and the state of the immune system at the time of potential re-administration. Moreover, several in vivo studies have demonstrated variable responses to re-administration of viral gene therapy in the same organism and using the same vector, with some subsequent doses indicating expression levels similar to or equivalent to the initially delivered dose, while others indicated no expression of the therapeutic construct. It is hypothesized that this variability is caused by the aforementioned immune and vector-related factors, leading to a lack of predictability for re-treatment, titration increases, or dose escalations. Targeted particles have been used for drug delivery, and virus-like particles have been explored as a means of reducing the tropism of gene therapy treatments. The targeted portion in such particles also prevents re-administration of gene therapy and thus improves titration after initial administration. Furthermore, when attempting to target tissues, most gene therapies are administered intravenously or directly to the organ requiring gene replacement. Intravenously administered gene therapy is primarily absorbed by the liver, regardless of whether viral or non-viral vectors are used, or whether targeted or non-targeted particles are applied. Gene therapy delivered to target organs can be effective in transducing target tissues, but it is sometimes associated with increased complications due to immunogenic responses within the organ, difficulty in accessing target tissues (e.g., the brain or retina), and the overall complexity of the procedure. Conversely, most protein replacement therapies or protein-based therapeutic interventions are administered subcutaneously, providing a route of administration with easy access, excellent bioavailability, and typically to tissues that can tolerate the damage without significantly impacting the overall function of the organism.

[0046] Furthermore, while gene therapies using non-immunogenic vectors have been explored, these treatments lack the ability to titrate the initial administered dose in a controlled and predictable manner. The inability to titrate gene therapy treatment after initial delivery hinders its use in many applications that require titration and de-titering of the therapeutic agent after an initial assessment of its safety and efficacy in a given patient. Conversely, many therapeutic agents require titration and de-titering to achieve effective efficacy; for example, GLP-1 receptor agonists (or GLP-1 analogs) and basal insulin are initially administered at low doses to assess patient-specific tolerability to the treatment, and the treatment dose is reviewed and adjusted after an initial assessment that can last from weeks to months to optimize its safety, tolerability, or efficacy. Other treatments require titration and de-titering of the initial dose due to the need to achieve efficacy and reduce safety-related risks. For example, anti-VEGF therapy for wet age-related macular degeneration is initially administered at high frequency or high mean in situ concentrations, which are eventually reduced (in dose or frequency) to minimize potential treatment-related side effects. Therefore, to extend the applicability of gene therapy to treatments that require or can benefit from dose adjustment after initial administration, dose-adjustable or titrable gene therapies have been developed, allowing for at least one, but in some embodiments multiple, optional dose-increasing and / or decreasing titrations after initial delivery. Finally, in some embodiments, the route of administration of the gene therapy is preferably optimal to allow for effective increasing and decreasing titrations, a safety profile of the therapy, and / or appropriate pharmacokinetics for a wide range of therapeutic applications. Therefore, this document provides: means for implementing and using adjustable gene therapy treatments that can be delivered at an initial dose and subsequently titrated or decreasing after initial administration; and related compositions and methods.

[0047] The therapeutic gene therapy formulations provided herein can be used to treat diseases or control disease symptoms. More specifically, the therapeutic formulations disclosed herein are for human or veterinary use and comprise at least one genetic construct or a genetic construct encoding at least one therapeutic gene or gene sequence capable of producing a beneficial effect in humans or veterinary medicine. Means are provided herein for the cis- or trans-delivery of a number of functional elements with a therapeutic gene or genetic construct, which enables or optimizes titration increases and / or decreases of the therapeutic formulation or, in particular, the therapeutic gene or genetic construct, after initial administration. In some embodiments, elements for adjustable-dose gene therapy are provided that can increase or decrease titration at least once after initial administration. The formulations provided herein can be used to optimize the safety, efficacy, and / or tolerability of the initial delivery dose. In some embodiments, means are provided for delivering gene therapy treatments into a subcutaneous space, which is typically well-vascularized and provides optimal pharmacokinetics for the subcutaneously delivered biological product; and means for reducing titration, such as by applying an agent or method 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. Such agents or methods may be one or more of ultrasound, focused ultrasound, high-frequency ultrasound, intermediate-frequency ultrasound, low-frequency ultrasound, ultrasound-mediated cavitation, ultracavitation, radio frequency, cooling, temperature flux, cooling effect, temperature reduction, electromagnetic radiation, and focused energy.

[0048] Generally, this document provides therapeutic gene therapy formulations for treating human or veterinary diseases or pathological conditions, which may also allow for increased and / or decreased titration of the delivery dose after initial administration. The formulation may comprise at least a target therapeutic genetic construct encoding at least one peptide, protein, or non-coding RNA, and a promoter sequence regulating the expression of the target therapeutic gene. Any of the formulations may be delivered in cis with a post-translational regulatory element. Any of the formulations may be delivered in cis or trans with a genetic sequence that can promote at least a portion of the therapeutic genetic construct to translocate to the target cell nucleus. Any of the formulations may comprise at least one genetic sequence that can reduce the expression of one or more elements from the genetic construct by acting on the cell containing the genetic construct, the genetic construct itself, or within the cell containing the genetic construct. Any of the genetic constructs may be at least partially encapsulated by a carrier or vector that does not elicit a memory immune response and / or is capable of neutralizing at least a portion of the therapeutic dose of a subsequent re-administration of the same or substantially similar composition via the usual route of administration. For therapeutic gene therapy systems described herein that do not include regulatory sequences for achieving reduced titration of therapeutic gene expression by: silencing, elimination of the genetic construct, or induction of apoptosis or overall reduction of gene expression in cells containing the construct; the reduction of therapeutic transgene expression can be achieved by applying an external stimulus that eliminates cells carrying the therapeutic construct, reduces cell viability, overall gene expression, overall transcription, translation, or protein secretion, or induces apoptosis or senescence in cells carrying the construct. In some embodiments, the cells may be removed, for example by surgical methods or other methods, such as liposuction.

[0049] Promoting translocation to the nucleus may include a sufficient amount of therapeutic construct translocated to the nucleus to achieve the efficacy of the construct without compromising the safety or tolerability of the treatment, and ideally at least 4% of the construct based on molar concentration, and most preferably at least 20% of the construct. Similarly, in some embodiments, the development of an adaptive immune response includes generating neutralizing antibodies or reactive T cells capable of eliminating or neutralizing subsequent doses in a manner that limits their efficacy, ideally neutralizing less than 50% of the dose, most preferably less than 10% of the dose, and preferably without significant construct or carrier-specific neutralization.

[0050] In some implementations, the downtiting is complete, while in others, the downtiting is incremental and may contain any amount between 2% and 15%, 90%, or 2% and 100% of the overall expression. Incremental downtiting may be performed once or in a series of stimuli to reduce expression by a fold of the initial downtiting or as needed to obtain optimal transgene expression.

[0051] The route of administration for delivering the formulation can typically be systemic or local, and can consist of one or more of any clinically established therapeutic routes of administration. More specifically, the route of administration can be local administration to one or more local tissues or organs, or systemic administration including enteral or parenteral administration. In some embodiments, the route of administration may include one or more of the following: intravenous, subcutaneous, subdermal, intra-articular, intraventricular, intravenous, intramuscular, subarachnoid, vaginal, rectal, inhalation, intravitreal, oral, buccal, sublingual, ocular, percutaneous, pulmonary, or peritoneal administration. In some embodiments, one of the preferred methods of gene therapy administration is subcutaneous administration, or administration to adipose tissue in the subcutaneous space. This allows for multiple rounds of titration increases, administration of numerous gene therapy constructs, and can be the safest means of reducing the titration of gene therapy while still providing optimal pharmacokinetics for treatment.

[0052] This document provides therapeutic formulations comprising a genetically encoded therapeutic gene, said therapeutic gene having or not having a functional element or genetic element involved in regulation. More specifically, the 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, the therapeutic formulation may comprise at least a portion of a gene, or more specifically at least the coding segment of a gene. In other embodiments, the target therapeutic gene may encode a modified furin-cleavable insulin, insulin or insulin analog, GLP-1 peptide, GLP-1 agonist, or GLP-1 agonist analog. In still other embodiments, the therapeutic formulation may encode at least a portion of at least one growth factor, cytokine, anti-inflammatory protein, complement protein, or protein of the general immune system or immune system modulator, and at least a portion of a receptor agonist or antagonist, a secretory protein or a portion thereof, or a receptor or a functional element thereof. In other embodiments, the therapeutic agent may encode at least a portion of a fusion protein consisting of one or more functional elements of at least two different proteins or peptides. Alternatively, the therapeutic agent may encode non-protein-coding elements of the human or animal genome, which typically include structural or regulatory elements such as transfer RNA (tRNA), ribosomal RNA (rRNA), long non-coding RNA (lncRNA), microRNA (miRNA), silencing RNA (siRNA), inducing RNA (iRNA), endogenous silencing inducing RNA, piwi-interacting RNA (piRNA), or other types of RNA or DNA capable of performing intracellular or extracellular functions, which may or may not involve encoding proteins.

[0053] Therapeutic genetic constructs may include at least one promoter that drives or regulates the expression of a therapeutic gene or target sequence, and one or more additional promoters that optionally drive or regulate the expression of dose-regulating elements, nuclear localization signals or helper elements, which are necessary for or provide supplemental functionality for therapeutic gene therapy.

[0054] In some embodiments, the promoter sequence intended to drive the expression of the therapeutic sequence may be one or more of mammalian promoters, constitutive promoters, inducible promoters, or tissue-specific promoters. In other embodiments, the promoter sequence may be at least partially derived from one or more of viral promoters, bacterial promoters, archaea promoters, promoters or regulatory sequences from the human or animal genome, and may optionally contain introns, untranslated regions, enhancers, or other regulatory elements required for gene expression or regulation. In some embodiments, the promoter sequence may include at least one or more of the following elements: chicken β-actin promoter and rabbit β-globin splice acceptor site (CAG) promoter, EF1α (EF1a) promoter, promoter of polyubiquitin C gene (UBC) gene promoter, CBh promoter composed of a modified form of CAG (a promoter composed of early enhancer elements of cytomegalovirus (CMV), promoter region, first exon and first intron of chicken β-actin gene and splice acceptor of rabbit β-globin gene), murine stem cell virus (MSCV) promoter, phosphoglycerate kinase PGK promoter, spleen focus forming virus (SFFV) promoter or simian 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 additionally be provided by one or more enhancer, regulator, operon, or repressor sequences, which may be delivered with the construct in cis or trans. In some embodiments, the promoter sequence may be at least partially composed of an inducible promoter that can upregulate or downregulate expression in response to external or internal stimuli (e.g., inflammation, heat, light, stress, administration of steroids, tetracyclines, antibiotics, rapamycin, ganciclovir, acyclovir), or may generally be induced by upregulation or downregulation of reactive oxygen species (ROS), reactive nitrogen species (NOS), or cytokine release. Furthermore, in other embodiments, the promoter sequence may be able to alter its expression level in response to internal or external stimuli by varying with the natural biological cycle. In some implementations, the promoter sequence may consist at least in part of a circadian or cyclic promoter whose activity level varies by at least 5%, or preferably at least 50%, over some cycles ranging from hours to months, and preferably from days to weeks.

[0055] In other embodiments, the promoter sequence designed to drive the expression of auxiliary elements (e.g., dose-regulating sequences or peptides that promote nuclear localization, transport, stability, or regulatory expression) may be one or more of mammalian promoters, constitutive promoters, inducible promoters, or tissue-specific promoters. In other embodiments, the promoter sequence may be at least partially derived from one or more of viral promoters, bacterial promoters, promoters, or regulatory sequences from the human or animal genome, and may optionally contain introns, untranslated regions, enhancers, or other regulatory elements required for expression or regulation. In some embodiments, the promoter sequence may contain at least one or more of the following elements: CAG promoter, EF1α (EF1a) promoter, UBC promoter, CBh promoter, MSCV promoter, hPGK promoter, SFFV promoter, or SV40 promoter. The promoter sequence may be a tetOn inducible promoter construct or a tetOff inducible promoter construct. Regulation may additionally be provided by one or more enhancer, regulator, operon, or repressor sequences, which may be delivered with the construct in cis or trans. In some embodiments, the promoter sequence may be at least partially composed of an inducible promoter that can upregulate or downregulate expression in response to external or internal stimuli (e.g., inflammation, heat, light, stress, administration of steroids, tetracyclines, antibiotics, rapamycin, ganciclovir, acyclovir), or may generally be induced by upregulation or downregulation of ROS, NOS, or cytokine release. Furthermore, in other embodiments, the promoter sequence may be able to alter its expression level in response to internal or external stimuli by varying with the natural biological cycle. In some embodiments, the promoter sequence may be at least partially composed of a circadian or cyclic promoter whose activity level varies by at least 5%, or preferably at least 50%, with some cycle ranging from hours to months, and preferably from days to weeks.

[0056] In some embodiments, to allow for additional functionality and / or optimal expression of the therapeutic gene and any associated regulatory elements or sequences, the construct may include one or more post-translational regulatory elements, which may include elements that regulate expression, conditionally regulate expression, stably transcribe RNA, or form secondary or tertiary structures with one or more elements of the therapeutic construct, genomic DNA, or other constructs delivered in cis or trans. Some embodiments may include one or more polyA signals, or signals encoding polyA or regulating polyA tail length, promoting or enhancing capping, promoting or enhancing ribozyme assembly or general assembly, or transcription or translation mechanisms (once the regulatory element is transcribed). In some embodiments, the post-translational regulatory elements included in the therapeutic construct may include at least a portion of a marmot hepatitis virus post-transcriptional regulatory element or WPRE, at least a portion of an optimized marmot hepatitis virus post-transcriptional regulatory element, a genetic sequence capable of enhancing the accumulation or stability of nuclear or cytoplasmic mRNA, at least a portion of an HIV type 1 Rev-Rev response element, at least a portion of a human hepatitis virus post-translational regulatory element, at least a portion of one or more viral post-translational regulatory elements capable of enhancing the expression of the target gene, or any other construct-coding element. In some embodiments, one or more post-transcriptional regulatory elements may be included in tandem with the construct, sequentially arranged, or delivered in trans with the construct. In other embodiments, the regulatory elements may be upstream or downstream of the therapeutic gene or RNA coding sequence and may optionally regulate the transcription, translation, or expression of accessory elements of the therapeutic construct.

[0057] In some embodiments, to allow for additional or optional expression, construct localization, intracellular durability of the construct, and / or prevention of innate or adaptive immune responses, the genetic sequence, elements of its vector, or general formulation may include one or more elements that promote the translocation of said sequence to the nucleus or other cellular compartments or organelles. These elements may at least partially comprise DNA, RNA, protein, peptide, lipid, polymer, cholesterol, or other molecules capable of inducing the aforementioned effects of expression optimization, construct localization, stability, durability, and immune response optimization. In some embodiments, the therapeutic construct comprises a genetic sequence delivered in cis or trans that promotes the translocation of at least a portion, ideally at least 4% molar, and ideally at least 20% molar, of the therapeutic construct to the target cell nucleus. In some embodiments, the genetic sequence comprises at least partially a protein-coding sequence having a dedicated promoter that encodes a protein that transports the therapeutic genetic construct to the nucleus. In other embodiments, the genetic sequence at least partially comprises a sequence encoding a sequence-specific DNA-binding protein coupled to a nuclear localization signal peptide, or at least partially comprises a DNA nuclear target sequence recognized by one or more specific transcription factors, or at least partially comprises an active DNA nuclear target sequence (DTS) in an input protein-mediated transport system. In other embodiments, the sequence promoting the translocation of a therapeutic construct to a nuclease at least partially comprises an SV40 DTS, 3NFDTS, Sox2 regulatory region 2 DTS sequence, or an input protein β(1), input protein 7, NF-κβ, or guanosine triphosphatase Ran interaction sequence. In other embodiments, the genetic sequence capable of inducing trans-delivered elements or genetic constructs to the nucleus encodes DNA capable of binding to intracellular transport proteins, or RNA capable of acting as an intermediate between intracellular transport proteins and trans-delivered elements or therapeutic constructs.

[0058] In some embodiments, to facilitate reduction titration after initial administration of gene therapy, the therapeutic construct may contain one or more elements capable of reducing the expression level of a therapeutic target gene, protein, or RNA. The therapeutic construct may contain a genetic sequence that, upon activation by an external stimulus, can reduce the expression of a therapeutic transgene by: altering expression levels; temporarily or permanently altering the tertiary structure of the therapeutic construct; eliminating the therapeutic construct from a cell; eliminating cells carrying the therapeutic construct; inducing cellular senescence; generally reducing transcription within cells containing the therapeutic construct; inducing apoptosis in cells containing the therapeutic construct; or inducing epigenetic changes in the therapeutic construct, its elements, or genomic DNA to alter the transcription, translation, or overall expression of the therapeutic gene or RNA-coding element contained in the therapeutic construct. In some embodiments, the sequence capable of reducing expression from the therapeutic construct is a suicide gene, or an induced suicide gene, or a suicide gene under an induced promoter. In other embodiments, the genetic sequence capable of reducing or decreasing expression from the therapeutic construct is an inducible system encoding an RNA molecule capable of reducing the expression of at least a portion of the therapeutic construct or a protein-coding gene regulated by an inducible promoter, said protein-coding gene encoding an epigenetically silenced protein capable of targeting at least a portion of the therapeutic construct. In some embodiments, the genetic sequence capable of reducing the expression of the therapeutic construct is delivered with the therapeutic construct in cis or trans and encodes one or more of the following elements: Herpes simplex virus thymidine kinase or HSV-TK suicide gene system, rapamycin-cystathin 9 or RapaCas9 suicide gene system, or other suicide gene systems capable of inducing programmed cell death or apoptosis in cells containing said construct. In other embodiments, the genetic sequence capable of reducing the expression of the therapeutic construct encodes one or more of miRNA, siRNA, shRNA, dsRNA, ncRNA, lncRNA, piwi-interacting RNA, PAT, eRNA, and circRNA. In other embodiments, the sequence encodes a DNA-binding protein with targeting capabilities, which can induce epigenetic changes in a sequence-specific manner through its domains or coupled domains to reduce expression from the therapeutic construct.In other embodiments, the sequence encodes at least a guide RNA and a CRISPR-dCas system coupled to a protein capable of inducing epigenetic silencing, or at least a guide RNA and a CRISPR-dCas9-KRAB, or at least a guide RNA and a CRISPR-dCas12b-KRAB (KRAB = a functional fragment of a krüppel-associated box protein of a protein analog or homology), or at least a guide RNA and a CRISPR-dCas8c-KRAB, or at least a guide RNA and a CRISPR-dCas8a-KRAB, or at least a guide RNA and a CRISPR-dCas8b-KRAB. In other embodiments, a genetic sequence capable of reducing the expression of a therapeutic construct to achieve dose-reduction titration encodes at least a transcription activator-like effector nuclease or TALEN, meganuclease, endonuclease, restriction enzyme, zinc finger protein, or other DNA-binding or RNA-binding protein.

[0059] In other embodiments, the means of reducing the titration of the therapeutic construct include within the genome, nucleus, mitochondria, or cytoplasm of the cells to which the therapeutic construct is delivered. In yet another embodiment, the method of reducing the titration is applied directly or indirectly to the cells, or to the vicinity of tissues containing the cells, or to at least some of the tissues containing the cells carrying the therapeutic construct, via external stimuli (e.g., ultrasound, heat, cold, heat flux, electromagnetic radiation, non-electromagnetic radiation, pulses, pressure, or aspiration). These stimuli may induce cell death, necrosis, apoptosis, inflammation, a decrease in intracellular transcription or translation levels, intracellular senescence, or a combination thereof.

[0060] The therapeutic constructs described herein may be delivered without a carrier, encapsulated in a carrier, partially encapsulated in a carrier, or delivered in connection 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 the following: liposomes, lipid nanoparticles, polymer nanoparticles, efflux bodies, microsomes, nanobody or efflux body-like structures, particles containing ionizable lipids, nanoparticles containing lipids and polymers, nanoparticles containing targeting ligands, nanoparticles chemically or physically conjugated to peptides, proteins, or functional sequences derived from peptides or proteins, lipid nanoemulsions, solid lipid nanoparticles, and nanostructured lipid carriers. In some embodiments, the carrier may comprise one or more of amphiphilic oligomers, phospholipids, sphingolipids, ionizable lipids, or cholesterol. In other embodiments, the carrier may be at least partially composed of a polyelectrolyte complex, cationic liposomes, stealth liposomes, cubosomes, lipid complexes, nanocells, or lipid bilayers. The carrier system or elements thereof may bind to one or more targeting ligands, structures or molecules, the targeting ligands, structures or molecules enhancing the stability of the carrier, the binding ligands, structures or molecules promoting cellular uptake, increasing the half-life of the carrier or therapeutic construct, promoting internalization, promoting endosome escape, controlling, regulating or promoting intracellular transport, or enhancing transport to the nucleus or other organelles.

[0061] Some specific examples of formulations and reagent concentration ranges that can be used to deliver genetic carriers are listed below:

[0062] Table 1.

[0063]

[0064] The degree of deacetylation of chitosan can range from about 0.5% to about 99.9%, or a mixture of multiple degrees of acetylation. The molecular weight of chitosan can range from 5,000 Da to 2.5 M Da.

[0065] Table 2.

[0066]

[0067] The molecular weight range of poly(ε-L-lysine) can be from 1,500 Da to 250,000 Da.

[0068] Table 3.

[0069]

[0070] Coatsomes can replace alternative polymers with similar structures within a comparable molar ratio range.

[0071] Table 4.

[0072]

[0073] SM-102 can replace other fatty acids or fatty esters with similar hydrophobicity and molecular weight but different saturation.

[0074] Possible variants of the assisting lipid include:

[0075] Table 5.

[0076]

[0077] Possible variants of PEGylated lipids include:

[0078] Table 6.

[0079]

[0080] Injection volume can range from 10 μL to 50 mL, depending on the disease, the animal or human receiving treatment, body weight, or other physiological factors. For a reference formulation containing 5 μg of DNA, 141.6 μg of lipid is used as the transporter in a 100 μL formulation.

[0081] Genetic constructs that can be used in formulations, as reporter genes to evaluate gene expression, or as reference sequences for regulatory sequences in combination with reporter or therapeutic genes are listed in the table below:

[0082] Table 7

[0083]

[0084] One embodiment includes a formulation comprising a modulated therapeutic gene therapy system designed for delivery to a subcutaneous space, and particularly to one or more cell types within the subcutaneous space, said therapeutic gene therapy system comprising: a genetic construct encoding at least one therapeutic gene, the genetic construct being regulated by at least one promoter element and optionally one or more regulatory elements; a delivery vector or carrier encapsulating at least a portion of said genetic construct; and means for increasing the expression level of the therapeutic transgene and / or for reducing (e.g., permanently) the expression level of the therapeutic transgene (e.g., by applying external stimulation). The delivery vector or carrier encapsulating at least a portion of said genetic construct may be low immunogenicity, minimal immunogenicity, or non-immunogenic, such that the same formulation or elements of the same formulation can be repeatedly administered to the same organism without inducing a neutralizing immune response that would reduce the efficiency of the re-administered dose by more than about 10%. In another embodiment, the delivery vector or carrier is non-immunogenic, thereby allowing the efficiency of the re-administered dose to remain substantially or substantially the same as the initially delivered dose. In another embodiment, this is independent of the time frame of subsequent dose administration. In some embodiments, the construct may carry at least one encoding or non-coding therapeutic gene for a specific purpose, which encodes 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 encoded 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 transgene encodes a gastric inhibitory peptide (GIP) or a gastric inhibitory peptide analog, a GLP-1 / GIP dual receptor agonist, a co-agonist, or an analog thereof. In another embodiment, the therapeutic gene is one or more of the following: growth factors, cytokines, anti-inflammatory proteins, pro-inflammatory proteins, complement proteins, receptor agonists or antagonists, hormones, peptides, fusion proteins consisting of one or more functional elements of different proteins, immunogens, vaccines, or subunits thereof or combinations thereof.

[0085] 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 may be used entirely by methods to increase or decrease titration, such methods being, for example, in addition to systemic or local administration of peptides, hormones, small molecules, or proteins or other chemical entities. In some embodiments, gene therapy is delivered to the subcutaneous fat pad.

[0086] In some embodiments, therapeutic gene therapy is formulated to deliver one or more of a circular single-stranded DNA construct, a circular double-stranded DNA construct, a linear single-stranded DNA construct, or a linear double-stranded DNA construct. In other embodiments, the genetic vector may typically consist of one or more of DNA, RNA, or a DNA-RNA hybrid or a chemical derivative thereof. The genetic sequence of the construct may contain at least one promoter, which may consist of one or more of the following: constitutive promoters, inducible promoters, tissue-specific promoters, or more specifically, CAG, EF1a, UBC, CBh, MSCV, hPGK, SFFV, or SV40 promoters, or combinations thereof. In other embodiments, the promoter may be a tetOn inducible promoter construct or a tetOff promoter construct, and may optionally contain one or more of an enhancer, regulator, operon, or repressor. In other embodiments, the promoter element may be an inducible promoter that can be upregulated or downregulated in response to external or internal stimuli (e.g., inflammation, heat, light, stress, administration of steroids, tetracyclines, antibiotics, rapamycin, ganciclovir, acyclovir), or may generally be induced by upregulation or downregulation of ROS, NOS, or cytokine release. In other embodiments, the promoter may be responsive to a circadian rhythm or be a cyclic promoter whose activity level changes by at least 5% over several cycles ranging from hours to months. The gene therapy construct may contain one or more post-translational regulatory elements, including a marmot hepatitis virus post-transcriptional regulatory element or WPRE, or an optimized marmot hepatitis virus post-transcriptional regulatory element or WPRE, or a cis-acting element capable of increasing cytoplasmic mRNA accumulation, or at least a portion of an HIV type 1 Rev-Rev response element, or at least a portion of a human hepatitis virus post-translational regulatory element, or at least a portion of one or more viral post-translational regulatory elements capable of increasing the expression of the target gene, or any other construct-coding element. Gene therapy constructs may contain one or more functional genetic sequences that promote at least 4% translocation of the genetic construct to the nucleus of a target cell containing the therapeutic construct. In some embodiments, the therapeutic construct may contain a protein-coding sequence having a dedicated promoter that encodes a protein that transports the therapeutic genetic construct to the nucleus, or encodes a functional genetic sequence that promotes at least 4% nuclear translocation of the genetic construct, optionally encoding a sequence-specific DNA-binding protein coupled to a nuclear localization signal peptide.Other means of translocating gene therapy constructs to the nucleus may include a DNA nuclear target sequence encoding a DNA nuclear target sequence recognized by one or more specific transcription factors, or a DNA nuclear target sequence (DTS) active in an input protein-mediated transport system, or at least a portion of an SV40 DTS, or at least a portion of a glucocorticoid response element (GRE) DTS, or at least a portion of a Sox2 regulatory region 2 DTS sequence, or at least a portion of an input protein β(1), input protein 7, NF-κβ, or guanosine triphosphatase Ran interaction sequence.

[0087] The gene therapies described herein can reduce titration through physical, chemical, or physiological stimulation. In some embodiments, titration reduction is facilitated by functional genetic sequences or elements in the gene therapy construct, such elements being induced by one or more chemical, physical, or physiological stimuli, and may be one or more of the following: 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 genetic construct; a protein-coding gene, under the regulation of an inducible promoter, encoding an epigenetically silenced protein capable of targeting at least a portion of the therapeutic genetic construct; or more specifically, one or more of the HSV-TK suicide gene system, the RapaCas9 suicide gene system; or one or more of miRNA, siRNA, shRNA, dsRNA, ncRNA, lncRNA, piwi-interacting RNA, PAT, eRNA, and circRNA. In other embodiments, the therapeutic genetic construct may encode at least a guide RNA and a CRISPR-dCas system, or a guide RNA and CRISPR-dCas9-KRAB, or a guide RNA and CRISPR-dCas12b-KRAB, or a guide RNA and CRISPR-dCas8c-KRAB, or a guide RNA and CRISPR-dCas8a-KRAB, or a guide RNA and CRISPR-dCas8b-KRAB, or TALEN, megnuclease, endonuclease, restriction enzyme, zinc finger protein, or other DNA-binding or RNA-binding protein, or combinations thereof, coupled with a protein capable of inducing epigenetic silencing. Elements regulating expression, whether used to increase or decrease titration, or for permanent or transient regulation, may include elements capable of altering the expression of one or more therapeutic transgenes in response to: temperature, or generally hot or cold, heat flux, ultrasound, focused energy, electromagnetic radiation, pulses, mechanical pressure, pressure reduction, vacuum, suction, vibration, agitation, electrical stimulation, chemical stimulation, changes in the physiological environment, changes in the level, concentration, gradient, or activity of paracrine, endocrine, or autocrine factors, changes in the level of local inflammation, or the presence or absence of inflammatory cells or molecules, or combinations thereof. In some embodiments, gene therapy may reduce titration by removing cells via liposuction, microliposuction, or microsurgery, or by applying ionizing or non-ionizing radiation. Regulatory elements may include genetic sequences that alter structure in response to internal or external stimuli, aptamers, aptazymes (e.g., K19 aptamer), protein-binding sequences (e.g., histone-binding sequences), sequences expressing ribosomes, RNA, proteins, or RNA-protein complexes, or combinations thereof.The aforementioned regulatory elements may include at least a segment of a proximal promoter, a distal promoter, an insulator, or a combination thereof, and are positioned to regulate therapeutic genes, dose-regulation mechanisms, both, or other segments of the expression system. These regulatory elements may be able to form secondary structures with themselves, other genomic sequences, or other sequences within the therapeutic construct. These secondary structures may act as means of regulating expression, stability, construct-to-nuclear transport, or combinations thereof.

[0088] In some embodiments, the genetic construct may be encapsulated in a delivery vector or carrier, which may be a carrier, capsule, membrane, capsid, structural element, or combination of one or more structural elements. The delivery vector or carrier may partially or completely encapsulate one or more genetic constructs and may be functionalized externally, internally, or on multiple surfaces. The delivery vector or carrier may at least partially comprise lipid nanoparticles, liposomes, micelles, cuboids, functionalized lipid nanoparticles, functionalized liposomes, stealth liposomes, solid lipid nanoparticles, lipid-polymer hybrid nanoparticles, transfections, endosomes, efflux bodies, cell membranes, vesicle systems, polymers, polymer nanoparticles, non-immunogenic or low-immunogenic viral vectors, proteins or peptides, or combinations thereof. One or more elements of the delivery vector or carrier 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, or enhance transport to the nucleus, or combinations thereof, or generally optimize physical, physiological, or chemical activity or stability. In some embodiments, gene therapy may be delivered intracellularly, within organelles, or within at least a portion of a cell. In other embodiments, the gene carrier may be delivered in cells or as part of a subcutaneously administered cell therapy, and has the potential for re-dosing and / or increasing or decreasing titration. The delivery vector or carrier may comprise one or more of ionizable lipids, ionizable elements, ionizable polymers, or ionizable oligomers or monomers, and may optionally comprise cationic or anionic elements, cholesterol, one or more amphiphilic polymers, polyethylene glycol or derivatives, one or more antibodies, nanobodies or antibody fragments, peptides or derivatives, DNA or derivatives, RNA or derivatives, or other chemical or physical elements or combinations thereof. Some embodiments include a delivery vector or carrier wherein at least a portion of the vector or carrier comprises nanoparticles or nanostructures chemically or physically conjugated to a peptide, protein, or a functional sequence derived from a peptide or protein. In other embodiments, the delivery carrier may be a nanoemulsion, a nanostructured lipid, or at least partially comprise an amphiphilic polymer or oligomer. In other embodiments, the delivery vector or carrier may at least partially comprise phospholipids, sphingolipids, ionizable lipids, polyelectrolyte polymers, polyelectrolyte complexes, or cholesterol. And in still other embodiments, the delivery vector or carrier may at least partially comprise metal or ceramic nanoparticles. Embodiments of any of the compositions or methods provided herein may require the use of one or more delivery vectors or carriers to deliver one or more genetic constructs with the aim of not inducing humoral or cell-mediated immune responses, not inducing memory immune responses, or inducing minimal immune responses that allow for re-dose of the therapeutic agent.At the initial dose level, the re-dose efficiency after the initial administration can be no less than 10%, and it elicits an immune response that does not lead to serious adverse reactions. Delivery vectors or carriers intended for use in the proposed gene therapy can be designed to be optimally or minimally immunogenic, such that any memory immune response generated by the vector or carrier or gene therapy cannot neutralize the formulation to reduce delivery efficiency by more than 90%, or ideally 50%, and most ideally 25% or 1%, such that the efficiency of subsequent doses is at least 10%, or ideally 50%, or most ideally 75% or 99%, or some value within the aforementioned efficiencies. Therefore, some embodiments provide delivery vectors or carriers with minimal immunogenicity, such that the memory immune response generated by the vector or carrier cannot neutralize at least 50%, at least 75%, at least 90%, or at least 99% of subsequent re-administered therapeutic formulations or substantially similar therapeutic formulations and / or via substantially the same route of administration. In some embodiments, the therapeutic gene therapy construct may use external stimuli to reduce titration, said external stimuli optionally inducing one or more of the following: cooling of the skin and subcutaneous tissue to cause adipocyte death, cooling of the skin and subcutaneous tissue to cause adipocyte senescence, a temperature change in the subcutaneous tissue sufficient to alter the expression of the therapeutic transgene, or a change in the physical environment of the adipocytes such that the change induces a reduction in gene expression from the therapeutic transgene. The one or more external stimuli may at least partially comprise high-frequency ultrasound, mid-frequency ultrasound, low-frequency ultrasound, or sound waves capable of inhibiting expression in adipocytes, inducing senescence in adipocytes, or inducing adipocyte death in at least a portion of the subcutaneous space; or electromagnetic waves capable of inhibiting expression in adipocytes, inducing senescence in adipocytes, or inducing adipocyte death in at least a portion of the subcutaneous space; or electrical signals capable of inhibiting expression in adipocytes, inducing senescence in adipocytes, or inducing adipocyte death in at least a portion of the subcutaneous space. The stimulus may be combined with or be chemically-based, including, for example, administration of a formulation containing one or more of the following: rapamycin or a derivative thereof, tetracycline or a derivative thereof, a combination of rapamycin and tetracycline or a derivative thereof, ganciclovir or a derivative thereof, doxycycline or a derivative thereof, or generally one or more pharmacologically active small molecules. Alternatively, the titration may be applied indirectly by delivering a formulation containing one or more peptides, with or without conjugation, or one or more hormones or analogues thereof, or more specifically, a formulation containing tamoxifen or a derivative thereof, which may then act directly on DNA or proteins expressed from the therapeutic formulation, or on receptors on cells containing genetic constructs, or by other secondary means.Alternative or combined external stimuli can be used to reduce titration by administering one or more agents that elicit an inflammatory response, formulations containing one or more types of steroids, formulations containing one or more functionalized or unfunctionalized antibodies, formulations containing one or more receptor ligands, formulations containing one or more antibody fragments, formulations containing one or more proteins, formulations containing one or more fusion proteins, formulations containing one or more RNA molecules, formulations containing one or more lipids, formulations containing one or more metabolites, administering agents that elicit an anti-inflammatory or immunomodulatory response, administering formulations containing one or more neurotransmitters, or combinations thereof. In some embodiments, the external stimulus can be achieved by administering a formulation containing one or more proteins that can interact directly or indirectly with a promoter, RNA, or DNA sequence, or by administering a formulation containing one or more proteins that can interact directly or indirectly with a promoter, RNA, or DNA sequence. In other embodiments, the external stimulus may be achieved by administering a formulation containing one or more RNA or DNA molecules that can interact directly or indirectly with a promoter, RNA, or DNA sequence, or by administering a formulation containing one or more RNA or DNA molecules that can interact directly or indirectly with a promoter, RNA, or DNA sequence.

[0089] The gene therapy constructs disclosed herein may have the ability to increase the dose or titrate the treatment after initial administration. This titration may be permanent or transient and may originate from an increase in transgene expression relative to the original dose, or from an additional dose of gene therapy that provides an absolute or relative increase relative to the original dose. In some embodiments, the dose increase or titration is achieved by administering a second dose of adjustable gene therapy, which is at least 1% of the original dose; or by administering a second dose of adjustable gene therapy, which is at least 2% of the original dose; or by administering a second dose, which is at least 5%, 10%, 15%, or 25% of the original dose. In other embodiments, the increase in therapeutic transgene expression is achieved by administering one or more additional doses of the gene therapy system. In still other embodiments, the means of increasing therapeutic transgene expression or titration may be by administering a formulation containing at least DNA, RNA, protein, or a combination thereof capable of temporarily or permanently increasing therapeutic transgene expression. Subsequent administration of therapeutic gene therapy may be performed partially or entirely to achieve titration enhancement; therefore, the means of enhancing transgene expression may consist of administering at least 10% of at least one element of any of the gene therapy systems provided herein. In some embodiments, titration or enhancement of therapeutic transgene expression may be achieved by administering small molecules capable of permanently or temporarily enhancing 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 enhance the expression of therapeutic transgenes or reduce the expression of elements capable of inhibiting therapeutic transgene expression.

[0090] Increased or decreased gene expression from the therapeutic construct can be temporary or permanent, and in the case of decrease, it can include less than 25%, 40%, 60%, 80%, or 90% of the total expression of the applied therapeutic agent, and in the case of increase, it can exceed 10%, 20%, 50%, or 100% of the total expression. In some embodiments, a permanent decrease can be complete or near-complete, exceeding 90% of the total expression of the applied therapeutic gene therapy, and in other cases, it can be regional, local, or affect only a portion of the delivered gene separated by construct type, responsive element, anatomical region, expressed transgene, or any other element distinguishing the portion.

[0091] Another optional element is the use of biomarkers to identify the location of treatment. Biomarkers are generally categorized as typically surface-based (defined as observable from a surface with minimal or no intervention) and / or internal (defined as observable from a surface after intervention). Biomarkers can be used to identify one or more of the following: type, location, dosage, formulation, date of administration, or any other relevant information about or related to the treatment. Biomarkers may have many functions, but are generally used to locate treatment for subsequent examination, improve titration site selection, reduce titration for initial or other treatments, select injection sites for drug therapy, administer inducers or stimulants to influence or prevent changes in the chemical, physical, or physiological environment affecting treatment. Biomarkers can also be used to store, preserve, transmit, or interact with information that is related to treatment in some way.

[0092] The markers (e.g., surface markers) may consist of one or more of skin tattoos, skin markings, skin tags, or other means of marking the skin with information, and can range from as simple as a dot to as complex as a barcode, 2D data matrix, or QR code. The markings may be permanent or semi-permanent, lasting for days, weeks, months, or years. These markings may be directly visible in sunlight or in ultraviolet light or any other spectral range, may be fluorescent, and require initial excitation before visualization, or may be visualized by applying energy from the electromagnetic spectrum, ultrasound, or mechanical energy (e.g., palpation) to the initial surface.

[0093] The marker (e.g., an internal marker) may be delivered with the formulation, encoded into a genetic construct within the formulation, or administered as part of the formulation, or may be applied after injecting the formulation. The aforementioned marker may be composed of one or more of tantalum, gold, platinum, barium sulfate, or other metals or metal salts, having or not having a carrier at least partially composed of silicone, polyvinyl alcohol, or other biostable or biodegradable polymers. In other embodiments, the marker may be at least partially composed of ceramics or composite materials. The marker may be passive, such as a radiopaque material, or actively emitting signals, or existing in an off or on state that can switch to the opposite or another state after stimulation is applied. The marker may be radiopaque, opaque or translucent to ultrasound or other detection or measurement techniques commonly used for visualization of the body or body structures. The marker may be atomic, molecular, or nanostructured and may be contained as part of the formulation. In another embodiment, the marker may be encoded into a genetic construct delivered as part of the formulation. For example, the biomarker may encode a fluorescent protein expressed by cells along with a therapeutic transgene, and said expression may be permanent, periodic, or activated by an external stimulus, such as heat, cold, radiation, light, application of energy, or systemic or generalized application to a treatment-associated area of ​​one or more molecular compounds, such as tetracycline or derivatives, rapamycin or derivatives, ganciclovir or derivatives, steroids or derivatives, tamoxifen or derivatives, or other therapeutic agents or molecular compounds or formulations, or combinations thereof. In another embodiment, the biomarker may be a bioluminescent protein, such as nanoluciferase or firefly luciferase, or any other bioluminescent protein that may constitutively luminescent in vivo or luminescent after application of a substrate, such as furimazine or furimazine derivatives, or any other small molecule, metal, polymer, or ceramic substrate for encoding the biomarker enzyme. The reporter gene may be constitutively expressed or expressed after the application of physical or chemical stimuli (e.g., heat or tetracycline, or typically energy in the electromagnetic spectrum, ultrasound, heat, cold, or any pharmacologically or chemically active substance).

[0094] The gene therapy is intended to treat diseases in humans or veterinarians. In some embodiments, the gene therapy may be classified as a treatment, and in other embodiments, it may be classified as enhancing or improving the health, physical condition, mental state, or physical or mental capabilities of a human or animal. In still other embodiments, gene therapy may be used to increase or alter the lifespan, healthy lifespan, or lifespan or healthy lifespan of a specific tissue, organ, cell, or combination thereof in a human or animal.

[0095] In some implementations, the target cell type is adipocytes, preadipocytes, progenitor cells capable of differentiating into adipocytes, stem cells capable of differentiating into adipocytes, resident cell types in subcutaneous tissue, or one or more of transient cells or stem cells that can become resident cells in subcutaneous tissue. Gene therapy is intended to treat or prevent any human or veterinary condition, but in some specific implementations it can be used to treat monogenic conditions, enzyme deficiencies, protein deficiencies (as a protein replacement therapy), metabolic disorders, autoimmune diseases, tumors, neurological disorders, cardiovascular pathologies, musculoskeletal disorders, hematological disorders, infectious diseases, skin diseases, immune system disorders, pulmonary diseases, kidney or bladder diseases, or diseases of other organs, tissues, systems, or combinations thereof. In some implementations, therapeutic gene therapy systems are designed to deliver therapeutic or preventative antibodies, therapeutic, preventative, prophylactic or fusion proteins, peptides, immunogens, antigens, enzymes, ribozymes, mRNA, non-coding RNA, miRNA, shRNA or other RNA, or molecules that can serve as vaccines or excipients by expression from a genetic construct.

[0096] The disclosed gene therapy treatment can be delivered in vials or pre-filled syringes or cartridges for subsequent application. Alternatively, the treatment can be administered via an autoinjector or patch injector, ultrasound-guided, or other guided techniques to ensure delivery to the correct location. Alternatively, and optionally, the treatment can be delivered via a set of needles or via a set or series of injections to distribute the construct more uniformly at the treatment site. The gene therapy treatment can be delivered via patches, microneedles, a set of microneedles, jet injection, on a carrier, or using other means to alter the integrity of the tissue through which it is delivered or into.

[0097] It should be understood that, within the scope of the compositions or methods provided herein, those skilled in the art may modify the formulations, materials, genetic constructs, sequences, biological and chemical components, biomarkers, or methods of use to the extent that the structures described herein perform the desired function and remain within the scope of the compositions or methods provided herein. Those skilled in the art may combine multiple components, elements, or features, with or without modification, to achieve the desired function of the aforementioned formulations.

[0098] Furthermore, all individual features and methods of use described herein, as well as various combinations and each combination of two or more such features and methods of use, are included within the scope of the compositions or methods provided herein, provided that such features and methods of use are not contradictory in such combinations. It should be understood that a person trained in the art can modify certain parts or combinations of such parts while still achieving the objectives of the compositions or methods provided herein.

[0099] Finally, it should be understood that the specific scope provided in this article is not restrictive and is for illustrative purposes only.

[0100] Example

[0101] Example 1

[0102] This embodiment outlines a minimal plasmid construct for delivery in lipid nanoparticle formulations, comprising an inducible promoter, a post-translational regulatory element, a target gene (GLP-1 receptor agonist), a nuclear targeting element, and a genetic regulatory element that enables the reduction of target gene expression after administration of ganciclovir.

[0103] Genetic construct map (excluding plasmid backbone):

[0104] Full-length construct sequence (excluding plasmid backbone):

[0105]

[0106]

[0107]

[0108]

[0109] AAV2 ITR:

[0110]

[0111] 3NF1:

[0112]

[0113] 3NF2:

[0114]

[0115] TRE promoter:

[0116]

[0117] Kozak sequence:

[0118]

[0119] GLP-1 receptor agonist (RA) nucleotide sequence:

[0120]

[0121] GLP-1 RA amino acid sequence:

[0122]

[0123] SV40 late pA:

[0124]

[0125]

[0126] CBh promoter:

[0127]

[0128] tTS nucleotide sequence:

[0129]

[0130] tTS amino acid sequence:

[0131]

[0132] T2A linker nucleotide sequence:

[0133]

[0134] T2A linker amino acid sequence:

[0135]

[0136] T2A nucleotide sequence:

[0137]

[0138] T2A amino acid sequence:

[0139]

[0140] rtTA nucleotide sequence:

[0141]

[0142] rtTA amino acid sequence:

[0143]

[0144] BGH pA:

[0145]

[0146] EF1a promoter:

[0147]

[0148]

[0149] HSV-TK sequence:

[0150]

[0151] HSV-TK amino acid sequence

[0152]

[0153] insulator:

[0154]

[0155] K19 aptamer enzyme:

[0156]

[0157] SV40 pA:

[0158]

[0159] Example 2

[0160] This embodiment outlines a hairpin DNA construct delivered within a polymeric lipid complex, comprising a constitutive promoter, a post-translational regulatory element, a target gene (furin-cleavable insulin), a nuclear targeting element, and a genetic regulatory element that enables the reduction of target gene expression following administration of a steroid and a second inducer.

[0161] Genetic construct map:

[0162] Full-length construct sequence (excluding plasmid backbone):

[0163]

[0164]

[0165]

[0166]

[0167] SV40 DTS:

[0168]

[0169] CAG promoter (CAGp):

[0170]

[0171] Frin protease-cleavable human insulin (FC-hINS):

[0172]

[0173] FC-hINS amino acid sequence:

[0174]

[0175] Optimized WPRE (oPRE):

[0176]

[0177] CMV promoter (CMVp):

[0178]

[0179] Synthetic intron IVS 8:

[0180]

[0181] SWITCH nucleic acid sequence:

[0182]

[0183]

[0184] SWITCH amino acid sequence:

[0185]

[0186] hGH pA:

[0187]

[0188] GAL4 UAS:

[0189]

[0190] Minimal Ad promoter (ADp):

[0191]

[0192] Example 3

[0193] This embodiment outlines a DNA microcircle construct delivered in liposomes that includes an inflammatory response promoter, a post-translational regulatory element, a target gene (interleukin-1 receptor antagonist), a nuclear targeting element, and a genetic regulatory element that enables the reduction of target gene expression after administration of rapamycin.

[0194] Genetic construct map (excluding plasmid backbone):

[0195] Full-length construct sequence (excluding plasmid backbone):

[0196]

[0197]

[0198]

[0199] 5xNFkB DTS:

[0200]

[0201] NFkB Response Element (RE):

[0202]

[0203] Minimum promoter (minP):

[0204]

[0205] MVM introns:

[0206]

[0207] Interleukin-1 receptor antagonist (IL-1 RA) sequence:

[0208]

[0209] The amino acid sequence of the interleukin-1 receptor antagonist (IL-1 RA):

[0210]

[0211] rapaCas9 sequence:

[0212]

[0213] rapaCas9 amino acid sequence:

[0214]

[0215] Example 4

[0216] This embodiment outlines a double-stranded DNA construct delivered within ligand-targeted polymer particles, comprising a constitutive promoter, a post-translational regulatory element, a target gene (hBDNF), a nuclear-targeting element, and a genetic regulatory element that enables the reduction of target gene expression following doxycycline administration.

[0217] Genetic construct map:

[0218] Full-length construct sequence (excluding plasmid backbone):

[0219]

[0220]

[0221]

[0222]

[0223] WPRE:

[0224]

[0225] Mature BDNF (mBDNF) sequence:

[0226]

[0227] mBDNF amino acid sequence:

[0228]

[0229] GRE DTS:

[0230]

[0231] Example 5

[0232] By combining the following lipid components / ml mixture, a therapeutic gene therapy formulation containing candidate genes intended for use as replacements for human proteins is produced.

[0233] Table 8.

[0234]

[0235] The formulation was combined with the genetic construct at an N / P ratio of 4 to 10 using impingement jet mixing, and the encapsulation efficiency was evaluated.

[0236] Table 9.

[0237]

[0238] Table 10.

[0239]

[0240] The biophysical properties of LNP particles were characterized using a dynamic light scattering instrument, as shown below.

[0241] Table 11.

[0242]

[0243] Will Figures 1A to 1B The genetic constructs shown are encapsulated in a formulation with an N / P ratio of 4 to 10. This formulation was then used to treat primary human adipocytes and preadipocytes at doses of 50 to 500 ng / well. Both constructs contain a suicide gene de-titering genetic element consisting of the rapaCasp9 gene fused to a K19 aptamer enzyme, which requires the presence of two regulators (tetracycline and rapamycin) for activation.

[0244] The firefly luciferase reporter gene, expressed from primary human adipocytes transduced with LNP102-npRB2.6v7, was used to quantify the effect of a single regulator, rapamycin, and the response of a rapamycin-induced suicide gene de-titering element, while the effective dose of another regulator, tetracycline, was fixed at 0.1 mM. Specifically, a simulated negative control was used to assess the absence of luminescence (cells without fLuc expression). A 0 nM rapamycin positive control was used to assess baseline fLuc expression without de-titering. Increasing the rapamycin concentration from 0 nM to 0.1 nM resulted in a significant dose-dependent decrease in transgene expression, as observed using quantitative bioluminescence analysis. Thus, within 76 hours of inducer activation, the induction of the de-titering suicide gene element reduced reporter gene expression in a dose-dependent manner. Figure 2 ).

[0245] In parallel, the titratable / downregulatory functionality of the construct in primary human adipocyte cultures transduced with LNP102-npRB2.6v3 was measured using an annexin NanoBiT 500x bioluminescence assay. Cultured cells were administered 0.01 to 1 nM rapamycin, with a second modulator, tetracycline, added at concentrations of 0 mM, 0.05 mM, or 0.1 mM. Although no apoptosis induction was observed in the absence of tetracycline indicating the functionality of the aptamer enzyme, significant dose-dependent apoptosis was observed after tetracycline administration at concentrations of 0.05 and 0.1 mM for all rapamycin concentrations. Following apoptosis, expression of the therapeutic transgene was effectively eliminated, enabling down-titering of gene therapy. Figure 3 ).

[0246] Example 6

[0247] Lipid-based carrier formulations were prepared and mixed at an N / P ratio of 7 for subsequent administration to C57BL / 6 mice or C6 albino background mice. A total of 2 to 10 μg of the formulation was injected into mice as either an “initial dose” or a “reinforced titration dose”, administered 2 to 4 weeks after the initial dose to allow for the formation of any potential immune responses. The administered construct sizes varied between 6203 and 3144 bp and included… Figure 1B and Figure 4 The constructor shown in the image.

[0248] Following subcutaneous injection of the initial dose, in vivo bioluminescence imaging (IVIS) was used to monitor serious adverse events, general safety, and durability of the signal (reporter transgene expression) in animals. IVIS confirmed the durability of the initial signal (transgene expression) to day 22. Figure 5A and Figure 5B The ability to titrate the initial dose in a predictable dose-dependent manner not only allows for re-dosing but also allows for controlled titrability increases in the initial treatment, such as... Figure 5A and Figure 5C As shown in the image.

[0249] Subsequently, combined treatments were performed to evaluate the long-term durability of reported transgene expression. A total of six IVIS measurements were performed over a 90-day period. Signals localized to the original injection site were clearly observed at all times and were statistically above background luminescence levels, indicating high stability and excellent long-term durability potential of gene therapy. Figures 6 to 7 ).

[0250] This example illustrates the minimum observable reduction in titration, ranging from about 2% to about 15%, and can be increased subsequently after the application of another stimulus.

[0251] Example 7

[0252] In this embodiment, 3 μg of LNP formulation N / P7 was prepared into 4 mL batches of 144 μL of 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid, 1-octylnonyl ester, 128 μL of 1,2-distearate phosphatidylcholine, 1184 μL of cholesterol, 1512 μL of 1,2-dimyristoyl-racemic-glycero-3-methoxy-polyethylene glycol (2,000), and 1032 μL of ethanol, which contained a 5496 nt genetic construct encoding the firefly luciferase reporter gene (regulated by the CBh promoter (hybrid cytomegalovirus enhancer / chicken β-actin)) and was subcutaneously delivered to C57BL / 6 albino mice. After 3 months of imaging to ensure signal stability, signal reduction titration was performed on animals via cryolipolysis using a 1.5 cm² diameter probe delivered a heat flux of 3 W / cm² at 0°C (previously confirmed to be able to cool subcutaneous tissue to approximately 6°C). Figure 8 After probe and skin equilibration, a series of three bi-daily treatments are applied, with a total cooling time of 10 minutes. Glycerin is applied to the probe tip to prevent crystal formation and potential damage to the dermis. Adipocyte reduction can be extrapolated to changes in therapeutic gene expression following treatment. If desired, additional treatments can be applied sequentially to further reduce subcutaneous adipocyte count, thereby completely shutting down gene expression.

[0253] The reporter gene can be replaced by any treatment-related transgene that has been identified, or is undergoing, a clinical evaluation assessing therapeutic efficacy by subcutaneous delivery as a protein or mRNA. Dosage, formulation, concentration, number of injections, promoter, or other regulatory sequences can be adjusted to optimize the transgene's therapeutic efficacy. If the treatment is administered to, for example, companion animals, humans, or horses, flux, probe area, time, cooling / heating profiles, and cooling aids can be adjusted to achieve cryolipolysis in non-rodent tissues.

[0254] Example 8

[0255] This example describes a method for reducing titration using the Lox-Cre system:

[0256] Genetic construct sequence (excluding plasmid backbone):

[0257]

[0258]

[0259]

[0260] 3NF1 DTS:

[0261]

[0262] 3NF2 DTS:

[0263]

[0264] EFS Starter:

[0265]

[0266] loxP site:

[0267]

[0268] NLuc encoded sequence:

[0269]

[0270] NLuc amino acid sequence:

[0271]

[0272] BGH poly A signal:

[0273]

[0274] Cre-ERT2 encoded sequence:

[0275]

[0276] Cre-ERT2 amino acid sequence:

[0277]

[0278] insulator:

[0279]

[0280] K19 aptamer enzyme:

[0281]

[0282] SV40 pA:

[0283]

[0284] Example 9

[0285] This embodiment describes a therapeutic gene therapy that reduces titration by applying a cooling device, ultrasound, or electromagnetic radiation, which can affect gene expression or viability in adipocytes or preadipocytes expressing the gene therapy.

[0286] Genetic construct sequence (excluding plasmid backbone):

[0287]

[0288]

[0289] 3NF1 DTS:

[0290]

[0291] 3NF2 DTS:

[0292]

[0293] CBh promoter:

[0294]

[0295]

[0296] NLuc encoded sequence:

[0297]

[0298] NLuc amino acid sequence:

[0299]

[0300] BGH poly A signal:

[0301]

[0302] Example 10

[0303] This embodiment describes a method for titrating or downregulating gene expression in a self-therapeutic construct by embedding a targeted epigenetic silencer, the expression and / or activation of which can be induced by the application of one or more exogenous stimuli.

[0304] Specifically, human or other target species (for example, mice) DNA methyltransferases (or other epigenetic silencers) having the following exemplary sequences.

[0305] Human DNMT3A (CD):

[0306]

[0307]

[0308] or

[0309] Mouse DNMT3L (CD):

[0310]

[0311] Coupled with targeting elements, such as zinc finger motifs or proteins having the following exemplary sequences.

[0312] ZN627_Hu:

[0313]

[0314] It can optionally be used with NLS sequences, for example: and flexible spacer regions or connector sequences, for example Combined to produce the following fusion proteins:

[0315]

[0316]

[0317] It is designed to target the following nucleotide sequences:

[0318] Target site (34th position downstream of TSS): As part of the following sequence of constructs:

[0319] ZF target area (total 400 nt, 200 nt upstream and 200 nt downstream of TSS):

[0320]

[0321] ZF:

[0322]

[0323] It can be expressed by non-codon-optimized or codon-optimized sequences, for example:

[0324]

[0325]

[0326] Or alternative examples of DNMT3A-3L-ZF-KRAB fusion:

[0327]

[0328] DNMT3A:

[0329]

[0330] DNMT3L:

[0331]

[0332]

[0333] connector SV40 NLS ZF

[0334]

[0335] for Combination sequences:

[0336]

[0337] .

Claims

1. A modifiable therapeutic gene therapy system for delivery to any of the tissues or cells provided herein, such as delivery to a subcutaneous space (e.g., delivery to one or more cell types in a subcutaneous space), the system comprising: a. A genetic construct encoding at least one therapeutic gene, said therapeutic gene being regulated by at least one promoter element and optionally one or more regulatory elements. b. A delivery vector or carrier encapsulating at least a portion of the genetic construct, optionally allowing for the reapplication of subsequent doses of the genetic construct (e.g., with at least 10% efficiency compared to the initial dose of the genetic construct), and c. Means for increasing or decreasing the expression level of therapeutic transgenes from said genetic construct.

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

3. The adjustable therapeutic gene therapy system of claim 1 or 2, wherein the means of reducing the therapeutic transgene expression level is by applying an external stimulus.

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

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

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

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

8. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the genetic construct encodes insulin or an insulin analogue.

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

10. The adjustable therapeutic gene therapy system of any one of claims 1 to 7, wherein the genetic construct encodes GLP-1, a GLP-1 agonist, or a GLP-1 agonist analogue.

11. The adjustable therapeutic gene therapy system of any one of claims 1 to 7, wherein the genetic construct encodes a growth factor.

12. The adjustable therapeutic gene therapy system of any one of claims 1 to 7, wherein the genetic construct encodes a cytokine.

13. The adjustable therapeutic gene therapy system of any one of claims 1 to 7, wherein the genetic construct encodes an anti-inflammatory protein.

14. The adjustable therapeutic gene therapy system of any one of claims 1 to 7, wherein the genetic construct encodes a complement protein.

15. The adjustable therapeutic gene therapy system of any one of claims 1 to 7, wherein the genetic construct encodes a receptor agonist.

16. The adjustable therapeutic gene therapy system of any one of claims 1 to 7, wherein the genetic construct encodes a receptor antagonist.

17. The adjustable therapeutic gene therapy system of any one of claims 1 to 7, wherein the genetic construct encodes a fusion protein (e.g., composed of one or more functional elements of different proteins).

18. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the subcutaneous space is the subcutaneous layer.

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

20. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the subcutaneous space is a layer between the dermis and the superficial fascia.

21. The adjustable therapeutic gene therapy system according to any of the preceding claims, wherein the genetic construct is composed of one or more circular single-stranded DNA constructs.

22. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the genetic construct comprises one or more circular double-stranded DNA constructs.

23. The adjustable therapeutic gene therapy system according to any of the preceding claims, wherein the genetic construct is composed of one or more linear single-stranded DNA constructs.

24. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the genetic construct is composed of one or more linear double-stranded DNA constructs.

25. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the genetic construct is at least partially composed of DNA or RNA.

26. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the genetic construct is composed of DNA and / or RNA.

27. The adjustable therapeutic gene therapy system according to any one of the preceding claims, wherein the external stimulus is composed of a chemical DNA derivative, an RNA derivative, or a combination of DNA and RNA derivatives.

28. The adjustable therapeutic gene therapy system of any one of claims 1 to 27, wherein the promoter element comprises a constitutive promoter.

29. The adjustable therapeutic gene therapy system of any one of claims 1 to 27, wherein the promoter element comprises an inducible promoter.

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

31. The adjustable therapeutic gene therapy system of 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. The adjustable therapeutic gene therapy system of any one of claims 1 to 27, wherein the promoter element comprises a tetOn inducible promoter construct.

33. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the promoter element comprises at least one promoter sequence having one or more of an enhancer, regulator, operator and / or repressor.

34. The adjustable therapeutic gene therapy system of any one of claims 1 to 27, wherein the promoter element comprises an inducible promoter that can be upregulated or downregulated in response to external or internal stimuli, such as inflammation, heat, light, stress, administration of steroids, tetracyclines, antibiotics, rapamycin, ganciclovir, acyclovir, or can be induced by upregulated or downregulated molecules (e.g., ROS, NOS, or cytokine release).

35. The adjustable therapeutic gene therapy system of any one of claims 1 to 27, wherein the promoter element comprises a circadian rhythm or cyclic promoter (e.g., which changes its activity level by at least 5% at some cycle from several hours to several months).

36. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the regulatory element comprises a post-translational regulatory element.

37. The adjustable therapeutic gene therapy system of claim 36, wherein the regulatory element comprises at least a portion or all of a marmot hepatitis virus posttranscriptional regulatory element or WPRE.

38. The adjustable therapeutic gene therapy system of claim 36, wherein the regulatory element comprises at least a portion or all of an optimized prairie hepatitis virus posttranscriptional regulatory element or WPRE.

39. The adjustable therapeutic gene therapy system of claim 36, wherein the regulatory element comprises a cis-acting element capable of increasing cytoplasmic mRNA accumulation.

40. The adjustable therapeutic gene therapy system of claim 36, wherein the regulatory element comprises at least a portion or all of an HIV type 1 Rev-Rev responsive element.

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

42. The adjustable therapeutic gene therapy system of claim 36, wherein the regulatory element comprises at least some or all of one or more viral post-translational regulatory elements capable of enhancing the expression of the target gene or any other element encoded by the genetic construct.

43. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the genetic construct comprises one or more functional genetic sequences.

44. The adjustable therapeutic gene therapy system of claim 43, wherein one or more functional genetic sequences promote translocation of the genetic construct (e.g., at least 4% of the genetic construct) to the target cell nucleus.

45. The adjustable therapeutic gene therapy system of claim 43, wherein the one or more functional genetic sequences comprise sequences encoding sequence-specific DNA-binding proteins coupled to nuclear localization signal peptides.

46. ​​The adjustable therapeutic gene therapy system of claim 43, wherein the one or more functional genetic sequences comprise a DNA nuclear target sequence recognized by one or more specific transcription factors.

47. The adjustable therapeutic gene therapy system of claim 43, wherein the one or more functional genetic sequences are contained in an active DNA nuclear target sequence (DTS) in an input protein-mediated transport system.

48. The adjustable therapeutic gene therapy system of claim 43, wherein the one or more functional genetic sequences comprise at least a portion or all of SV40 DTS.

49. The adjustable therapeutic gene therapy system of claim 43, wherein the one or more functional genetic sequences comprise at least a portion or all of the glucocorticoid response element (GRE) DTS.

50. The adjustable therapeutic gene therapy system of claim 43, wherein the one or more functional genetic sequences comprise the Sox2 regulatory region 2 DTS sequence.

51. The adjustable therapeutic gene therapy system of claim 43, wherein one or more functional genetic sequences comprise an input protein β(1), an input protein 7, NF-κβ, or a guanosine triphosphatase Ran interaction sequence.

52. The adjustable therapeutic gene therapy system of claim 43, wherein one or more functional genetic sequences (e.g., reducing expression from the genetic construct) comprise an induced suicide gene.

53. The adjustable therapeutic gene therapy system of claim 43, wherein one or more functional genetic sequences (e.g., reducing expression from the genetic construct) comprise an inducible expression system encoding an RNA molecule that reduces the expression of at least a portion of the therapeutic genetic construct.

54. The adjustable therapeutic gene therapy system of claim 43, wherein one or more functional genetic sequences (e.g., reduced expression from the genetic construct) encode proteins capable of targeted epigenetic silencing.

55. The adjustable therapeutic gene therapy system of claim 43, wherein one or more functional genetic sequences (e.g., reducing expression from the genetic construct) encode the HSV-TK suicide gene or system.

56. The adjustable therapeutic gene therapy system of claim 43, wherein one or more functional genetic sequences (e.g., reducing expression from the genetic construct) encode the RapaCas9 suicide gene or system.

57. The adjustable therapeutic gene therapy system of claim 43, wherein the one or more functional genetic sequences (e.g., reducing expression from the genetic construct) encode one or more of miRNA, siRNA, shRNA, dsRNA, ncRNA, lncRNA, piwi-interacting RNA, PAT, eRNA, and / or circRNA.

58. The adjustable therapeutic gene therapy system of claim 43, wherein one or more functional genetic sequences (e.g., reducing expression from said genetic construct) encode at least a guide RNA and a CRISPR-dCas system (e.g., coupled with a protein that induces epigenetic silencing).

59. The adjustable therapeutic gene therapy system of claim 43 or 58, wherein one or more functional genetic sequences (e.g., reducing expression from said genetic construct) encode at least guide RNA and CRISPR-dCas9-KRAB.

60. The adjustable therapeutic gene therapy system of claim 43 or 58, wherein one or more functional genetic sequences (e.g., reducing expression from said genetic construct) encode at least guide RNA and CRISPR-dCas12b-KRAB.

61. The adjustable therapeutic gene therapy system of claim 43 or 58, wherein one or more functional genetic sequences (e.g., reducing expression from said genetic construct) encode at least guide RNA and CRISPR-dCas8c-KRAB.

62. The adjustable therapeutic gene therapy system of claim 43 or 58, wherein one or more functional genetic sequences (e.g., reducing expression from said genetic construct) encode at least guide RNA and CRISPR-dCas8a-KRAB.

63. The adjustable therapeutic gene therapy system of claim 43 or 58, wherein one or more functional genetic sequences (e.g., reducing expression from said genetic construct) encode at least guide RNA and CRISPR-dCas8b-KRAB.

64. The adjustable therapeutic gene therapy system of claim 43, wherein one or more functional genetic sequences (e.g., reducing expression from the genetic construct) encode at least TALEN, meganuclease, endonuclease, restriction enzyme, zinc finger protein, or other DNA-binding or RNA-binding protein.

65. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the regulatory element alters the expression of the genetic construct in response to temperature.

66. The adjustable therapeutic gene therapy system of any one of claims 1 to 64, wherein the regulatory element alters the expression of the genetic construct in response to heat.

67. The adjustable therapeutic gene therapy system of any one of claims 1 to 64, wherein the regulatory element alters the expression of the genetic construct in response to cold.

68. The adjustable therapeutic gene therapy system of any one of claims 1 to 64, wherein the regulatory element alters the expression of the genetic construct in response to ultrasound.

69. The adjustable therapeutic gene therapy system of any one of claims 1 to 64, wherein the regulatory element alters the expression of the genetic construct in response to electrical stimulation.

70. The adjustable therapeutic gene therapy system of any one of claims 1 to 64, wherein the regulatory element alters the expression of the genetic construct in response to a chemical stimulus.

71. The adjustable therapeutic gene therapy system of any one of claims 1 to 64, wherein the regulatory element alters the expression of the genetic construct in response to changes in the physiological environment.

72. The adjustable therapeutic gene therapy system of any one of claims 1 to 64, wherein the regulatory element alters the expression of the genetic construct in response to paracrine, endocrine, or autocrine factors.

73. The adjustable therapeutic gene therapy system of any one of claims 1 to 64, wherein the regulatory element alters the expression of the genetic construct in response to the presence of inflammation.

74. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the regulatory element comprises a proximal promoter.

75. The adjustable therapeutic gene therapy system of any one of claims 1 to 73, wherein the regulatory element comprises a distal promoter.

76. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the regulatory element comprises an insulator.

77. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the regulatory element forms a secondary structure with the genetic construct or other genetic sequence.

78. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the delivery vector or carrier comprises lipid nanoparticles (e.g., functionalized lipid nanoparticles, solid lipid nanoparticles, lipid-polymer hybrid nanoparticles).

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

80. The adjustable therapeutic gene therapy system of any one of claims 1 to 78, wherein the delivery vector or carrier comprises one or more types of cells (e.g., functional cells), cell components, or cell membranes.

81. The adjustable therapeutic gene therapy system of any one of claims 1 to 78, wherein the delivery vector or vector comprises a cubosome, transfection body, endosome, efflux body, or vesicle system.

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

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

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

85. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the delivery vector or vector is functionalized to reduce immunogenicity.

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

87. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the delivery vector or vector is functionalized to promote endocytosis, pinocytosis or enhance transport to the cell nucleus.

88. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the delivery vector or carrier comprises ionizable lipids.

89. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the delivery vector or carrier comprises an ionizable element.

90. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the delivery vector or carrier comprises an ionizable polymer.

91. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the delivery vector or carrier comprises cholesterol.

92. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the delivery vector or carrier comprises a cationic component.

93. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the delivery vector or vector comprises an amphiphilic polymer.

94. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the delivery vector or carrier comprises polyethylene glycol.

95. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the delivery vector or carrier comprises an antibody, nanobody, or antibody fragment.

96. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the delivery vector or vector comprises a peptide.

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

98. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the delivery vector or carrier comprises a nanoemulsion, a nanostructured lipid, or is at least partially composed of an amphiphilic polymer or oligomer.

99. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the delivery vector or carrier comprises phospholipids, sphingolipids, polyelectrolyte polymers, or polyelectrolyte complexes.

100. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the delivery vector or carrier comprises metal or ceramic nanoparticles.

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

102. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the delivery vector or vector does not elicit a memory immune response.

103. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the delivery vector or carrier is minimally immunogenic (e.g., in test subjects, such that the memory immune response generated by the vector or carrier cannot neutralize at least 10% of the vector or carrier subsequently administered via the same route of administration).

104. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the delivery vector or carrier is minimally immunogenic (e.g., in test subjects, such that the memory immune response generated by the vector or carrier cannot neutralize at least 50% of the vector or carrier subsequently administered via the same route of administration).

105. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the delivery vector or carrier is minimally immunogenic (e.g., in test subjects, such that the memory immune response generated by the vector or carrier cannot neutralize at least 75% of the vector or carrier subsequently administered via the same route of administration).

106. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the delivery vector or carrier is minimally immunogenic (e.g., in test subjects, such that the memory immune response generated by the vector or carrier cannot neutralize at least 90% of any subsequent administration of the vector or carrier via the same route of administration).

107. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein the delivery vector or carrier is minimally immunogenic (e.g., in test subjects, such that the memory immune response generated by the vector or carrier cannot neutralize at least 99% of any subsequent administration of the vector or carrier via the same route of administration).

108. The adjustable therapeutic gene therapy system according to any one of the preceding claims, wherein external stimulation causes cooling of the skin and subcutaneous tissue (e.g., to cause fat cell death).

109. The adjustable therapeutic gene therapy system according to any of the preceding claims, wherein external stimulation causes cooling of the skin and subcutaneous tissue to lead to fat cell aging.

110. The adjustable therapeutic gene therapy system of any of the preceding claims, wherein an external stimulus causes a temperature change in subcutaneous tissue, said temperature change being sufficient to alter the expression of the genetic construct.

111. The adjustable therapeutic gene therapy system according to any one of the preceding claims, wherein the external stimulation comprises high-frequency ultrasound.

112. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulation comprises intermediate frequency ultrasound.

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

114. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulation comprises high-frequency ultrasound-mediated cavitation.

115. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulation comprises mid-frequency ultrasound-mediated cavitation.

116. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulation comprises low-frequency ultrasound-mediated cavitation.

117. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulus comprises ultrasound with a frequency in the range of 1 to 10 kHz.

118. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulation comprises ultrasound with a frequency in the range of 10 to 28 kHz.

119. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulation comprises ultrasound with a frequency in the range of 28 to 40 kHz.

120. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulation comprises ultrasound with a frequency in the range of 40 to 60 kHz.

121. The adjustable therapeutic gene therapy system according to any one of claims 1 to 110, wherein the external stimulation comprises ultrasound in the frequency range of 1 to 320 kHz.

122. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulus comprises sound waves (e.g., in at least a portion of the subcutaneous space, capable of inhibiting expression in adipocytes, inducing senescence in adipocytes, or inducing adipocyte death).

123. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulus comprises electromagnetic waves (e.g., in at least a portion of the subcutaneous space, capable of inhibiting expression in adipocytes, inducing senescence in adipocytes, or inducing adipocyte death).

124. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulus comprises an electrical signal (e.g., capable of inhibiting expression in adipocytes, inducing senescence in adipocytes, or inducing adipocyte death, for example, in at least a portion of the subcutaneous space).

125. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulation comprises the administration of a formulation containing rapamycin or a rapamycin analog or a rapamycin derivative.

126. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulation comprises the administration of a formulation containing tetracycline or a derivative thereof.

127. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulation comprises administration of rapamycin or a rapamycin analog or a rapamycin derivative in combination with tetracycline or a tetracycline derivative.

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

129. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulation comprises the administration of doxycycline or a doxycycline derivative.

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

131. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulation comprises the administration of one or more peptides having or not having conjugations.

132. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulation comprises the administration of one or more hormones or analogues thereof.

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

134. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulus comprises the administration of one or more agents that induce an inflammatory response.

135. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulation comprises the administration of one or more types of steroids.

136. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulus comprises the administration of one or more functionalized or unfunctionalized antibodies.

137. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulation comprises the administration of one or more receptor ligands.

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

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

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

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

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

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

144. The modulating therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulus comprises administration of an agent that induces an anti-inflammatory response or an immunomodulatory response.

145. The adjustable therapeutic gene therapy system of any one of claims 1 to 110, wherein the external stimulation comprises the administration of one or more neurotransmitters.

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

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

148. A method for therapeutic transgenic expression, comprising at least once administering the adjustable therapeutic gene therapy system as described in any of the preceding claims.

149. A method of expressing a therapeutic transgene, comprising administering to any of the tissues or cells provided herein, such as to a subcutaneous space (e.g., to one or more cell types in a subcutaneous space): a genetic construct encoding at least one therapeutic gene, said therapeutic transgene being regulated by at least one promoter element and optionally one or more regulatory elements; and optionally, a delivery vector or vector encapsulating at least a portion of said genetic construct.

150. The method of claim 149, wherein the genetic construct is as defined in any of the preceding claims, or any of the genetic constructs described herein.

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

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

153. The method of any one of claims 148 to 152, wherein the method further comprises killing, reducing, removing, or inducing cellular senescence in the subcutaneous space where the genetic construct has been applied.

154. The method of any one of claims 148 to 152, wherein the method further comprises reducing transgene expression from the genetic construct.

155. The method of any one of claims 148 to 152, wherein the method further comprises enhancing transgene expression from the genetic construct.

156. The method of any one of claims 148 to 155, wherein an external stimulus is applied to cells in the subcutaneous space where the genetic construct has been applied.

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

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

159. The method of any one of claims 148 to 158, wherein the modulating gene therapy system and / or genetic construct comprises means for upregulating or downregulating transgene expression, optionally, said means being any of the methods provided herein for such purposes.

160. The method of any one of claims 148 to 159, wherein the method further comprises administering a second dose of the following to a subcutaneous space (e.g., to one or more cell types in the subcutaneous space): i) a modulated gene therapy system or ii) the genetic construct encoding at least one therapeutic gene, the therapeutic gene being regulated by at least one promoter element and optionally one or more regulatory elements; and optionally, a delivery vector or vector encapsulating at least a portion of the genetic construct.

161. The method of any one of claims 148 to 160, wherein the expression of the transgene is permanently reduced (e.g., relative to the baseline expression level of the applied transgene).

162. The method of any one of claims 148 to 161, wherein the application is used to treat a disease in an object (e.g., a human object).

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

164. The method of any one of claims 148 to 163, wherein the application is used to strengthen or enhance the health, physical condition, mental state, or physical or mental capacity of a person or animal.

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

166. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, wherein the target cell is an adipocyte.

167. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, wherein the target cell is a preadipocyte.

168. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, wherein the target cell is a progenitor cell capable of differentiating into an adipocyte.

169. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, wherein the target cell is a stem cell capable of differentiating into an adipocyte.

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

171. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, wherein the target cell is a transient cell or stem cell, but capable of becoming a resident cell in the subcutaneous tissue.

172. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, used for treating monogenic diseases.

173. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, used for treating enzyme dysfunction disorders.

174. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, used for protein replacement therapy.

175. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, used for treating metabolic disorders.

176. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, used for treating autoimmune diseases.

177. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, used for treating tumors.

178. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, used for treating neurological disorders.

179. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, used for treating cardiovascular pathological conditions.

180. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, for treating musculoskeletal disorders.

181. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, used for treating hematological disorders.

182. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, used for treating skin conditions.

183. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, used for treating immune system disorders.

184. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, used for treating pulmonary system diseases.

185. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, for treating kidney or bladder diseases.

186. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, for delivering therapeutic antibodies by expression from said genetic construct.

187. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, for delivering therapeutic proteins by expression from said genetic construct.

188. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, for delivering a fusion protein by expression from said genetic construct.

189. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, for delivering peptides via expression from said genetic construct.

190. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, for delivering an immunogen via expression from said genetic construct.

191. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, for delivering a vaccine by expression from said genetic construct.

192. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims further includes, or the application thereof, one or more biomarkers for subsequent localization, visualization, analysis, achievement of improved titration, achievement of degraded titration, selection of subsequent doses, or overall visualization, or means thereof.

193. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims further includes, or the application thereof, the following: surface or internal markers at the delivery site for subsequent localization, visualization, analysis, achievement of increased titration, achievement of decreased titration, selection of subsequent doses, or overall visualization, or means thereof.

194. The adjustable therapeutic gene therapy system or method of claim 192 or 193, wherein the one or more biomarkers comprise any of the biomarkers described herein (e.g., one or more of a metal, ceramic, polymer or composite material, or the genetic code encoding a reporter protein, optionally which can be visualized directly or after stimulation is applied to allow or enhance visualization).

195. The adjustable therapeutic gene therapy system or method of claim 194, wherein the stimulus is any of the stimuli described herein (e.g., comprising one or more of the following: energy, cold, heat, application of one or more chemicals, treatments, molecules or atoms, to alter the physical, chemical or physiological state of the system, thereby enabling biomarker reporting, visualization, readout or interaction to provide information about the treatment).

196. The adjustable therapeutic gene therapy system or method according to any one of the preceding claims, wherein the administration occurs more than once, for example, multiple times, during treatment.

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