Compositions, systems, and methods for reducing adipose tissue

Abstract

Polynucleotide constructs are provided for adipocyte-specific production of cytotoxic proteins. Also provided are formulations and systems, including lipid-based delivery vectors, for delivering the polynucleotide constructs, and methods for making and using such nucleic acid-based polynucleotide constructs, formulations, and systems to reduce the proliferation and / or viability of adipocytes. The compositions, systems, and methods disclosed herein can be used to reduce adipocyte or fat volume in visceral, subcutaneous, and abdominal fat.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 441,611, filed January 27, 2023, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Since 1975, global obesity has nearly tripled, and in 2016, more than 1.9 billion adults were overweight, of which over 650 million were obese. Excess fat accumulation is associated with an increased relative risk of many diseases and health conditions, as well as psychological and emotional distress. Overweight or obese individuals have an increased risk of heart disease, stroke, diabetes, musculoskeletal disease, and several cancers, including endometrial, breast, ovarian, prostate, liver, gallbladder, kidney, and colon cancer.

[0003] Conventional interventions such as diet and exercise are often ineffective due to poor compliance and other factors, and lipid-lowering disorders may be associated with genetic or hormonal factors that are difficult to address. Alternative interventions involving surgical procedures can be invasive and risky, therefore, there is a need for therapeutic strategies to reduce adipose tissue. Summary of the Invention

[0004] Disclosed herein, in some embodiments, is a polynucleotide construct for selective killing of human adipocytes, the polynucleotide construct comprising (a) a transcriptional promoter that is preferentially or specifically active in human adipocytes, and (b) an introduced gene encoding a cytotoxic protein, wherein expression of the cytotoxic protein is regulated by the transcriptional promoter.

[0005] In some embodiments, the polynucleotide construct comprises DNA. In some embodiments, the polynucleotide construct comprises double-stranded DNA. In some embodiments, the polynucleotide construct is a plasmid. In some embodiments, the polynucleotide construct is a plasmid. In some embodiments, the polynucleotide construct is a plasmid. In some embodiments, the transcription promoter comprises the ADIPOQ promoter or a functional fragment thereof. In some embodiments, the transcription promoter comprises the FABP4, PLIN1, PPARγ, PPARγ1, PPARγ2, CD36, LPL, LEP, CIDEC, TUSC5, CIDEA, or LIPE promoter, or a functional fragment thereof. In some embodiments, the transcription promoter comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or 100% sequence identity to any one of SEQ ID NOs: 1 and 56-72. In some embodiments, the promoter is at least 50% more active in human adipocytes than in human non-adipocyte control cells. In some embodiments, the control cell is a myocyte, hepatocyte, bone cell, erythrocyte, neuron, leukocyte, lymphocyte, or fibroblast. In some embodiments, the cytotoxic protein induces non-inflammatory cell death when expressed in human adipocytes. In some embodiments, the cytotoxic protein induces apoptosis when expressed in human adipocytes. In some embodiments, the cytotoxic protein comprises a caspase or its catalytic domain. In some embodiments, the caspase comprises an inducible caspase or its catalytic domain. In some embodiments, the caspase comprises a rapamycin-inducible caspase. In some embodiments, the rapamycin-inducible caspase comprises an FKBP-rapamycin-binding (FRB) domain. In some embodiments, the rapamycin-inducible caspase comprises an FK506-binding protein (FKBP) domain. In some embodiments, the FKBP domain is an FKBP12 domain.In some embodiments, the rapamycin-inducible caspase comprises, from N-terminus to C-terminus, an FRB domain, an FKBP12 domain, and a caspase or a functional fragment thereof. In some embodiments, the caspase is a non-inducible caspase. In some embodiments, the caspase is an auto-activating caspase. In some embodiments, the caspase comprises caspase 9 or its catalytic domain. In some embodiments, the caspase comprises caspase 1 or its catalytic domain. In some embodiments, the caspase comprises caspase 3 or its catalytic domain. In some embodiments, the cytotoxic protein comprises caspase 8, BAX, DFF40, HSV-TK, cytosine deaminase, or a catalytic domain thereof. In some embodiments, the cytotoxic protein comprises an amino acid sequence having at least 80% sequence identity or sequence similarity to any one of SEQ ID NOs: 3-13 and 52-55. In some embodiments, the adipocytes are white adipocytes. In some embodiments, the adipocytes are not brown adipocytes. In some embodiments, the polynucleotide construct further comprises a safety element that reduces expression of the cytotoxic protein in control cells that are not human adipocytes. In some embodiments, the control cells are myocytes, hepatocytes, bone cells, erythrocytes, neurons, leukocytes, lymphocytes, or fibroblasts. In some embodiments, expression of the safety element is driven by a regulatory element that is active in the control cells but less active or substantially inactive in human adipocytes. In some embodiments, the safety element comprises a regulatory RNA that targets and degrades transcription encoding the cytotoxic protein. In some embodiments, the safety element comprises a target site of a regulatory RNA, e.g., an endogenous or engineered regulatory RNA. In some embodiments, the regulatory RNA is an siRNA or miRNA. In some embodiments, the safety element comprises a transcriptional repressor that reduces expression mediated by a transcriptional promoter.

[0006] Disclosed herein, in some aspects, is a lipid-based delivery vector (LDV) comprising a polynucleotide construct according to any one of the preceding embodiments.

[0007] In some embodiments, the LDV comprises a fusion-associated small transmembrane (FAST) protein. In some embodiments, the FAST protein comprises the ectodomain of a first reovirus FAST protein and the endodomain of a second reovirus FAST protein. In some embodiments, the FAST protein comprises p10, p13, p14, p15, p16, p22, or a functional domain thereof. In some embodiments, the FAST protein comprises a fusion of a first domain from a p14 FAST protein or a p10 FAST protein with a second domain from a p14 FAST protein or a p15 FAST protein. In some embodiments, the FAST protein comprises the ectodomain of p14 and the endodomain of p15. In some embodiments, the LDV comprises an ionizable lipid. In some embodiments, the molar ratio of ionizable lipid to polynucleotide construct is between about 2:1 and 25:1. In some embodiments, the molar ratio is about 5:1, about 7.5:1, about 10:1, or about 15:1. In some embodiments, the ionizable lipid comprises Dlin-KC2-DMA (KC2), DODMA, DODAP, DOBAQ, DOTMA, 18:1 EPC, DOTAP, DDAB, 18:0 EPC, 18:0 DAP, or 18:0 TAP. In some embodiments, the LDV is configured to deliver the polynucleotide construct to human adipocytes upon contacting the human adipocytes with the LDV. In some embodiments, the LDV is configured to deliver the polynucleotide construct to human adipocytes upon administration of the LDV to a subject. In some embodiments, the LDV is formulated for non-targeted delivery to human adipocytes and non-adipocytes.

[0008] Disclosed herein, in some aspects, is a cell comprising the polynucleotide construct of any one of the preceding embodiments.

[0009] Disclosed herein, in some aspects, is a method for reducing the viability of a population of adipocytes, the method comprising contacting the population of adipocytes with an LDV of any one of the preceding embodiments under conditions that promote uptake of a polynucleotide construct by the adipocytes.

[0010] In some embodiments, the adipocytes comprise white adipocytes. In some embodiments, the adipocytes are human adipocytes. In some embodiments, during the contacting step, the LDV containing the polynucleotide construct is present at a concentration of at least 1 nM. In some embodiments, at least about 1% of the white adipocytes in the population are killed. In some embodiments, up to about 95% of the white adipocytes in the population are killed. In some embodiments, about 5% to 80% of the white adipocytes are killed.

[0011] Disclosed herein, in some aspects, is a method for reducing adipose tissue volume, the method comprising administering to a subject an effective amount of an LDV of any one of the preceding embodiments.

[0012] In some embodiments, the LDV is administered systemically. In some embodiments, the LDV is administered locally. In some embodiments, the LDV is administered via injection into adipose tissue. In some embodiments, the LDV is administered into visceral fat. In some embodiments, the LDV is administered into subcutaneous fat. In some embodiments, the LDV is administered into abdominal fat. In some embodiments, the adipose tissue volume is reduced by at least about 5%. In some embodiments, the adipose tissue volume is reduced by up to about 95%. In some embodiments, the adipose tissue volume is reduced by about 5% to about 80%. In some embodiments, the adipose tissue is white adipose tissue. In some embodiments, the reduction in adipose tissue volume is determined by DEXA scanning to quantify adipose tissue before and after administering the LDV containing the polynucleotide construct. In some embodiments, the cytotoxic protein is an inducible caspase, and the method further comprises administering an inducer of caspase to the subject. In some embodiments, the cytotoxic protein is a rapamycin-inducible caspase, and the method further comprises administering rapamycin or a structural analog thereof to the subject. In some embodiments, LDV is administered to the subject two or more times. In some embodiments, rapamycin or a structural analog thereof is administered to the subject two or more times. In some embodiments, the method treats cellulite in the subject. In some embodiments, the method treats a metabolic disorder in the subject. In some embodiments, the method treats Dercam's disease in the subject.

[0013] Disclosed herein, in some aspects, is a system for selective killing of human adipocytes, the system comprising a polynucleotide construct of any one of the preceding embodiments, rapamycin or a structural analog thereof, and optionally an LDV of any one of the preceding embodiments. [Brief explanation of the drawings]

[0014] [Figure 1]FIG. 1 provides images of H&E cross sections of human tissue explants showing reduced fat content after treatment with a chemical inducer of polynucleotide construct delivery, iCasp9 expression, and dimerization. [Figure 2] FIG. 2 quantifies the percent change in adipose tissue area in H&E cross sections of human tissue explants following therapeutic delivery of polynucleotide constructs, iCasp9 expression, and treatment with chemical inducers of dimerization. [Figure 3] FIG. 3 shows the in vivo expression of a reporter gene in adipose tissue of mice administered an LDV formulation containing a polynucleotide construct with reporter gene expression driven by the adiponectin promoter. [Figure 4] FIG. 4 shows the in vivo expression of a reporter gene in adipose tissue of mice administered an LDV formulation containing a polynucleotide construct. [Figure 5] FIG. 5 illustrates the collection of skin and fat pad samples near the site of injection of the polynucleotide construct. [Figure 6] FIG. 6 illustrates reporter gene expression in adipose tissue of mice administered an LDV formulation containing a polynucleotide construct. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present disclosure provides polynucleotide constructs, systems and methods for selectively reducing the proliferation and / or survival of adipocytes, which may be referred to as "target cells."

[0016] The provided compositions, systems, and methods can be used to reduce the proliferation and / or survival of adipocytes, thereby reducing adipose tissue volume. Adipocytes can be associated with excess fat, lipedema, lipidemia, metabolic disorders, and other diseases. Polynucleotide constructs are provided for target cell-specific expression of therapeutic proteins, e.g., cytotoxic proteins. The polynucleotide constructs can utilize intracellular features, including transcriptional regulatory features, that are present in target cells but absent or substantially reduced in control or non-target cells. Such polynucleotide constructs are used in systems that include vectors for delivery of polynucleotide constructs to target cells, including lipid-based delivery vectors (LDVs).

[0017] I. Polynucleotide Constructs Disclosed herein are polynucleotide constructs and systems and methods comprising the polynucleotide constructs. The polynucleotide constructs may include expression regulatory regions for driving or controlling the expression of one or more transgenes, such as transgenes encoding cytotoxic proteins for inducing adipocyte death (e.g., apoptosis), extracellular matrix remodeling factors, and / or safety elements.

[0018] The polynucleotide constructs disclosed herein may be or comprise DNA. The polynucleotide constructs may be or comprise double-stranded DNA. For example, the polynucleotide constructs disclosed herein may be or comprise a plasmid, such as a nanoplasmid. In some embodiments, the polynucleotide constructs disclosed herein are or comprise a minicircle, midge, MIP, or doggybone. The polynucleotide constructs disclosed herein may be or comprise a circular polynucleotide. The polynucleotide constructs disclosed herein may be or comprise a linear polynucleotide. The polynucleotide constructs disclosed herein may comprise RNA, for example, circular RNA.

[0019] In some embodiments, the polynucleotide constructs or polynucleotides disclosed herein are not single-stranded DNA. In some embodiments, the polynucleotide constructs or polynucleotides disclosed herein lack components of a viral genome or lack viral packaging elements, e.g., lack 5' and / or 3' inverted terminal repeats (ITRs).

[0020] In some embodiments, the polynucleotide constructs or polynucleotides disclosed herein are non-integrated, e.g., do not integrate into the genome of the host cell.

[0021] A. Promoter and expression control region The polynucleotide constructs, systems, and methods disclosed herein can utilize cell- or cell type-specific transcriptional regulatory mechanisms inherent in target cells, such as adipocytes (e.g., white adipocytes). The polynucleotide constructs can be used for targeted production of cytotoxic proteins in adipocytes (e.g., white adipocytes).

[0022] The polynucleotide constructs disclosed herein may include an expression control region. The expression control region may include, for example, a promoter (e.g., an adipocyte-specific promoter), an enhancer, an intron, an exon, or a functional fragment thereof, or a combination thereof. The polynucleotide construct may include multiple expression control regions, for example, two or more expression control regions.

[0023] The transcription promoter may be a promoter found in a naturally occurring genome. In some embodiments, the promoter is not found in a naturally occurring genome. In some embodiments, the promoter is an engineered promoter. The promoter may be a minimal promoter or a functional fragment of a larger promoter effective to drive expression of a downstream transgene in a target cell.

[0024] The polynucleotide constructs disclosed herein may include a transcription promoter that is activated in target cells, such as adipocytes (e.g., white adipocytes). The transcription promoter may be specifically, selectively, or preferentially activated in target cells (e.g., adipocytes) compared to control cells (e.g., non-adipocytes, such as muscle cells, hepatocytes, bone cells, erythrocytes, neurons, leukocytes, lymphocytes, monocytes, or fibroblasts). In some embodiments, the control cells may include brown adipocytes. The promoter may be specifically, selectively, or preferentially derepressed in target cells compared to control cells. Transcription promoters that may be suitably used in the polynucleotide constructs, systems, and methods of the present disclosure include transcription promoters that are capable of driving expression of a transgene in target cells (i.e., adipocytes) but are incapable of, or exhibit a substantially reduced ability to, drive expression of that transgene in control cells.

[0025] In some embodiments, the transcription promoter causes the level of expression in an adipocyte (e.g., a white adipocyte) to be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, or less than the level of expression in a control cell (e.g., a hepatocyte, bone cell, erythrocyte, neuron, leukocyte, lymphocyte, monocyte, fibroblast, or brown adipocyte) disclosed herein. The expression level of the adipocytes and the control cells can be at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 250-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, or at least 10000-fold higher. The adipocytes and the control cells can each be primary cells. In some embodiments, the transcription promoter drives an expression level in adipocytes (e.g., white adipocytes) that is detectable in adipocytes (e.g., white adipocytes), and the expression is undetectable or below the detection limit in the control cells. Expression can be determined, for example, by a detectable reporter gene (e.g., a fluorescent or luminescent protein or substrate), ELISA, Western blot, etc.

[0026] The transcription promoters used in the compositions, systems, or methods disclosed herein can be responsive to one or more factors that are specifically or preferentially produced in target cells, such as adipocytes.

[0027] The transcription promoter itself may be the primary mechanism by which adipocytes are preferentially targeted in the systems or methods disclosed herein. In some embodiments, the polynucleotide constructs disclosed herein that utilize adipocyte-specific (e.g., white adipocyte-specific) promoters overcome the safety and / or efficacy limitations associated with technologies that rely on targeted delivery of therapeutic compounds. For example, adipocyte-specific promoters can reduce off-target effects, such as those resulting from transgene expression in non-adipocytes, and the use of delivery vectors such as the LDVs disclosed herein can improve delivery of polynucleotide constructs to adipocytes compared to alternative delivery vectors.

[0028] In some embodiments, the transcriptional promoters disclosed herein reduce or eliminate the need for targeted delivery vectors that target adipocytes, for example, using proteins or antibodies directed to adipocyte-specific surface molecules. For example, the system can use selective promoters (e.g., adipocyte-selective promoters) for expression only or preferentially in a desired cell type without selective uptake of the polynucleotide construct by adipocytes over other control cells disclosed herein.

[0029] The transcription promoters used in the compositions, systems, and methods disclosed herein can be promoters known to be active in adipocytes (eg, white adipocytes).

[0030] The transcription promoter used in the compositions, systems, and methods disclosed herein can be an adiponectin promoter or a functional fragment thereof, such as a core or minimal adiponectin promoter. In humans, adiponectin is encoded by the ADIPOQ gene. Adiponectin can function as an adipokine secreted by adipocytes. Adiponectin can function as a homeostatic factor for regulating glucose levels, lipid metabolism, and insulin sensitivity. In humans, adiponectin is located on chromosome 3 (3q27). Adipocytes can exhibit high levels of adiponectin expression, which can contribute to adipocyte differentiation. In adipocytes, CCAAT / enhancer-binding protein α (C / EBFα), peroxisome proliferator-activated receptor γ (PPARγ), sterol regulatory element-binding protein (SREBP)-1c, forkhead box 1, and specificity protein 1 can upregulate adiponectin expression and participate in promoting adipogenesis and increasing lipid content and insulin-induced glucose transport, whereas reactive oxygen species, TNFα, and IL-6 can downregulate adiponectin expression.

[0031] The transcription promoter used in the compositions, systems, and methods disclosed herein can be a mammalian adiponectin promoter or a functional fragment thereof, such as a core or minimal mammalian adiponectin promoter.The transcription promoter used in the compositions, systems, and methods disclosed herein can be a human adiponectin promoter or a functional fragment thereof, such as a core or minimal human adiponectin promoter.In some embodiments, the adiponectin promoter comprises about 100, about 200, about 500, about 1000, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, or about 2500 base pairs upstream of the human adiponectin transcription start site. In some embodiments, the adiponectin promoter comprises at least about 100, at least about 200, at least about 500, at least about 1000, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, or at least about 2500 base pairs upstream of the human adiponectin transcription start site. In some embodiments, the polynucleotide constructs or expression control regions disclosed herein comprise or utilize an adiponectin enhancer element. An exemplary adiponectin promoter or fragment thereof is provided in SEQ ID NO: 1.

[0032] The transcriptional promoter used in the compositions, systems, and methods disclosed herein can be the fatty acid binding protein 4 (FABP4) promoter or a functional fragment thereof, such as the core or minimal FABP4 promoter. FABP4, also known as adipocyte P2 (aP2), is an adipocyte-specific fatty acid-binding protein that plays a role in fatty acid uptake, transport, and metabolism. FABP4 is highly expressed in adipocytes, and its expression is highly induced during adipocyte differentiation. Its expression is negligible in preadipocytes and increases substantially during adipocyte differentiation. The promoter / enhancer region of FABP4, particularly the 540-bp enhancer, may be important for its adipose-specific expression. FABP4 is involved in regulating glucose and lipid metabolism in relation to inflammation and metabolic processes. FABP4 expression is transcriptionally regulated by peroxisome proliferator-activated receptor (PPAR) γ agonists, fatty acids (FAs), dexamethasone, and insulin.

[0033] The transcription promoter used in the compositions, systems, and methods disclosed herein can be a mammalian FABP4 promoter or a functional fragment thereof, such as a core or minimal mammalian FABP4 promoter. The transcription promoter used in the compositions, systems, and methods disclosed herein can be a human FABP4 promoter or a functional fragment thereof, such as a core or minimal human FABP4 promoter. In some embodiments, the polynucleotide construct or expression control region disclosed herein comprises a FABP4 enhancer. In some embodiments, the polynucleotide construct or expression control region disclosed herein comprises a FABP4 (aP2) minimal promoter, for example, comprising an approximately 540-bp adipose-specific aP2 enhancer linked at bp-63 upstream of the basal aP2 promoter (e.g., as provided in SEQ ID NO: 63). This mini-promoter can, for example, exhibit increased specificity for adipocytes over cardiac and skeletal muscle cells.

[0034] In some embodiments, the FABP4 promoter comprises about 100, about 200, about 500, about 1000, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, or about 2500 base pairs upstream of the human FABP4 transcription start site. In some embodiments, the FABP4 promoter comprises about 5400 or about 5403 base pairs upstream of the human FABP4 transcription start site. In some embodiments, the FABP4 promoter comprises at least about 100, at least about 200, at least about 500, at least about 1000, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, or at least about 2500 base pairs upstream of the human FABP4 transcription start site. In some embodiments, the polynucleotide constructs or expression control regions disclosed herein comprise or utilize a FABP4 enhancer element. Exemplary FABP4 promoters or fragments thereof are provided in SEQ ID NOS: 61-63. In some embodiments, the FABP4 promoter is as deposited in GenBank AJ627200.1, as described in US2020 / 0102361A1, or as described by Rival et al. in "Human adipocyte fatty acid-binding protein (aP2) gene promoter-driven reporter assay discriminates nonlipogenic peroxisome proliferator-activated receptor γ ligands." Journal of Pharmacology and Experimental Therapeutics 311.2 (2004): 467-475, each of which is incorporated herein for such disclosure.

[0035] The transcription promoter used in the compositions, systems, and methods disclosed herein can be the perilipin 1 (PLIN1) promoter or a functional fragment thereof, such as the core or minimal PLIN1 promoter. PLIN1 is a lipid droplet-associated protein that may play a role in protecting lipid droplets from hormone-sensitive lipase. PLIN1 has been described as being primarily expressed in adipose tissue. For example, PLIN1 has been described as having strong expression specificity in adipose tissue, including both subcutaneous and omental fat (e.g., two-fold higher expression in subcutaneous fat compared to visceral fat).

[0036] The transcription promoter used in the compositions, systems, and methods disclosed herein can be a mammalian PLIN1 promoter or a functional fragment thereof, such as a core or minimal mammalian PLIN1 promoter. The transcription promoter used in the compositions, systems, and methods disclosed herein can be a human PLIN1 promoter or a functional fragment thereof, such as a core or minimal human PLIN1 promoter. In some embodiments, the PLIN1 promoter comprises about 500, about 1000, about 1500, about 1600, about 1700, about 1731, about 1800, about 1900, or about 2000 base pairs upstream of the human PLIN1 transcription start site. In some embodiments, the PLIN1 promoter comprises about 100, about 200, about 500, about 1000, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, or about 2500 base pairs upstream of the human FABP4 transcription start site. In some embodiments, the PLIN1 promoter comprises at least about 100, at least about 200, at least about 500, at least about 1000, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, or at least about 2500 base pairs upstream of the human PLIN1 transcription start site. In some embodiments, a polynucleotide construct or expression control region disclosed herein comprises or utilizes a PLIN1 enhancer element. Exemplary PLIN1 promoters or fragments thereof are provided in SEQ ID NOs: 56 and 57. In some embodiments, the PLIN1 promoter is as described in Bialesov et al. "Epigenetic regulation of PLIN 1 in obese women and its relation to lipolysis." Scientific reports 7.1 (2017): 10152, which is incorporated herein for such disclosure.

[0037] The transcription promoter used in the compositions, systems, and methods disclosed herein can be the peroxisome proliferator-activated receptor gamma (PPARγ) promoter or a functional fragment thereof, such as the core or minimal PPARγ1 or PPARγ2 promoter. PPARγ is a nuclear receptor and a prominent regulator of adipocyte differentiation. It is activated upon ligand binding and regulates the transcription of target genes involved in lipid storage and metabolism by binding to specific PPAR response elements (PPREs) in DNA. Mutations in PPARγ are associated with diseases such as partial lipodystrophy, and its dysfunction results in the absence of discernible adipose tissue. Alternatively spliced ​​transcript variants (i.e., PPARγ1 and PPARγ2) encoding distinct isoforms have been described. Forced expression of mouse PPARγ2 in fibroblasts can be sufficient to drive adipocyte differentiation, suggesting its role as an adipocyte-specific transcription factor. PPARγ mRNA has been described to be at least 5-fold or at least 20-fold more abundant in adipose tissue than in most other tissues.

[0038] The transcription promoter used in the compositions, systems, and methods disclosed herein can be a mammalian PPARγ promoter or a functional fragment thereof, such as a core or minimal mammalian PPARγ promoter. The transcription promoter used in the compositions, systems, and methods disclosed herein can be a human PPARγ promoter or a functional fragment thereof, such as a core or minimal human PPARγ promoter. The transcription promoter used in the compositions, systems, and methods disclosed herein can be a mammalian PPARγ1 promoter or a functional fragment thereof, such as a core or minimal mammalian PPARγ1 promoter. The transcription promoter used in the compositions, systems, and methods disclosed herein can be a human PPARγ1 promoter or a functional fragment thereof, such as a core or minimal human PPARγ1 promoter. The transcription promoter used in the compositions, systems, and methods disclosed herein can be a mammalian PPARγ2 promoter or a functional fragment thereof, such as a core or minimal mammalian PPARγ2 promoter. The transcription promoter used in the compositions, systems, and methods disclosed herein can be the human PPARγ2 promoter or a functional fragment thereof, such as the core or minimal human PPARγ2 promoter.

[0039] In some embodiments, the PPARγ promoter comprises about 100, about 200, about 500, about 1000, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, or about 2500 base pairs upstream of the human PPARγ transcription start site. In some embodiments, the PPARγ promoter comprises at least about 100, at least about 200, at least about 500, at least about 1000, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, or at least about 2500 base pairs upstream of the human PPARγ transcription start site. In some embodiments, the polynucleotide constructs or expression control regions disclosed herein comprise or utilize a PPARγ enhancer. Exemplary PPARγ promoters or fragments thereof are provided in SEQ ID NOs: 58-60. In some embodiments, PPARγ is expressed as described in Zhu et al. "Structural organization of mouse peroxisome proliferator-activated receptor gamma (mPPAR gamma) gene: alternative promoter use and different splicing yield two mPPAR gamma isoforms." Proceedings of the National Academy of Sciences 92.17 (1995): 7921-7925; Fajas et al. "The organization, promoter analysis, and expression of the human PPARγ gene.")” Journal of Biological Chemistry 272.30 (1997): 18779-18789, or as deposited in GenBank as AF012873.1 or AF012874.1, each of which is incorporated herein for such disclosure.

[0040] The transcription promoter used in the compositions, systems, and methods disclosed herein can be the CD36 promoter or its functional fragments, such as the core or minimal CD36 promoter.CD36, also known as fatty acid translocase, is a multifunctional membrane glycoprotein that is involved in various cellular processes, including fatty acid uptake and cell adhesion, and acts as a class B scavenger receptor.Research using the Genotype-Tissue Expression (GTEx) database has classified CD36 as an adipose tissue-enhanced gene, showing that its expression is more than five times higher in adipose tissue than in most other tissues.

[0041] The transcription promoter used in the compositions, systems, and methods disclosed herein can be a mammalian CD36 promoter or a functional fragment thereof, such as a core or minimal mammalian CD36 promoter. The transcription promoter used in the compositions, systems, and methods disclosed herein can be a human CD36 promoter or a functional fragment thereof, such as a core or minimal human CD36 promoter. In some embodiments, the CD36 promoter comprises about 100, about 200, about 500, about 1000, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, or about 2500 base pairs upstream of the human CD36 transcription start site. In some embodiments, the CD36 promoter comprises at least about 100, at least about 200, at least about 500, at least about 1000, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, or at least about 2500 base pairs upstream of the human CD36 transcription start site. In some embodiments, a polynucleotide construct or expression control region disclosed herein comprises or utilizes a CD36 enhancer element. Exemplary CD36 promoters or fragments thereof are provided in SEQ ID NOs: 64 and 65. In some embodiments, the CD36 promoter is as deposited in GenBank AF266759.1 or as described in Armesilla and Vega. "Structural organization of the gene for human CD36 glycoprotein.")” Journal of Biological Chemistry 269.29 (1994):18985-18991, or as described in Zingg et al. “Novel 5' exon of scavenger receptor CD36 is expressed in cultured human vascular smooth muscle cells and atherosclerotic plaques.” Arteriosclerosis, thrombosis, and vascular biology 22.3(2002):412-417, each of which is incorporated herein for such disclosure.

[0042] The transcription promoter used in the compositions, systems, and methods disclosed herein can be a lipoprotein lipase (LPL) promoter or a functional fragment thereof, such as a core or minimal LPL promoter. Lipoprotein lipase (LPL) is a central enzyme in lipid metabolism, primarily functioning in the hydrolysis of triglycerides in chylomicrons and very low-density lipoproteins (VLDL) into free fatty acids and glycerol, and is important for lipid clearance, utilization, and storage in the body. In adipose tissue, LPL is key to lipid uptake, adipocyte differentiation, and maturation, acting as the main enzyme for the entry and re-esterification of free fatty acids. Its regulation is complex and changes in response to physiological stimuli such as nutritional changes, and is highly responsive to fasting and exercise.

[0043] The transcription promoter used in the compositions, systems, and methods disclosed herein can be a mammalian LPL promoter or a functional fragment thereof, such as a core or minimal mammalian LPL promoter.The transcription promoter used in the compositions, systems, and methods disclosed herein can be a human LPL promoter or a functional fragment thereof, such as a core or minimal human LPL promoter.In some embodiments, the LPL promoter comprises about 100, about 200, about 500, about 1000, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, or about 2500 base pairs upstream of the human LPL transcription start site. In some embodiments, the LPL promoter comprises at least about 100, at least about 200, at least about 500, at least about 1000, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, or at least about 2500 base pairs upstream of the human LPL transcription start site. In some embodiments, the polynucleotide constructs or expression control regions disclosed herein comprise or utilize an LPL enhancer element. Exemplary LPL promoters or fragments thereof are provided in SEQ ID NOs: 66 and 67. In some embodiments, the LPL promoter is as described in GenBank at X68111.1, or as described in Enerback et al., "Characterization of the human lipoprotein lipase (LPL) promoter: evidence of two cis-regulatory regions, LP-α and LP-β, of importance for the differentiation-linked induction of the LPL gene during adipogenesis.")" Molecular and Cellular Biology 12.10 (1992): 4622-4633 Molecular and Cellular Biology 12.10 (1992): 4622-4633, each of which is incorporated herein for such disclosure.

[0044] The transcription promoter used in the compositions, systems, and methods disclosed herein can be the leptin (LEP) promoter (also known as OB) or a functional fragment thereof, such as the core or minimal LEP promoter. The leptin gene (LEP), which encodes the hormone leptin, is expressed primarily in adipocytes and plays a key role in regulating food intake and energy expenditure, primarily via the central nervous system. The human LEP(OB) gene promoter, which controls leptin expression, may require only 217 bp of 5' sequence for basal adipose tissue-specific expression, and a CCAAT enhancer-binding protein α (C / EBPα) site within this proximal promoter plays an important role in high-level expression in preadipocytes and adipocytes.

[0045] The transcription promoter used in the compositions, systems, and methods disclosed herein can be a mammalian LEP promoter or a functional fragment thereof, such as a core or minimal mammalian LEP promoter. The transcription promoter used in the compositions, systems, and methods disclosed herein can be a human LEP promoter or a functional fragment thereof, such as a core or minimal human LEP promoter. In some embodiments, a minimal LEP promoter (e.g., approximately 217 bp of 5' sequence) is used in the disclosed compositions, methods, or systems. In some embodiments, the polynucleotide constructs or expression control regions disclosed herein include or utilize a CCAAT enhancer-binding protein alpha (C / EBPα) site, for example, within the LEP promoter. In some embodiments, the leptin promoter can drive gene expression in vivo, but is not solely responsible for the adipose tissue specificity of leptin expression. A 30-bp region approximately 4.5 kb upstream of the LEP transcription start site has been identified as an enhancer in mature adipocytes, suggesting that multiple regions, particularly the 3' end of the gene, may contribute to its regulation. In some embodiments, the polynucleotide constructs or expression control regions disclosed herein comprise or utilize an LEP enhancer element (e.g., an enhancer approximately 30 base pairs from approximately 4.5 kb upstream of the LEP transcription start site).

[0046] In some embodiments, the LEP promoter comprises about 100, about 200, about 500, about 1000, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, or about 2500 base pairs upstream of the human LEP transcription start site. In some embodiments, the LEP promoter comprises at least about 100, at least about 200, at least about 500, at least about 1000, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, or at least about 2500 base pairs upstream of the human LEP transcription start site. In some embodiments, the polynucleotide constructs or expression control regions disclosed herein comprise or utilize a LEP enhancer element. An exemplary LEP promoter or fragment thereof is provided in SEQ ID NO: 68. In some embodiments, the LEP promoter is as deposited in GenBank U48621.1 or as described in Miller et al. "The adipocyte specific transcription factor C / EBPalpha modulates human ob gene expression." Proceedings of the National Academy of Sciences 93.11 (1996):5507-5511, each of which is incorporated herein for such disclosure.

[0047] The transcription promoter used in the compositions, systems, and methods disclosed herein can be the cell death-inducing DFFA-like effector C (CIDEC) promoter or a functional fragment thereof, such as the core or minimal CIDEC promoter. CIDEC, also known as FSP27, is a protein associated with lipid droplets in adipocytes and plays an important role in lipid droplet formation and potentially adipocyte apoptosis. It is a member of the cell death-inducing DNA fragmentation factor-like effector family, which is important in apoptosis, is regulated by insulin, and positively correlates with insulin sensitivity. CICEC expression is undetectable in 3T3-L1 preadipocytes but dramatically increases (15,000-fold) in mature adipocytes, resembling the expression pattern of PPARγ2. Analysis of 65 human tissues revealed that CIDEC is primarily expressed in mature adipocytes, with the highest expression in subcutaneous adipose tissue, followed by adipose tissue of unspecified origin and omentum. This fat-restricted expression pattern was also demonstrated by RT-PCR analysis of isolated adipocytes, adipose tissue, and various human tissues, indicating that CIDEC expression is largely restricted to adipocytes within adipose tissue. Comparative microarray and semi-quantitative PCR analysis identified CIDEC as one of the few fat-specific genes in humans and mice with higher expression in subcutaneous than visceral adipose tissue. The 2.5 kb 5' flanking sequence of the CIDEC gene confers adipocyte-specific expression, with specificity achievable with only 176 bp of 5' flanking sequence. However, in some embodiments, higher expression levels were achieved with approximately 1,950 bp of upstream flanking sequence, indicating additional enhancer-like elements.

[0048] The transcription promoter used in the compositions, systems, and methods disclosed herein can be a mammalian CIDEC promoter or a functional fragment thereof, such as a core or minimal mammalian CIDEC promoter. The transcription promoter used in the compositions, systems, and methods disclosed herein can be a human CIDEC promoter or a functional fragment thereof, such as a core or minimal human CIDEC promoter. In some embodiments, various upstream fragments of the human CIDEC gene, such as the approximately -1,800 / +21, -1561 / +217, or -269 / -1 fragments, can be used as promoters or as part of expression control regions (which in some embodiments include several bases past the transcription start site) in the compositions, systems, or methods disclosed herein.

[0049] In some embodiments, the CIDEC promoter comprises about 100, about 176, about 200, about 500, about 1000, about 1500, about 1600, about 1700, about 1800, about 1900, about 1950, about 2000, about 2100, about 2200, about 2500, or about 4500 base pairs upstream of the human CIDEC transcription start site. In some embodiments, the CIDEC promoter comprises at least about 100, at least about 200, at least about 500, at least about 1000, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, or at least about 2500 base pairs upstream of the human CIDEC transcription start site. In some embodiments, a polynucleotide construct or expression control region disclosed herein comprises or utilizes a CIDEC enhancer element. An exemplary CIDEC promoter, or fragment thereof, is provided in SEQ ID NO: 71. In some embodiments, the CIDEC promoter is as described in Danesch and Ringold. "Cloning and transcriptional regulation of a novel adipocyte-specific gene, FSP27. CAAT-enhancer-binding protein (C / EBP) and C / EBP-like proteins interact with sequences required for differentiation-dependent expression." Journal of Biological Chemistry 267.10 (1992):7185-7193, or as described in Tan et al. "TNF-α downregulates CIDEC via MEK / ERK pathway in human adipocytes.")” Obesity 24.5(2016):1070-1080, or as described in Chen et al. “FTO promotes SREBP1c maturation and enhances CIDEC transcription during lipid accumulation in HepG2 cells.” Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids 1863.5(2018):538-548, or as described in Yasumoto et al. “Hepatitis B virus prevents excessive viral production via reduction of cell death-inducing DFF45-like effectors.” Journal of General Virology 98.7(2017):1762-1773, each of which is incorporated herein for such disclosure.

[0050] The transcriptional promoter used in the compositions, systems, and methods disclosed herein can be the tumor suppressor candidate 5 (TUSC5) promoter or a functional fragment thereof, such as the core or minimal TUSC5 promoter. TUSC5 is a cold-repressed gene first identified in brown adipose tissue (BAT) transcriptome flow cytometry analysis and found to be highly expressed in mouse white adipose tissue (WAT) and BAT, as well as human adipocytes. It is significantly increased during adipogenesis and is a target gene of PPARγ, which binds to its promoter region. TUSC5 regulates insulin-stimulated glucose uptake and transport in adipose tissue by regulating GLUT4 recycling. Its expression correlates with improved insulin sensitivity in obese patients. TUSC5 has been identified as an adipose-specific gene, highly expressed in certain adipose tissues and minimally expressed in other tissues.

[0051] The transcription promoter used in the compositions, systems, and methods disclosed herein can be a mammalian TUSC5 promoter or a functional fragment thereof, such as a core or minimal mammalian TUSC5 promoter. The transcription promoter used in the compositions, systems, and methods disclosed herein can be a human TUSC5 promoter or a functional fragment thereof, such as a core or minimal human TUSC5 promoter. In some embodiments, the fragment is about 200, about 500, about 1000, about 1200, about 1400, about 1600, or about 1800 base pairs upstream of the Tusc5 gene, e.g., the -1415 / +276 fragment. In some embodiments, the TUSC5 promoter used as part of a promoter or expression control region in the compositions, systems, or methods disclosed herein includes base pairs from upstream and / or downstream of the TUSC5 transcription start site, e.g., the -1415 / +276 fragment. In some embodiments, the TUSC5 promoter comprises about 100, about 200, about 500, about 1000, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, or about 2500 base pairs upstream of the human TUSC5 transcription start site. In some embodiments, the TUSC5 promoter comprises at least about 100, at least about 200, at least about 500, at least about 1000, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, or at least about 2500 base pairs upstream of the human TUSC5 transcription start site. In some embodiments, the polynucleotide constructs or expression control regions disclosed herein comprise or utilize a TUSC5 enhancer element. An exemplary TUSC5 promoter or fragment thereof is provided in SEQ ID NO: 72.

[0052] The transcription promoter used in the compositions, systems, and methods disclosed herein can be the cell death-inducing DNA fragmentation factor alpha-like effector A (CIDEA) promoter or a functional fragment thereof, such as the core or minimal CIDEA promoter. CIDEA is a lipid droplet-associated protein in adipocytes that regulates triglyceride deposition. CIDEA can be expressed in human white and brown adipose tissue, with its expression highly influenced by body fat status. In some embodiments, CIDEA expression is decreased in obesity and inversely correlated with metabolic syndrome index. CIDEA as an adipose-specific gene in humans.

[0053] The transcription promoter used in the compositions, systems, and methods disclosed herein can be a mammalian CIDEA promoter or a functional fragment thereof, such as a core or minimal mammalian CIDEA promoter. The transcription promoter used in the compositions, systems, and methods disclosed herein can be a human CIDEA promoter or a functional fragment thereof, such as a core or minimal human CIDEA promoter. In some embodiments, the CIDEA promoter comprises about 100, 200, 500, 1000, 1500, 1600, 1700, 1800, 1900, 2000, or 2500 base pairs upstream of the human CIDEA transcription start site. In some embodiments, the CIDEA promoter comprises at least about 100, at least about 200, at least about 500, at least about 1000, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, or at least about 2500 base pairs upstream of the human CIDEA transcription start site. In some embodiments, a polynucleotide construct or expression control region disclosed herein comprises or utilizes a CIDEA enhancer element. An exemplary CIDEA promoter or fragment thereof is provided in SEQ ID NO: 69. In some embodiments, the CIDEA promoter is as described in Pettersson, et al. "Characterization of the human CIDEA promoter in fat cells." International journal of obesity 32.9 (2008):1380-1387, which is incorporated herein for such disclosure.

[0054] The transcription promoter used in the compositions, systems, and methods disclosed herein can be a lipase E (LIPE) promoter, or a functional fragment thereof, such as a core or minimal LIPE promoter. LIPE, known as hormone-sensitive lipase (HSL), is highly expressed in adipose tissue and may be important for adipose tissue lipolysis, releasing free fatty acids for energy consumption. LIPE has been identified as an adipose tissue-specific gene expressed in subcutaneous adipose tissue.

[0055] The transcription promoter used in the compositions, systems, and methods disclosed herein can be a mammalian LIPE promoter or a functional fragment thereof, such as a core or minimal mammalian LIPE promoter.The transcription promoter used in the compositions, systems, and methods disclosed herein can be a human LIPE promoter or a functional fragment thereof, such as a core or minimal human LIPE promoter.In some embodiments, the LIPE promoter comprises about 100, about 200, about 500, about 1000, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, or about 2500 base pairs upstream of the human LIPE transcription start site. In some embodiments, the LIPE promoter comprises at least about 100, at least about 200, at least about 500, at least about 1000, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, or at least about 2500 base pairs upstream of the human LIPE transcription start site. In some embodiments, the polynucleotide construct or expression control region disclosed herein comprises or utilizes a LIPE enhancer element. An exemplary LIPE promoter or fragment thereof is provided in SEQ ID NO:70. In some embodiments, the LIPE promoter is as deposited in GenBank AJ222693.1 or as described in Grober et al. "Characterization of the promoter of human adipocyte hormone-sensitive lipase." Biochemical Journal 328.2 (1997):453-461, each of which is incorporated herein for such disclosure.

[0056] In some embodiments, the transcription promoter is the Ap2 or UCP1 (uncoupling protein 1) promoter, or a functional fragment thereof.

[0057] Table 1 provides exemplary promoter element sequences.

[0058] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12]

[0059] The transcription promoters disclosed herein may be at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, or at least about 90% of any one of SEQ ID NOs: 1 and 56 to 72. %, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity to the nucleotide sequence.

[0060] The transcription promoters disclosed herein may be at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, or at least about 88% of at least 100 consecutive nucleotides of any one of SEQ ID NOS: 1 and 56-72. %, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity to the nucleotide sequence.

[0061] The transcription promoters disclosed herein may be at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 1109%, at least about 1111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least about 124%, at least about 125%, at least about 126%, at least about 127%, at least about 128%, at least about 129%, at least about 130%, at least about 131%, at least about 132%, at least about 133%, at least about 134%, at least about 135%, at least about %, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity to the nucleotide sequence.

[0062] The transcription promoters disclosed herein may be at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 1109%, at least about 1111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least about 124%, at least about 125%, at least about 126%, at least about 127%, at least about 128%, at least about 129%, at least about 130%, at least about 131%, at least about 132%, at least about 133%, at least about 134%, at least about 135%, at least about %, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity to the nucleotide sequence.

[0063] The transcription promoter disclosed herein may have a transcriptional activity of up to about 70%, up to about 71%, up to about 73%, up to about 74%, up to about 75%, up to about 76%, up to about 77%, up to about 78%, up to about 79%, up to about 80%, up to about 81%, up to about 82%, up to about 83%, up to about 84%, up to about 85%, up to about 86%, up to about 87%, up to about 88%, up to about 89%, up to about 90%, up to about 91%, up to about 92%, up to about 93%, up to about 94%, up to about 95%, up to about 96%, up to about 97%, up to about 98%, up to about 99%, up to about 100%, up to about 101%, up to about 102%, up to about 103%, up to about 104%, up to about 105%, up to about 106%, up to about 107%, up to about 108%, up to about 109%, up to about 1109%, up to about 1111%, up to about 112%, up to about 113%, up to about 114%, up to about 115%, up to about 116%, up to about 117%, up to about 118%, up to about 119%, up to about 120%, up to about 121%, up to about 122%, up to about 123%, up to about 124%, up to about 125%, up to about 126%, up to about 127%, up to about 128%, up to about 129%, up to about 130%, up to about 131%, up to about 132%, up to about 133%, up to about 134%, up to about 135%, up to about 13 It may comprise, consist essentially of, or consist of a nucleotide sequence having about 89%, up to about 90%, up to about 91%, up to about 92%, up to about 93%, up to about 94%, up to about 95%, up to about 95.5%, up to about 96%, up to about 96.5%, up to about 97%, up to about 97.5%, up to about 98%, up to about 98.5%, up to about 99%, or up to about 99.5% sequence identity.

[0064] In some embodiments, the transcription promoter comprises, consists essentially of, or consists of a nucleotide sequence having about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%, or about 100% sequence identity to any one of SEQ ID NOs: 1 and 56-72.

[0065] In some embodiments, the transcription promoter comprises, consists essentially of, or consists of the nucleotide sequence of any one of SEQ ID NOs: 1 and 56-72.

[0066] In some embodiments, the transcription promoter comprises one or more insertions, deletions, and / or substitutions relative to any one of SEQ ID NOs: 1 and 56-72.

[0067] For example, the transcription promoter can comprise a nucleotide sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 nucleotide insertion relative to any one of SEQ ID NOs: 1 and 56-72.

[0068] In some embodiments, the transcription promoter comprises a nucleotide sequence having an insertion of at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 nucleotides relative to any one of SEQ ID NOs: 1 and 56-72.

[0069] In some embodiments, the transcription promoter comprises a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 nucleotide insertion relative to any one of SEQ ID NOs: 1 and 56-72.

[0070] The one or more insertions may be at the 5' end, the 3' end, within the nucleotide sequence, or a combination thereof. The one or more insertions may be contiguous, discontinuous, or a combination thereof.

[0071] In some embodiments, the transcription promoter comprises a nucleotide sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 nucleotide deletions relative to any one of SEQ ID NOs: 1 and 56-72.

[0072] In some embodiments, the transcription promoter comprises a nucleotide sequence having at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, or at most 50 nucleotide deletions relative to any one of SEQ ID NOs: 1 and 56-72.

[0073] In some embodiments, the transcription promoter comprises a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 nucleotide deletion relative to any one of SEQ ID NOs: 1 and 56-72.

[0074] The one or more deletions can be at the 5' end, the 3' end, within the nucleotide sequence, or a combination thereof. The one or more deletions can be contiguous, non-contiguous, or a combination thereof.

[0075] In some embodiments, the transcription promoter comprises a nucleotide sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 nucleotide substitutions relative to any one of SEQ ID NOs: 1 and 56-72.

[0076] In some embodiments, the transcription promoter comprises a nucleotide sequence having at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, or at most 50 nucleotide substitutions relative to any one of SEQ ID NOs: 1 and 56-72.

[0077] In some embodiments, the transcription promoter comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 nucleotide substitutions relative to any one of SEQ ID NOs: 1 and 56-72.

[0078] The one or more substitutions can be at the 5' end, the 3' end, within the nucleotide sequence, or a combination thereof. The one or more substitutions can be consecutive, non-consecutive, or a combination thereof.

[0079] A transcription promoter (or promoter) can describe a region of DNA involved in initiating or increasing transcription of a particular gene, such as a transgene disclosed herein. A promoter can be located near the transcription start site of a gene, on the same strand, and upstream of the coding sequence. A promoter can be, for example, approximately 100 to 1000 base pairs in length. A promoter can contain specific DNA sequences and response elements that provide initial binding sites for RNA polymerase and proteins called transcription factors that recruit RNA polymerase. A promoter can be part of an expression control system that includes other regulatory regions, such as enhancers, silencers, and / or boundary elements / insulators to direct the transcription level of a given gene. An enhancer can be a regulatory element located distant from the transcription start site. In some embodiments, enhancers that are naturally distant from the transcription start site are located relatively close to the transcription start site in the polynucleotide constructs disclosed herein.

[0080] A transcription promoter can contain a transcription factor binding site. A transcription promoter can contain two or more transcription factor binding sites.

[0081] A transcription promoter can be, comprise, consist essentially of, or consist of a core promoter, e.g., the minimum portion of a promoter required to initiate transcription. A core promoter can include, for example, (1) a transcription start site (TSS), (2) an RNA polymerase binding site (e.g., an RNA polymerase II binding site in the promoter of a gene encoding a messenger RNA), (3) a general transcription factor binding site (e.g., a TATA box with the consensus sequence TATAAA can be a binding site for TATA-binding protein (TBP)), (4) a B recognition element (BRE), (5) a proximal promoter (e.g., approximately 250 bp) containing regulatory elements, (6) a transcription factor binding site (e.g., an E box with the sequence CACGTF, which is a binding site for basic helix-loop-helix (bHLH) transcription factors including BMAL11-Clock and cMyc), and / or (7) a distal promoter containing additional regulatory elements.

[0082] The transcription promoter disclosed herein can be, for example, (1) an AT-based class promoter, (2) a CG-based class promoter, (3) an ATCG compact class promoter, (4) an ATCG-balanced class promoter, (5) an ATCG middle class promoter, (6) an ATCG-less class promoter, (7) an AT-less class promoter, (8) a CG spike class promoter, (9) a CG-less class promoter, or (10) an AT spike class promoter.

[0083] The transcription promoters disclosed herein can be unidirectional promoters. The transcription promoters disclosed herein can be bidirectional promoters.

[0084] Transcription factors can be sequence-specific DNA-binding factors that bind to specific sequences within a transcriptional promoter, thereby regulating transcription of genes (e.g., transgenes) that are operably adjacent to the promoter and downstream of it. Transcription factors can include activators, which promote transcription, and repressors, which block or negatively regulate transcription by reducing RNA polymerase recruitment or binding. Transcription factors can contain (1) one or more DNA-binding domains (DBDs) that facilitate sequence-specific binding to cognate transcription factor binding sites (e.g., response elements) within the transcriptional promoter, (2) one or more signal-sensing domains (SSDs), which may contain ligand-binding domains that respond to external signals; and / or (3) one or more transactivation domains (TADs), which contain binding sites for other proteins, including transcriptional coregulators.

[0085] Transcription factors can be classified according to the structural characteristics of their DNA-binding domains. They may contain a basic helix-loop-helix domain, a basic leucine zipper (bZIP) domain, a bipartite response regulator C-terminal effector domain, a GCC box domain, a helix-turn-helix domain, a homeodomain, a lambda repressor-like domain, a serum response factor-like (srf-like) domain, a paired box domain, a winged helix domain, a zinc finger domain, a multi-Cys2His2 zinc finger domain, a Zn2Cys6 domain, and / or a Zn2Cys8 nuclear receptor zinc finger domain.

[0086] In some embodiments, the promoters disclosed herein improve the selectivity of expression of the polynucleotide constructs disclosed herein compared to mRNA payloads that can be translated by any cell to which they are delivered.

[0087] In some embodiments, the promoters disclosed herein are inducible promoters. In some embodiments, the promoters disclosed herein are not inducible promoters. In some embodiments, the promoters disclosed herein are constitutive promoters (e.g., induce substantially constitutive expression in adipocytes or white adipocytes). In some embodiments, the promoters disclosed herein are not constitutive promoters.

[0088] The expression regulatory region disclosed herein may comprise an appropriate number of promoters. The expression regulatory region may comprise at least one, at least two, at least three, at least four, or at least five promoters. The expression regulatory region may contain up to one, up to two, up to three, up to four, or up to five promoters. The expression regulatory region may comprise one, two, three, four, or five promoters.

[0089] B. Cytotoxic proteins The polynucleotide construct may include a transgene encoding a therapeutic protein, such as a cytotoxic protein. Expression of the transgene may be driven by a transcription promoter disclosed herein. The transgene may be operably linked to a transcription promoter disclosed herein. The transgene may be under the regulatory control of a transcription promoter disclosed herein.

[0090] A cytotoxic protein can reduce, prevent, and / or substantially eliminate the growth or survival of cells that express it, eg, adipocytes, such as white adipocytes.

[0091] In some embodiments, the cytotoxic protein induces a non-inflammatory form of cell death. In some embodiments, the cytotoxic protein induces a programmed form of cell death. In some embodiments, the cytotoxic protein induces cell death by apoptosis. In some embodiments, the cytotoxic protein induces an inflammatory form of cell death.

[0092] The cytotoxic protein can comprise, consist essentially of, or consist of a caspase or its catalytic domain. The cytotoxic protein can be a caspase, e.g., caspase 1, caspase 3, caspase 8, or caspase 9. The cytotoxic protein can comprise the catalytic domain of a caspase, e.g., the catalytic domain of caspase 1, caspase 3, caspase 8, or caspase 9. Proteins of the caspase family can execute genetic programs that result in cell death.

[0093] The cytotoxic protein may be, comprise, consist essentially of, or consist of caspase 1 or its catalytic domain. The cytotoxic protein may be, comprise, consist essentially of, or consist of caspase 3 or its catalytic domain. The cytotoxic protein may be, comprise, consist essentially of, or consist of caspase 8 or its catalytic domain. The cytotoxic protein may be, comprise, consist essentially of, or consist of caspase 9 or its catalytic domain.

[0094] The cytotoxic protein may be, comprise, consist essentially of, or consist of an uninducible caspase, such as uninducible caspase 1, caspase 3, caspase 8, or caspase 9, or an uninducible protein comprising the catalytic domain of caspase 1, caspase 3, caspase 8, or caspase 9.

[0095] The cytotoxic protein may be, comprise, consist essentially of, or consist of an autoactivating caspase, such as autoactivating caspase 1, autoactivating caspase 3, autoactivating caspase 8, or autoactivating caspase 9, or an autoactivating protein containing the catalytic domain of caspase 1, caspase 3, caspase 8, or caspase 9. Autoactivating caspases can be activated in the absence of an inducer, e.g., a chemical inducer of dimerization (CID) such as rapamycin. Autoactivating caspases can be advantageously used, for example, to induce apoptosis in rapidly dividing cells, where the inducible caspase protein is diluted prior to administration of the inducer.

[0096] The cytotoxic protein may be, comprise, consist essentially of, or consist of an inducible caspase, e.g., inducible caspase 1, inducible caspase 3, inducible caspase 8, or inducible caspase 9, or an inducible protein comprising the catalytic domain of caspase 1, caspase 3, caspase 8, or caspase 9.

[0097] Inducible cytotoxic proteins, such as the inducible caspases disclosed herein, can be inactive until contacted with chemical or biological compounds that activate cytotoxic proteins.Inducible cytotoxic proteins can provide an additional layer of regulation for the activity of cytotoxic proteins beyond, for example, the promoter that is preferentially active in adipocytes or white adipocytes.For example, to induce apoptosis of adipocytes, it may be necessary to contact adipocytes with an inducer (for example, rapamycin or its structural analogue) and / or administer the inducer to a subject.

[0098] The inducible cytotoxic proteins, such as the inducible caspases disclosed herein, can be activated by contact with a macrolide. The inducible cytotoxic proteins, such as the inducible caspases disclosed herein, can be activated by contact with rapamycin or a structural analog thereof. The inducible cytotoxic proteins, such as the inducible caspases disclosed herein, can be activated by contact with another inducer, such as AP20187.

[0099] The inducible cytotoxic protein may comprise caspase 9 fused to human FK506 binding protein (FKBP) to allow conditional dimerization with the small molecule AP20187, which may be a synthetic analog of FK506.

[0100] The inducible cytotoxic protein can be a rapamycin-induced cytotoxic protein. For example, the cytotoxic protein can comprise, consist essentially of, or consist of a rapamycin-induced caspase, such as rapamycin-induced caspase 1, rapamycin-induced caspase 3, rapamycin-induced caspase 8, or rapamycin-induced caspase 9, or a rapamycin-induced protein containing the catalytic domain of caspase 1, caspase 3, caspase 8, or caspase 9.

[0101] Rapamycin-inducible cytotoxic proteins can utilize a dual rapamycin induction system for caspases 3 and 9 that utilizes RU486 and a chemical dimerization inducer (CID).

[0102] Rapamycin-inducible cytotoxic proteins can utilize the rapamycin-inducible caspase-8 system by using the ARIAD™ homodimerization system (FKC8; ARIAD Pharmaceuticals).

[0103] The rapamycin-induced cytotoxic protein may comprise full-length rapamycin-induced caspase-9. For example, the rapamycin-induced cytotoxic protein may comprise a caspase recruitment domain (CARD; GenBank NM001 229) linked to two 12 kDa human FK506-binding proteins. The FK506-binding protein may be, for example, FKBP12 (GenBank AH002818), optionally containing the F36V mutation. A linker (e.g., a Ser-Gly-Gly-Gly-Ser linker, or another linker disclosed herein) may link the FK506-binding protein and / or FKBP to caspase-9.

[0104] The rapamycin-induced cytotoxic protein may contain a dimerization domain, such as an FKBP, FK506, and / or FRB-binding protein domain, that binds to rapamycin or its structural analogs. Exemplary genes (e.g., human genes) encoding FKBP domains include AIP, AIPL1, FKBP1A, FKBP1B, FKBP2, FKBP3, FHBP5, FKBP6, FKBP7, FKBP8, FKBP9L, FKBP10, FKBP11, FKBP14, FKBP15, FKBP52, and LOC541473.

[0105] Rapamycin and rapamycin analogs can induce dimerization (e.g., heterodimerization) by creating an interface between the FRB domain of mTOR and FKBP12. This association results in FKBP12 blocking access to the mTOR active site, thereby inhibiting its function. Although mTOR is a very large protein, the precise small segment of mTOR required for interaction with rapamycin is known and can be used in rapamycin-inducible cytotoxic proteins to promote dimerization and activation. Dimerization mediated by rapamycin or its structural analogs can be used to induce dimerization of rapamycin-inducible cytotoxic proteins (e.g., caspases), including multi-domain rapacaspase proteins. Dimerization can be, for example, heterodimerization (e.g., of domains on different polypeptide chains or the same polypeptide chain) or homodimerization (e.g., of domains on different polypeptide chains or the same polypeptide chain).

[0106] Rapamycin-inducible cytotoxic proteins, including rapamycin-inducible caspases, can include (i) an FRB domain (e.g., from, based on, or derived from mTOR), (ii) an FKBP12 domain, and (iii) a caspase or a functional fragment thereof. Heterodimerization between the FRB domain of a first rapa-caspase fusion protein and the FKB12 ​​domain of a second rapa-caspase fusion protein can activate caspase activity. In some embodiments, the first heterodimerization domain of the rapamycin-inducible cytotoxic proteins disclosed herein includes an FK506-binding protein (FKBP), and the second heterodimerization domain includes an FRB domain (e.g., from, based on, or derived from mTOR). Rapamycin-inducible cytotoxic proteins can include one polypeptide chain (e.g., having a heterodimerizing domain) or two dimerizing polypeptide chains.

[0107] Rapamycin or a structural analog thereof can bind with high affinity to the FKBP12 protein, forming a drug-protein conjugate, which then binds to a second protein or domain, such as the FKBP-rapamycin binding (FRB) domain or a derivative thereof. The FRB domain of mTOR can comprise a polypeptide of approximately 89 amino acids.

[0108] Rapamycin-induced cytotoxic proteins can be activated by rapamycin. Rapamycin-induced cytotoxic proteins can be activated by structural analogs of rapamycin, such as FK506, C-20-methyltolylrapamycin (MaRap), C16(S)-butylsulfonamidorapamycin (C16-BS-Rap), C16-(S)-7-methylindoleramycin (AP21976 / C16-AiRap), C16-(S)-3-methylindoleramycin (C16-iRap), sirolimus, tacrolimus, everolimus, temsirolimus, or deforolimus. Rapamycin structural analogs can be functionalized at C16 and / or C20 of rapamycin. Contacting cells expressing a rapamycin-inducible cytotoxic protein with rapamycin or a structural analog thereof can promote dimerization of the rapaCasp9 protein, which, in some embodiments, induces apoptosis in target cells, such as adipocytes.

[0109] Table 2 provides non-limiting examples of cytotoxic proteins and domains thereof disclosed herein. FKBP12 may describe the amino acid sequence of FKBP12. dCasp9 may describe the catalytic domain of Casp9. L1 may describe a one-repeat linker. FMD-2A may describe the foot and mouth disease 2A-like peptide ERAV. Optionally, another 2A peptide disclosed herein can be used, for example, instead. FRB may describe the FRB domain of mTOR. L3 may describe a two-repeat linker. FRBw may describe codon wobble FRB. Optionally, another linker disclosed herein, or repeats thereof, can be used in place of the linker sequence in any of the sequences in Table 2.

[0110] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0111] The cytotoxic proteins disclosed herein may have a cytotoxicity of at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least about 124%, at least about 125%, at least about 126%, at least about 127%, at least about 128%, at least about 129%, at least about 130%, at least about 131%, at least about 132%, at least about 133%, at least about 134%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or similarity.

[0112] The cytotoxic proteins disclosed herein may be at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87% of at least 100 consecutive amino acids of any one of SEQ ID NOS: 3-13 and 52-55. , at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or similarity.

[0113] The cytotoxic proteins disclosed herein may be at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87% of at least 250 consecutive amino acids of any one of SEQ ID NOS: 3-13 and 52-55. , at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or similarity.

[0114] The cytotoxic proteins disclosed herein may have a cytotoxicity of up to about 70%, up to about 71%, up to about 72%, up to about 73%, up to about 74%, up to about 75%, up to about 76%, up to about 77%, up to about 78%, up to about 79%, up to about 80%, up to about 81%, up to about 82%, up to about 83%, up to about 84%, up to about 85%, up to about 86%, up to about 87%, up to about 88%, up to about 89%, up to about 90%, up to about 91%, up to about 92%, up to about 93%, up to about 94%, up to about 95%, up to about 96%, up to about 97%, up to about 98%, up to about 99%, up to about 100%, up to about 101%, up to about 102%, up to about 103%, up to about 104%, up to about 105%, up to about 106%, up to about 107%, up to about 108%, up to about 109%, up to about 110%, up to about 111%, up to about 112%, up to about 113%, up to about 114%, up to about 115%, up to about 116%, up to about 117%, up to about 118%, up to about 119%, up to about 120%, up to about 121%, up to about 122%, up to about 123%, up to about 124%, up to about 125%, up to about 126%, up to about 127%, up to about 128%, up to about 129%, up to about 130%, up to about 131%, up to about 132%, up to about 133%, up to about 134%, up to about It may comprise, consist essentially of, or consist of an amino acid sequence having about 88%, up to about 89%, up to about 90%, up to about 91%, up to about 92%, up to about 93%, up to about 94%, up to about 95%, up to about 95.5%, up to about 96%, up to about 96.5%, up to about 97%, up to about 97.5%, up to about 98%, up to about 98.5%, up to about 99%, or up to about 99.5% sequence identity or similarity.

[0115] In some embodiments, the cytotoxic protein comprises, consists essentially of, or consists of an amino acid sequence having about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%, or about 100% sequence identity or similarity to any one of SEQ ID NOs: 3-13 and 52-55.

[0116] In some embodiments, the cytotoxic protein comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 3-13 and 52-55.

[0117] In some embodiments, the cytotoxic protein comprises one or more insertions, deletions, and / or substitutions relative to any one of SEQ ID NOs: 3-13 and 52-55.

[0118] For example, the cytotoxic protein can comprise an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acid insertions relative to any one of SEQ ID NOs: 3-13 and 52-55.

[0119] In some embodiments, the cytotoxic protein comprises an amino acid sequence having an insertion of at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, or at most 50 amino acids relative to any one of SEQ ID NOs: 3-13 and 52-55.

[0120] In some embodiments, the cytotoxic protein comprises a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acid insertion relative to any one of SEQ ID NOs: 3-13 and 52-55.

[0121] The one or more insertions may be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The one or more insertions may be contiguous, discontinuous, or a combination thereof.

[0122] In some embodiments, the cytotoxic protein comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acid insertions relative to any one of SEQ ID NOs: 3-13 and 52-55.

[0123] In some embodiments, the cytotoxic protein comprises an amino acid sequence having at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, or at most 50 amino acid deletions relative to any one of SEQ ID NOs: 3-13 and 52-55.

[0124] In some embodiments, the cytotoxic protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acid deletions relative to any one of SEQ ID NOs: 3-13 and 52-55.

[0125] The one or more deletions can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The one or more deletions can be contiguous, non-contiguous, or a combination thereof.

[0126] In some embodiments, the cytotoxic protein comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acid substitutions relative to any one of SEQ ID NOs: 3-13 and 52-55.

[0127] In some embodiments, the cytotoxic protein comprises an amino acid sequence having at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, or at most 50 amino acid substitutions relative to any one of SEQ ID NOs: 3-13 and 52-55.

[0128] In some embodiments, the cytotoxic protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acid substitutions relative to any one of SEQ ID NOs: 3-13 and 52-55.

[0129] The one or more substitutions may be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The one or more substitutions may be consecutive, non-consecutive, or a combination thereof.

[0130] The cytotoxic protein may be, may comprise, may consist essentially of, or may consist of BAX, DFF40, Herpes Simplex Virus Thymidine Kinase (HSV-TK), cytosine deaminase, or a catalytic domain thereof.

[0131] The cytotoxic protein may be, may comprise, may consist essentially of, or may consist of inducible (e.g., rapamycin-inducible) BAX, DFF40, Herpes Simplex Virus Thymidine Kinase (HSV-TK), cytosine deaminase, or a catalytic domain thereof.

[0132] The cytotoxic protein may be, may comprise, may consist essentially of, or may consist of non-inducible BAX, DFF40, Herpes Simplex Virus Thymidine Kinase (HSV-TK), cytosine deaminase, or a catalytic domain thereof.

[0133] The cytotoxic protein may be, may comprise, may consist essentially of, or may consist of autoactivated BAX, DFF40, Herpes Simplex Virus Thymidine Kinase (HSV-TK), cytosine deaminase, or a catalytic domain thereof.

[0134] DNA fragmentation factor (DFF) can be a complex of the DNase DFF40 (CAD) and its chaperone / inhibitor DFF45 (ICAD-L). In its inactive form, DFF can be a heterodimer composed of a 45 kDa chaperone inhibitor subunit (DFF45 or ICAD) and a 40 kDa latent endonuclease subunit (DFF40 or CAD). Upon caspase-3 cleavage of DFF45, DFF40 forms an active endonuclease homo-oligomer. Active DFF can induce DNA fragmentation. DNA binding by DFF is mediated by a nuclease subunit that can also form a stable DNA complex after release from DFF. The nuclease subunit inhibits DNA cleavage but not DNA binding. DFF45 can also be cleaved and inactivated by caspase-7. The cleaved DFF45 fragment dissociates from DFF40, allowing DFF40 to oligomerize and form a large complex that cleaves DNA by introducing double-strand breaks. Histone H1 confers DNA-binding ability to DFF and stimulates the nuclease activity of DFF40.

[0135] Thymidine kinase (TK) is an ATP-thymidine 5'-phosphotransferase that can be present in living cells and certain viruses, including herpes simplex virus (HSV), varicella-zoster virus (VZV), and Epstein-Barr virus (EBV). Thymidine kinase converts deoxythymidine to deoxythymidine 5'-monophosphate (TMP), which is phosphorylated by thymidylate kinase and nucleoside diphosphate kinase to deoxythymidine diphosphate and deoxythymidine triphosphate, respectively. Deoxythymidine triphosphate can be incorporated into cellular DNA by DNA polymerases and viral reverse transcriptases. Once incorporated into DNA, certain dNTP analogs, such as synthetic analogs of 2'-deoxyguanosine (e.g., ganciclovir), cause premature termination of DNA synthesis, inducing cellular apoptosis. In some embodiments, the disclosed polynucleotide constructs and systems can utilize a transgene encoding HSV-TK. After administering to a human a polynucleotide construct or system using a transgene encoding HSV-TK, a 2'-deoxynucleotide analog, such as 2'-deoxyguanosine, can be administered to the human. HSV-TK efficiently converts the 2'-deoxynucleotide analog into a dNTP analog, which, when incorporated into DNA, can induce apoptosis in target cells.

[0136] Cytosine deaminase (CD) catalyzes the hydrolytic conversion of cytosine in DNA to uracil and ammonia. When a CD-modified site is recognized by an endonuclease, the phosphodiester bond is cleaved and repaired by incorporating a new cytosine in normal cells. In the presence of 5-fluorocytosine (5-FC), cytosine deaminase converts 5-FC to 5-fluorouracil (5-FU), which can inhibit target cell proliferation. Therefore, transgenic expression of CD in target cells can reduce the growth and / or survival of target cells.

[0137] In some embodiments, the cytotoxic protein induces cell death by activating an intrinsic cell death pathway (eg, activating one or more caspases).

[0138] Non-limiting examples of cytotoxic proteins that can be used include those described in US20170354682A1, WO2008 / 154644, US2011 / 0286980, US20230065562A1, Stavrou, Mol. Therapy 26(5):1266-1276(2018), Xie et al., Cancer Res 61(18):186-91(2001), Carlotti et al., Cancer Gene Ther 12(7):627-39(2005), Lowe et al., Gene Ther 8(18):1363-71(2001), and Shariat et al., Cancer Res 61(6):2562-71(2001), Liu et al., J Biol Chem 274(20):13836-40(1999), Shah et al., Genesis 45(4):104-199 (2007), Straathof et al., Blood 105(11):4247-4254(2005), Carlotti et al., Cancer Gene Ther 12(7):627-39(2005), Clackson et al., Proc. Natl. Acad. Sci. USA 95:10437-10442(1998), Gargett T, Brown MP. The inducible caspase-9 suicide gene system as a “safety switch” to limit on-target, off-tumor toxicities of chimeric antigen receptor T cells. Front Pharmacol. 2014 Oct 28;5:235, Zhou X, Brenner MK. Improving the safety of T-cell therapies using an inducible caspase-9 gene. Exp Hematol.2016 Nov;44(11):1013-1019, Falcon et al. Combinatorial suicide gene strategies for the safety of cell therapies. Front Immunol. 2022 Sep 14;13:975233, and Bouquet et al. "RapaCaspase-9-based suicide gene applied to the safety of IL-1RAP CAR-T cells." Gene Therapy (2023):1-8, each of which is incorporated herein by reference in its entirety.

[0139] C. Extracellular matrix remodeling factors The polynucleotide construct disclosed herein can include a transgene encoding an extracellular matrix remodeling factor.The extracellular matrix remodeling factor can function, for example, in rebuilding the extracellular matrix after adipocyte apoptosis.The transgene encoding the extracellular matrix remodeling factor can be under the regulatory control of the same promoter as the cytotoxic protein or a different promoter.The transgene encoding the extracellular matrix remodeling factor and the transgene encoding the cytotoxic protein can be part of a fusion protein and can be separated by a 2A cleavable linker or a self-cleaving linker as disclosed herein.

[0140] In some embodiments, the extracellular matrix remodeling factor is a matrix metalloproteinase, such as MMP-1, MMP-3, MMP-8, MMP-10, MMP-11, MMP-12, MMP-13, MMP-21, MMP-27, MMP-7, MMP-26, MMP-2, MMP-9, MMP-14, MMP-15, MMP-16, MMP-17, MMP-24, or MMP-25. In some embodiments, the extracellular matrix remodeling factor is a thrombospondin-associated motif (ADAMTS) protein, e.g., ADAMTS-1, ADAMTS-4, ADAMTS-5, ADAMTS-8, ADAMTS-9, ADAMTS-15, ADAMTS-20, ADAMTS-2, ADAMTS-3, ADAMTS-14, ADAMTS-13, ADAMTS-7, ADAMTS-12, ADAMTS-6, ADAMTS-10, ADAMTS-16, ADAMTS-17, ADAMTS-18, or ADAMTS-19. In some embodiments, the extracellular matrix remodeling factor is a serine proteinase, e.g., plasmin or cathepsin G. In some embodiments, the extracellular matrix remodeling factor is a cysteine ​​protease, e.g., cathepsin B or cathepsin L.

[0141] D. Safety elements The polynucleotide constructs disclosed herein may contain safety elements to regulate the expression or activity of a transgene, therapeutic protein, or cytotoxic protein disclosed herein in an appropriate cell (e.g., an adipocyte or white adipocyte), and / or to limit the expression or activity of a transgene, therapeutic protein, or cytotoxic protein in a control cell (e.g., a non-adipocyte, e.g., a muscle cell, hepatocyte, bone cell, erythrocyte, neuron, leukocyte, lymphocyte, monocyte, or fibroblast, a hematopoietic lineage cell, a cell of the central or peripheral nervous system, a dorsal root ganglion neuron, or a cell that is not a white adipocyte, e.g., a brown adipocyte) or tissue (e.g., skeletal muscle, cardiac muscle, pancreas, gastrointestinal tract).

[0142] In some embodiments, the safety element is a regulatory RNA, e.g., a small interfering RNA (siRNA). In some embodiments, the siRNA is identical, substantially identical, or a variant thereof (e.g., may include a complement, reverse complement, and / or RNA equivalent thereof) that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identical to a portion of any one of SEQ ID NOs: 2 and 14-17. In some embodiments, the regulatory RNA may be a microRNA (miRNA). In some embodiments, the miRNA is identical to, or substantially identical to, a portion of any one of SEQ ID NOS: 2 and 14-17, or a variant thereof (e.g., which may include a complement, reverse complement, and / or its RNA equivalent) that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identical to a portion of any one of SEQ ID NOS: 2 and 14-17. The portion may be at least 10, at least 15, at least 20, or at least 30 nucleotides. The portion may be at most 20, at most 30, at most 50, or at most 100 nucleotides.

[0143] In some embodiments, the safety element comprises a target site for a regulatory RNA, miRNA, or siRNA, for example, in an untranslated region.

[0144] MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression. They function by binding to complementary sequences on target messenger RNAs (mRNAs), resulting in either mRNA degradation or translation inhibition. This interaction with mRNA occurs primarily in the 3' untranslated region (3'UTR). In gene therapy, miRNAs have been utilized to improve transgene specificity and minimize off-target effects. By incorporating tissue-specific miRNA target sites (miRTs) into the 3'UTR of transgene mRNAs, expression can be selectively inhibited in specific tissues where these miRNAs are abundant, enhancing the precision of the gene therapy applications and adipocyte targeting systems and methods disclosed herein. Examples and characteristics of tissue-specific miRNA target sequences that can be used in the compositions and methods disclosed herein are provided in Table 3, and exemplary sequences are provided in Table 4.

[0145] In some embodiments, the safety element or site targeted by the safety element comprises, is identical to, is substantially identical to, is the complement, or is the reverse complement, or is the RNA equivalent of any one of SEQ ID NOs: 73-90, or is a variant thereof (e.g., may comprise the complement, reverse complement, and / or its RNA equivalent) that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identical to a portion of any one of SEQ ID NOs: 73-90. The portion may be at least 10, at least 15, at least 20, or at least 30 nucleotides. The portion may be at most 20, at most 30, at most 50, or at most 100 nucleotides.

[0146] The polynucleotide constructs or systems disclosed herein may use one safety element or multiple safety elements, e.g., two, three, four, five, or more regulatory RNAs and / or sites targeted by regulatory RNAs.

[0147] [Table 3]

[0148] Table 4: Exemplary miRNA sequences. Optionally, oligonucleotides are provided that can be annealed to generate sequences containing multiple copies of the miR-TS. The underline indicates the miR target site (TS) complementary to the tissue-specific miRNA.

[0149] [Table 4]

[0150] Non-limiting examples of regulatory RNAs and regulatory RNA target sites that can be utilized include those described in the following publications: Brown et al., "Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer." Nat Med. 2006 May;12(5):585-91; Brown et al., "A microRNA-regulated lentiviral vector mediates stable correction of hemophilia B mice." Blood. 2007 Dec 15;110(13):4144-52; Wolff et al., "Effect of tissue-specific promoters and microRNA recognition elements on stability of transgene expression after hydrodynamic naked plasmid DNA delivery." Hum Gene Ther. 2009 Apr;20(4):374-88, Geisler et al. Application of mutated miR-206 target sites enables skeletal muscle-specific silencing of transgene expression of cardiotropic AAV9 vectors. Mol Ther. 2013 May;21(5):924-33, Greig et al.Intramuscular injection of AAV8 in mice and macaques is associated with substantial hepatic targeting and transgene expression. PLoS One. 2014 Nov 13;9(11):e112268, Qiao C et al. Liver-specific microRNA-122 target sequences incorporated in AAV vectors efficiently inhibits transgene expression in the liver. Gene Ther. 2011 Apr;18(4):403-10, Geisler et al. microRNA122-regulated transgene expression increases the specificity of cardiac gene transfer upon intravenous delivery of AAV9 vectors. Gene Ther. 2011 Feb, 18(2):199-209, Trepel et al. Treatment of multifocal breast cancer by systemic delivery of dual-targeted adeno-associated viral vectors. Gene Ther. 2015 Oct;22(10):840-7, Xie et al.MicroRNA-regulated, systemically delivered rAAV9: a step closer to CNS-restricted transgene expression. Mol Ther. 2011 Mar;19(3):526-35. Won et al. Targeted anticancer effect through microRNA-181a regulated tumor-specific hTERT replacement. Cancer Lett. 2015 Jan 28;356(2 Pt B):918-28. Colin et al. Engineered lentiviral vector targeting astrocytes in vivo. Glia. 2009 Apr 15;57(6):667-79. Ruiz et al. MicroRNA-Detargeted Mengovirus for Oncolytic MicroRNA-detargeted mengovirus for oncolytic virotherapy. J Virol. 2016 Mar 28;90(8):4078-4092, Hordeaux et al. MicroRNA-mediated inhibition of transgene expression reduces dorsal root ganglion toxicity by AAV vectors in primates. Sci Transl Med. 2020 Nov 11;12(569):eaba9188, and Baertsch et al. MicroRNA-mediated multi-tissue detargeting of oncolytic measles virus.Cancer Gene Ther. 2014 Sep;21(9):373-80, each of which is incorporated herein by reference for such disclosure.

[0151] In some embodiments, the safety element can induce transcriptional repression. In some embodiments, the safety element can be a transcriptional repressor, such as Snail (SNAI1) protein, B-cell lymphoma 6 (BCL6) protein, nuclear factor of activated T cells, cytoplasmic 4 (NFATC4) protein, or activating transcription factor 3 (ATF-3) protein.

[0152] In some embodiments, expression of the safety element (or regulatory RNA targeting a target site in the safety element) is driven by a non-adipocyte promoter (e.g., a promoter that is not active, substantially inactive, or minimally active in adipocytes may be active in one or more other cell types, e.g., a control cell type disclosed herein). The promoter may be an endogenous, exogenous, or engineered promoter. In some embodiments, the regulatory RNA targeting a target site in the safety element is endogenously expressed in one or more control (e.g., non-adipocyte) tissues. In some embodiments, the non-adipocyte promoter may be a hepatocyte-specific promoter, e.g., an albumin (ALB) promoter, an alpha-fetoprotein (AFP) promoter, or a transthyretin (TTR) promoter. In some embodiments, the non-adipocyte promoter may be a leukocyte-specific promoter, e.g., a lymphocyte-specific protein-1 (LSP1) promoter, or a CD11a (ITGAL) promoter.

[0153] E. Further Embodiments of Polynucleotide Constructs The polynucleotide construct may include a polyadenylation (poly(A)) signal that can direct the formation of the 3' end of the mRNA and the addition of poly(A) to the 3' end of the mRNA. In some embodiments, the poly(A) signal may be or include BGH pA (CpG-free long). In some embodiments, the poly(A) signal may be or include a β-globin poly(A) signal.

[0154] The polynucleotide construct may lack an origin of replication. The polynucleotide construct may include an origin of replication.

[0155] The polynucleotide construct may include an antibiotic resistance gene that may allow for positive selection of cells (e.g., bacterial cells) containing the disclosed polynucleotide construct during the vector purification process. In some embodiments, the polynucleotide construct lacks an antibiotic resistance gene.

[0156] The polynucleotide construct may include a base of mobility (BOM) region that can function in bacterial conjugation and horizontal transfer of extrachromosomal plasmids. In some embodiments, the polynucleotide construct lacks a BOM region.

[0157] In some embodiments, the polynucleotides or expression constructs disclosed herein comprise natural, synthetic, and / or artificial nucleotide analogs or bases. In some embodiments, the synthetic or artificial nucleotide analogs or bases comprise modifications to one or more of the deoxyribose moiety, ribose moiety, phosphate moiety, nucleoside moiety, or combinations thereof.

[0158] In some embodiments, the transgene (e.g., encoding a cytotoxic protein, extracellular matrix remodeling factor, or safety element disclosed herein) is codon-optimized. In some embodiments, the transgene (e.g., encoding a cytotoxic protein, extracellular matrix remodeling factor, or safety element) is a codon-optimized version of a transgene or transgene sequence disclosed herein.

[0159] Codon optimization can be used, for example, to increase expression in human cells, such as adipocytes. Codon-optimized coding regions can be designed by a variety of different methods, including published, publicly available, or commercially available methods. Because the genetic code is degenerate (i.e., each amino acid can be coded for by an average of three different codons), DNA sequences can be modified by synonymous nucleotide substitutions without changing the amino acid sequence of the encoded protein.

[0160] Exemplary codon optimization methods are described, for example, in U.S. Pat. No. 7,561,972; U.S. Patent Nos. 7,561,972, 7,561,973 and 7,888,112, and International Patent Application Publication No. WO2015 / 012924, which are incorporated herein by reference for their disclosure.The transgene sequence encoding a product (for example, the cytotoxic protein, extracellular matrix remodeling factor or safety element disclosed herein) can be modified with synonymous codon sequences.Codon optimization can include the use of any suitable available codon frequency table, including those disclosed or referenced in U.S. Patent Nos. 7,561,972, 7,561,973 and 7,888,112 and International Patent Application Publication No. WO2015 / 012924, which are incorporated herein by reference for their disclosure. Codons can be selected for a particular tissue or cell type, for example, white adipose tissue or white adipocytes.

[0161] In some embodiments, the entire length of the product open reading frame (ORF) is modified. In some embodiments, only a fragment of the ORF is altered. By using one of these methods, codon frequencies can be applied to any given polypeptide sequence to generate a nucleic acid fragment of a codon-optimized coding region that encodes the polypeptide.

[0162] In some embodiments, the polynucleotide constructs or polynucleotides disclosed herein may be or comprise single-stranded DNA.

[0163] Polynucleotide constructs can be assembled by various methods, such as automated solid-phase synthesis. Polynucleotide constructs can be constructed using standard solid-phase DNA / RNA synthesis. Polynucleotide constructs can also be constructed using synthetic procedures. Polynucleotide constructs can be synthesized manually or in a fully automated manner. Polynucleotide constructs can be recombinant nucleic acids. In some cases, the synthesis procedure can include a 5'-hydroxyl oligonucleotide, which is first converted to the corresponding 5'-H-phosphonate monoester, then oxidized to an activated 5'-phosphorimidazolidate in the presence of imidazole, and finally reacted with pyrophosphate on a solid support. This procedure can include a post-synthesis purification step, such as PAGE, HPLC, MS, or any combination thereof. Polynucleotides can be commercially purchased.

[0164] II. Target cells The target cells in which the transcriptional promoters disclosed herein are active can be adipocytes, such as white adipocytes (e.g., adipose / fat cells). White adipocytes can form as a result of excess calorie accumulation.

[0165] In some embodiments, the target cell is not a brown adipocyte. Brown adipocytes can generate heat by burning calories, e.g., in the process of non-shivering thermogenesis. In some embodiments, the target cell can be a single adipocyte. A plurality of adipocytes can be target cells, e.g., as a population of target cells. In some embodiments, the target cell comprises a brown adipocyte.

[0166] The target cell or cells can be a population of adipocytes in adipose tissue. In some embodiments, the target cell population can be a population of adipocytes in adipose tumors (e.g., lipomas).

[0167] The target cell or cells may be present in subcutaneous fat. The target cell or cells may be present in abdominal fat. The target cell or cells may be present in visceral fat. The target cell or cells may be present in liver fat.

[0168] The target cell or cells may be located in a specific part of the subject's body, e.g., a body cavity or anatomical region. In some embodiments, the adipocyte or cells targeted by a composition, system, or method disclosed herein is located in the abdominal cavity of the subject. In some embodiments, the adipocyte or cells targeted by a composition, system, or method disclosed herein is located in the abdominal cavity of the subject. In some embodiments, the adipocyte or cells targeted by a composition, system, or method disclosed herein is located in the thoracic, pelvic, thoracic, or pericardial cavity of the subject. In some embodiments, the adipocyte or cells targeted by a composition, system, or method disclosed herein is located in the leg of the subject. In some embodiments, the adipocyte or cells targeted by a composition, system, or method disclosed herein is located in the arm of the subject. In some embodiments, the adipocyte or cells targeted by a composition, system, or method disclosed herein is located in the torso of the subject. In some embodiments, the adipocyte or adipocytes targeted by the compositions, systems, or methods disclosed herein are located in the back of a subject. In some embodiments, the adipocyte or adipocytes targeted by the compositions, systems, or methods disclosed herein are located in the neck of a subject. In some embodiments, the adipocyte or adipocytes targeted by the compositions, systems, or methods disclosed herein are located in the head of a subject. In some embodiments, the adipocyte or adipocytes targeted by the compositions, systems, or methods disclosed herein are located in the liver of a subject. In some embodiments, the adipocyte or adipocytes targeted by the compositions, systems, or methods disclosed herein are located in omental fat, mesenteric fat, splenic fat, portal fat, or gonadal fat.

[0169] In some embodiments, the adipocyte or adipocytes targeted by the compositions, systems, or methods disclosed herein are located within multiple body cavities or anatomical regions, hi some embodiments, the adipocyte or adipocytes targeted by the compositions, systems, or methods disclosed herein are dispersed throughout the body of a subject.

[0170] III. Delivery Vectors The compositions, systems, and methods of the present disclosure can include or utilize, for example, a delivery vector for delivery of a polynucleotide construct or polynucleotide encoding a cytotoxic protein.

[0171] The delivery vector disclosed herein may be a lipid-based delivery vector (LDV). The LDV disclosed herein can facilitate delivery of the polynucleotide construct or polynucleotide disclosed herein and expression of the transgene of interest after in vivo administration to a subject. For example, the LDV disclosed herein can facilitate expression of a cytotoxic protein, extracellular matrix remodeling factor, or safety element after in vivo administration. The LDV disclosed herein can utilize an effective and re-administerable delivery platform that allows for high tolerability compared to alternative formulations or approaches. The LDV may include a lipid membrane and / or lipid bilayer. The LDV may exclude enveloped viral vectors.

[0172] The LDVs disclosed herein can include one or more (e.g., two or more, three or more, four or more, five or more, 1, 2, 3, 4, 5, or 6) lipids, such as 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG).2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), Dlin-KC2-DMA(KC2), DOBAQ, 18:1 EPC, DDAB, 18:0 EPC, 18:0 DAP, La-dioleoylphosphatidylcholine (DOPC), cholesterol, DF4C11PE (rac-2,3-di[11-(F-butyl)undecanoyl]glycero-1-phosphoethanolamine, distearoyl La-phosphatidylethanolamine [DS(9-yne)PE], 18:0 TAP, dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidic acid (DPPA); dimyristoylphosphatidine The phosphate group is selected from dimethicone, dimethyl phosphate (DMPA), distearoyl phosphatidic acid (DSPA), dipalmitoyl phosphatidylserine (DPPS), dimyristoyl phosphatidylserine (DMPS), distearoyl phosphatidylserine (DSPS), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), and 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA).

[0173] The LDV disclosed herein can comprise one or more ionizable lipids.The charge of the ionizable lipid can depend on the pH of the surrounding environment.Ionizable lipids include but are not limited to 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), DLin-MC3-DMA (MC3), DLin-KC2-DMA (KC2), DOBAQ, 18:1 EPC, DDAB, 18:0 EPC, 18:0 DAP, and 18:0 TAP.

[0174] In some embodiments, the ionizable lipids in the LDVs disclosed herein comprise, consist essentially of, or consist of DODAP. In some embodiments, the ionizable lipids in the LDVs disclosed herein comprise, consist essentially of, or consist of DODMA. In some embodiments, the ionizable lipids in the LDVs disclosed herein comprise, consist essentially of, or consist of DODAP and DODMA. In some embodiments, the ionizable lipids (e.g., the combinations or ratios referred to herein) do not include cationic lipids such as DOTMA and / or DOTAP.

[0175] The LDVs disclosed herein may contain one or more cationic lipids. Non-limiting examples of cationic lipids include 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). In some embodiments, the cationic lipids are used in LDVs at a sufficiently low amount to reduce proinflammatory responses (e.g., Th1-type cytokines or type I interferons) to the LDV compared to control lipid nanoparticles. In some embodiments, the LDVs are free of or substantially devoid of cationic lipids. In some embodiments, the LDVs are free of or substantially devoid of DOTAP. In some embodiments, the LDVs are free of or substantially devoid of DOTMA. In some embodiments, the LDVs are free of or substantially devoid of cationic lipids except for DOTAP. In some embodiments, the LDVs are free of or substantially devoid of cationic lipids except for DOTMA. In some embodiments, the LDV does not contain or is substantially devoid of cationic lipids except for DOTAP and DOTMA. In some embodiments, the cationic lipid in the LDV disclosed herein comprises DOTMA, consists essentially of DOTMA, or consists of DOTMA. In some embodiments, the cationic lipid in the LDV disclosed herein comprises DOTAP, consists essentially of DOTAP, or consists of DOTAP. In some embodiments, the cationic lipid in the LDV disclosed herein comprises, consists essentially of, or consists of DOTAP and DOTMA.

[0176] The LDVs disclosed herein may contain one or more helper lipids. Non-limiting examples of helper lipids include 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and La-dioleoylphosphatidylcholine (DOPC). In some embodiments, the helper lipid in the LDVs disclosed herein comprises, consists essentially of, or consists of DOPE. In some embodiments, the LDVs are free of, or substantially free of, DOPE and / or DOPC.

[0177] The LDV disclosed herein can contain one or more PEGylated lipids. A non-limiting example of a PEGylated lipid is 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG). In some embodiments, the PEGylated lipid in the LDV disclosed herein comprises, essentially consists of, or consists of DMG-PEG. In some embodiments, the LDV does not contain or is substantially free of DMG-PEG.

[0178] The LDVs disclosed herein may contain cholesterol. In some embodiments, the LDVs are free of or substantially devoid of cholesterol.

[0179] In some embodiments, the LDVs disclosed herein comprise a lipid combination at a molar ratio appropriate for reducing toxicity, immunogenicity, or proinflammatory responses associated with administration of the delivery vector. For example, the LDV may comprise a lipid combination at a molar ratio appropriate for reducing the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interferon-γ (IFN-γ), interleukin-6 (IL-6), type I interferon, or a combination thereof, associated with administration of the delivery vector. In some embodiments, the LDVs disclosed herein comprise a lipid combination at a molar ratio appropriate for reducing complement activation-associated pseudoallergy (CARPA). The reduction may be, for example, in comparison to control lipid nanoparticles containing a higher proportion of cationic lipid. The reduction may be determined by experiments administering empty LDVs or substantially non-immunogenic cargo (e.g., a polynucleotide construct or polynucleotide encoding a reporter such as GFP). In some embodiments, the lipid combination in the LDV makes the LDV or system more suitable for high-dose and / or systemic administration compared to control lipid nanoparticles. In some embodiments, the LDVs disclosed herein exhibit a wider distribution upon systemic administration than control lipid nanoparticles or viral vectors. In some embodiments, the LDVs disclosed herein exhibit reduced liver accumulation upon systemic administration than control lipid nanoparticles or viral vectors.

[0180] The LDV disclosed herein can show superior properties for the delivery of DNA expression constructs or polynucleotides compared to control lipid nanoparticles.For example, in some embodiments, the LDV disclosed herein requires fewer cationic components to neutralize the anionic charge of DNA compared to control lipid nanoparticles.

[0181] In some embodiments, the LDVs disclosed herein comprise DODAP. In some embodiments, the LDVs disclosed herein comprise DODMA. In some embodiments, the LDVs disclosed herein comprise DODAP and DODMA.

[0182] In some embodiments, the LDV disclosed herein comprises cationic lipid:ionizable lipid:helper lipid:PEGylated lipids in the molar ratios disclosed herein. The cationic lipid may comprise or consist of DOTAP. The ionizable lipid may comprise or consist of DODAP. The ionizable lipid may comprise or consist of DODMA. The ionizable lipid may comprise or consist of DOTAP. The ionizable lipid may comprise or consist of DODAP and DODMA. The ionizable lipid may comprise or consist of DODAP and DOTAP. The ionizable lipid may comprise or consist of DODMA and DOTAP. The ionizable lipid may comprise or consist of DODAP, DODMA, and DOTAP. The helper lipid may comprise or consist of DOPE. The PEGylated lipid may comprise or consist of DMG-PEG.

[0183] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of cationic lipids:ionizable lipids:helper lipids:PEGylated lipids in a molar ratio of about 24:42:30:4: cationic lipids:ionizable lipids:helper lipids:PEGylated lipids.

[0184] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of cationic lipids:ionizable lipids:helper lipids:PEGylated lipids in a molar ratio of about 6:60:30:4: cationic lipids:ionizable lipids:helper lipids:PEGylated lipids.

[0185] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of cationic lipids:ionizable lipids:helper lipids:PEGylated lipids in a molar ratio of about 0:66:30:4: cationic lipids:ionizable lipids:helper lipids:PEGylated lipids.

[0186] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of cationic lipids:ionizable lipids:helper lipids:PEGylated lipids in a molar ratio of about 3:63:30:4: cationic lipids:ionizable lipids:helper lipids:PEGylated lipids.

[0187] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of cationic lipids:ionizable lipids:helper lipids:PEGylated lipids in a molar ratio of about 49.5:24.75:23.75:2.

[0188] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of cationic lipids:ionizable lipids:helper lipids:PEGylated lipids in a molar ratio of about 49.5:38.5:10:2: cationic lipids:ionizable lipids:helper lipids:PEGylated lipids.

[0189] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of cationic lipids:ionizable lipids:helper lipids:PEGylated lipids in a molar ratio of about 61.7:26.3:19:3: cationic lipids:ionizable lipids:helper lipids:PEGylated lipids.

[0190] In some embodiments, the LDVs disclosed herein comprise an ionizable lipid, cholesterol, a helper lipid, and a PEGylated lipid. In some embodiments, the lipids in the LDVs disclosed herein comprise, consist essentially of, or consist of an ionizable lipid, cholesterol, a helper lipid, and a PEGylated lipid in a molar ratio of about 49.5:38.5:10:2. In some embodiments, the lipids in the LDVs disclosed herein comprise, consist essentially of, or consist of an ionizable lipid, cholesterol, a helper lipid, and a PEGylated lipid in a molar ratio of about 49.5:24.75:23.75:2. In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of ionizable lipids, cholesterol, helper lipids, and PEGylated lipids in a molar ratio of about 61.7:26.3:19:3.

[0191] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DOTAP, DODAP, DOPE, and DMG-PEG, hi some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DOTAP, DODAP, DOPE, and DMG-PEG in a molar ratio or molar percentage of about 24:42:30:4.

[0192] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DOTAP, DODMA, DOPE, and DMG-PEG, hi some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DOTAP, DODMA, DOPE, and DMG-PEG in a molar ratio or molar percentage of about 24:42:30:4.

[0193] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DOTAP, DODAP, DODMA, DOPE, and DMG-PEG, hi some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DOTAP, DODAP, DODMA, DOPE, and DMG-PEG in a molar ratio or molar percentage of about 24:21:21:30:4.

[0194] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DOTAP, DODAP, DOPE, and DMG-PEG, hi some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DOTAP, DODAP, DOPE, and DMG-PEG in a molar ratio or molar percentage of about 6:60:30:4 or 3:63:30:4.

[0195] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DODAP, DOPE, and DMG-PEG, hi some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DODAP, DOPE, and DMG-PEG in a molar ratio or molar percentage of about 66:30:4.

[0196] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DODAP, cholesterol, DOPE, and DMG-PEG in a molar ratio or molar percentage of about 49.5:24.75:23.75:2, about 49.5:38.5:10:2, or about 61.7:26.3:19:3.

[0197] Delivery vectors such as LDVs may contain fusogenic proteins to enhance fusion of LDVs with the plasma membrane of target cells. Non-limiting examples of fusogenic proteins include fusion-associated small transmembrane (FAST) protein, herpes simplex virus glycoprotein H, and amphipathic anionic peptides derived from the N-terminal segment of the HA-2 subunit of influenza virus hemagglutinin, such as IFN7 and E5CA.

[0198] The fusogenic protein may be a fusion-associated small transmembrane (FAST) protein or may comprise a functional fragment of a FAST protein. FAST proteins can function receptor-independently and at physiological pH. In some embodiments, the use of FAST proteins in LDVs allows for a minimal molar ratio of cationic and / or ionizable lipids to be used to neutralize the anionic charge of the nucleic acid, reducing or substantially eliminating the role of ionizable lipids in the delivery process (e.g., endosomal escape). In some embodiments, the incorporation of FAST proteins into LDVs enhances intracellular delivery of the polynucleotide constructs or polynucleotides disclosed herein. In some embodiments, the use of FAST proteins in LDVs allows for the omission or use of lower concentrations of cholesterol, for example, compared to control lipid nanoparticles.

[0199] Non-limiting examples of FAST proteins are provided in WO2012040825A1, which is incorporated herein by reference for such disclosure.

[0200] The FAST protein family includes six members (p10, p13, p14, p15, p16, and p22) named according to their molecular weight in daltons.

[0201] In some embodiments, the FAST protein utilized in the compositions, systems, or methods disclosed herein is a native FAST protein found in the Reoviridae family, e.g., the Aquareovirus or Orthoreovirus genus. Non-limiting examples of orthoreoviruses include BRV (Baboon orthoreovirus), MRV (Mammalian orthoreovirus), NBV (Nelson Bay orthoreovirus), BrRV (Broome orthoreovirus), RRV (Reptilian orthoreovirus), and ARV (Avian orthoreovirus). In some embodiments, the FAST protein utilized in the compositions, systems, or methods disclosed herein comprises a FAST protein or domain thereof from ARV p10, BrRv p13, RRV p14, BRV p15, AqV p16, or AqV p22.

[0202] FAST proteins can contain an N-terminal ectodomain located extracellularly or on the exterior of the membrane or LDV. The ectodomain can be, for example, approximately 19-40 residues with a myristoylation motif or a myristic acid moiety on a glycine, such as the penultimate N-terminal glycine. The FAST protein ectodomain can contain a hydrophobic patch.

[0203] FAST proteins may contain a transmembrane domain that acts as a reverse signal-anchor sequence in the membrane or LDV to induce a bitropic Nout / Cin type I topology.

[0204] FAST proteins can include a C-terminal endodomain on the cytoplasmic or internal side of the membrane or LDV. The FAST protein endodomain can be, for example, approximately 40 to 140 residues long with a membrane-destabilizing fusion peptide motif. The FAST protein endodomain can include a membrane-proximal polybasic motif. The FAST protein endodomain can include a membrane-proximal membrane curvature sensor (e.g., an amphipathic alpha helix, such as a helix-kink-helix membrane curvature sensor) that drives pore formation. The FAST protein endodomain can include a hydrophobic patch.

[0205] The FAST protein may contain a proline hinge loop. The FAST protein may contain a type II polyproline helix. The FAST protein may contain a conserved region that functions as a fusion peptide, for example, by promoting rapid lipid bilayer destabilization and membrane fusion. The FAST protein may contain a palmitoylated cysteine ​​residue. The FAST protein may contain a hydrophobic patch.

[0206] The structure-function relationships between different FAST proteins suggest that overlapping structural motifs can be exchanged between specific FAST proteins to generate functional chimeric FAST fusion protein. In some embodiments, the chimeric FAST proteins disclosed herein exhibit superior fusion activity compared to wild-type FAST protein.

[0207] Chimeric FAST proteins can be synthesized that combine domains from different FAST proteins, such as p10, p14, and / or p15 peptides, to form a functional fusogenic protein.

[0208] The FAST protein used in the LDV disclosed herein may include an ectodomain or functional portion thereof from a p14 FAST protein, a transmembrane domain from a p14 FAST protein, and an endodomain or functional portion thereof from a p15 FAST protein. Such FAST proteins may be referred to as "p14endo15" or "p14e15" FAST proteins. In some embodiments, the fusion activity of p14e15 is mediated by an efficient p14 ectodomain fusion peptide and myristic acid moiety that promote lipid mixing with target cell membranes, followed by a p15 endodomain fusogenic lipid packing sensor (FLiP) motif that partitions into the LDV membrane to promote pore formation and liposome-cell fusion activity.

[0209] The FAST protein used in the LDV disclosed herein may include an ectodomain or a functional portion thereof derived from a p14 FAST protein, a transmembrane domain derived from a p15 FAST protein, and an endodomain or a functional portion thereof derived from a p14 FAST protein. Such a FAST protein may be referred to herein as "p14TM15."

[0210] The FAST protein used in the LDV disclosed herein may include an ectodomain or a functional portion thereof from a p14 FAST protein, a transmembrane domain from a p15 FAST protein, and an endodomain or a functional portion thereof from a p15 FAST protein. Such a FAST protein may be referred to as "p15ecto14" or "p15e14."

[0211] The FAST protein used in the LDV disclosed herein may include p10, p13, p14, p15, p16, p22, or chimeric fusion proteins thereof. In some embodiments, the FAST protein is a p14 / p15 chimera, a p10 / p14 chimera, or a p10 / p15 chimera. In some embodiments, the FAST protein includes (i) the p14 ectodomain and transmembrane domain and the p15 endodomain, (ii) the p14 ectodomain and the p15 transmembrane domain and endodomain, or (iii) the p14 ectodomain and endodomain and the p15 transmembrane portion.

[0212] In some embodiments, the FAST protein has a sequence similar to any one of SEQ ID NOs: 18-22, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about The amino acid sequence may have 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or similarity.

[0213] In some embodiments, the FAST protein has at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 1109%, at least about 1111, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121 %, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity.

[0214] Table 5 provides exemplary FAST protein sequences.

[0215] [Table 5]

[0216] The LDVs disclosed herein can include one or more ionizable lipids and one or more FAST proteins (e.g., chimeric FAST proteins). In some embodiments, the use of FAST proteins in LDVs allows for the use of specific ionizable lipids and allows for preferred ratios of ionizable lipids, helper lipids, and PEGylated lipids.

[0217] The delivery vector may comprise a cell-penetrating peptide.

[0218] The molar ratio of ionizable lipid to polynucleotide can be between about 2.5:1 and about 20:1. In some embodiments, the molar ratio of ionizable lipid to polynucleotide is about 5:1, about 7.5:1, about 10:1, or about 15:1.

[0219] In some embodiments, the molar ratio of ionizable lipids to polynucleotide is between about 4:1 and about 7.5:1. In some embodiments, the molar ratio of ionizable lipids to polynucleotide is between about 2.5:1 and about 7:5:1. In some embodiments, the molar ratio of ionizable lipids to polynucleotide is between about 3:1 and about 7.5:1.

[0220] In some embodiments, the molar ratio of ionizable lipid to polynucleotide is between about 5:1 and about 10:1.

[0221] In some embodiments, the molar ratio of ionizable lipid to polynucleotide is between about 5:1 and about 12:1.

[0222] In some embodiments, the molar ratio of ionizable lipids to polynucleotide is between about 2.5:1 and about 15:1. In some embodiments, the molar ratio of ionizable lipids to polynucleotide is between about 5:1 and about 15:1. In some embodiments, the molar ratio of ionizable lipids to polynucleotide is between about 7.5:1 and about 15:1. In some embodiments, the molar ratio of ionizable lipids to polynucleotide is between about 2.5:1 and about 15:1.

[0223] In some embodiments, the molar ratio of ionizable lipid to polynucleotide is between about 5:1 and about 20:1.

[0224] In some embodiments, the molar ratio of ionizable lipid to polynucleotide is about 5:1.

[0225] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DOTAP:DODAP:DOPE:DMG-PEG in a molar ratio or molar percentage of about 24:42:30:4, and the molar ratio of ionizable lipid to polynucleotide (e.g., pDNA) is between about 5:1 and about 10:1.

[0226] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DOTAP:DODMA:DOPE:DMG-PEG in a molar ratio or molar percentage of about 24:42:30:4, and the molar ratio of ionizable lipid to polynucleotide (e.g., pDNA) is between about 4:1 and about 7.5:1.

[0227] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DOTAP:DODMA:DOPE:DMG-PEG in a molar ratio or molar percentage of about 24:42:30:4, and the molar ratio of ionizable lipid to polynucleotide (e.g., mRNA) is between about 2.5:1 and about 7.5:1.

[0228] In some embodiments, the lipids of the LDV disclosed herein comprise, consist essentially of, or consist of DOTAP:DODAP:DODMA:DOPE:DMG-PEG in a molar ratio or molar percentage of about 24:21:21:30:4, and the molar ratio of ionizable lipid to polynucleotide (e.g., mRNA) is between about 3:1 and about 7.5:1.

[0229] In some embodiments, the lipids of the LDV disclosed herein comprise, consist essentially of, or consist of DOTAP:DODAP:DODMA:DOPE:DMG-PEG in a molar ratio or molar percentage of about 24:21:21:30:4, and the molar ratio of ionizable lipid to polynucleotide (e.g., mRNA) is between about 2.5:1 and about 7.5:1.

[0230] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DOTAP:DODAP:DOPE:DMG-PEG in a molar ratio or molar percentage of about 3:63:30:4, and the molar ratio of ionizable lipid to polynucleotide (e.g., pDNA) is between about 7.5:1 and about 15:1.

[0231] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DOTAP:DODAP:DOPE:DMG-PEG in a molar ratio or molar percentage of about 3:63:30:4, and the molar ratio of ionizable lipid to polynucleotide (e.g., pDNA) is about 5:1 to about 12:1.

[0232] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DOTAP:DODAP:DOPE:DMG-PEG in a molar ratio or molar percentage of about 6:60:30:4, and the molar ratio of ionizable lipid to polynucleotide (e.g., pDNA) is between about 5:1 and about 15:1.

[0233] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DOTAP:DODAP:DOPE:DMG-PEG in a molar ratio or molar percentage of about 6:60:30:4, and the molar ratio of ionizable lipid to polynucleotide (e.g., mRNA) is between about 5:1 and about 15:1.

[0234] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DODAP:DOPE:DMG-PEG in a molar ratio or molar percentage of about 66:30:4, and the molar ratio of ionizable lipid to polynucleotide (e.g., pDNA) is between about 5:1 and about 20:1.

[0235] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DODAP:DOPE:DMG-PEG in a molar ratio or molar percentage of about 66:30:4, and the molar ratio of ionizable lipid to polynucleotide (e.g., mRNA) is between about 5:1 and about 20:1.

[0236] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DODAP:cholesterol:DOPE:DMG-PEG in a molar ratio or molar percentage of about 49.5:24.75:23.75:2, and the molar ratio of ionizable lipid to polynucleotide (e.g., pDNA) is between about 5:1 and about 15:1.

[0237] In some embodiments, the lipids of the LDV disclosed herein comprise, consist essentially of, or consist of DODAP:cholesterol:DOPE:DMG-PEG in a molar ratio or molar percentage of about 49.5:24.75:23.75:2, and the molar ratio of ionizable lipid to polynucleotide (e.g., mRNA) is between about 2.5:1 and about 15:1.

[0238] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DODAP:cholesterol:DOPE:DMG-PEG in a molar ratio or molar percentage of about 49.5:38.5:10:2, and the molar ratio of ionizable lipid to polynucleotide (e.g., pDNA) is between about 5:1 and about 15:1.

[0239] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DODAP:cholesterol:DOPE:DMG-PEG in a molar ratio or molar percentage of about 49.5:38.5:10:2, and the molar ratio of ionizable lipid to polynucleotide (e.g., mRNA) is between about 2.5:1 and about 15:1.

[0240] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of a molar ratio or molar percentage of DODAP:cholesterol:DOPE:DMG-PEG of about 61.7:26.3:19:3, and the molar ratio of ionizable lipid to polynucleotide (e.g., pDNA) is between about 5:1 and about 15:1.

[0241] In some embodiments, the lipids of the LDVs disclosed herein comprise, consist essentially of, or consist of DODAP:cholesterol:DOPE:DMG-PEG in a molar ratio or molar percentage of about 61.7:26.3:19:3, and the molar ratio of ionizable lipid to polynucleotide (e.g., mRNA) is between about 2.5:1 and about 15:1.

[0242] In some embodiments, the LDVs comprise vesicles less than about 80 nm in size.

[0243] In some embodiments, the LDV is not targeted and can facilitate the delivery of polynucleotide constructs to various cell types, including, for example, target cells and non-target cells (e.g., adipocytes and non-adipocytes, e.g., white adipocytes and control cells that are not white adipocytes). The LDV can be capable of non-targeted delivery or can be configured for non-targeted delivery. Specificity of expression in target cells during non-targeted delivery can be facilitated by an expression regulatory region, such as a cell type-specific promoter.

[0244] In some embodiments, LDVs can be targeted to promote preferential delivery of polynucleotide constructs to target cell types or populations, such as adipocytes or white adipocytes. Delivery vectors can target receptors that are specifically or preferentially expressed on target cells, such as adipocyte-specific receptors or surface proteins, or receptors or surface proteins that show higher expression on adipocytes compared to control cells. The specificity of expression in target cells during targeted or non-targeted delivery can be further enhanced by expression regulatory regions, such as cell type-specific promoters.

[0245] An exemplary method for making lipid formulations used in generating LDVs may include heating the lipids disclosed herein to 37°C, combining the lipids in the ratios disclosed herein, mixing (e.g., vortexing), dehydrating the lipid mixture (e.g., in a rotary evaporator under vacuum at 60 rpm for 2 hours), rehydrating with 100% ethanol, and sonicating at 37°C.

[0246] For LDV generation, the NanoAssemblr Benchtop microfluidics mixing device can be used to mix organic and aqueous solutions to create the LDV. The organic solution can contain or consist of a lipid formulation. The aqueous solution can contain or consist of the nucleic acid cargo, 5FAST protein (e.g., 5 nM), and acetate buffer (e.g., 10 mM, pH 4.0). The Benchtop NanoAssemblr run protocol can include a total flow rate of 12 mL / min and an aqueous-to-organic flow ratio of 3:1. LDVs can be dialyzed in 8000 MWCO dialysis tubing with one end clipped. The loaded tubing can be rinsed with 5 mL of double-distilled water and dialyzed in 500 mL of dialysis buffer (ENT1844) with gentle agitation (60 rpm) at ambient temperature for 1 hour, then repeated twice with fresh dialysis buffer. LDVs can be concentrated using a 100 kDa Ultra filter. The LDV can be filter sterilized through a 0.2 μm Acrodisc Supor filter.

[0247] Non-limiting examples of LDVs are provided in WO2022067446A1, which is incorporated herein by reference for such disclosure.

[0248] In some embodiments, the lipid-based delivery vector is or comprises a lipid nanoparticle (LNP). LNPs can be formulated with cationic and / or ionizable lipids, which neutralize the anionic charge of nucleic acids and promote endosomal escape of the encapsulated nucleic acid through charge-mediated lipid bilayer disruption. LNPs can contain combinations of different classes of lipids, such as cationic or ionizable lipids (CILs), structured lipids (e.g., phospholipids and sterol lipids), and PEG-conjugated lipids (PEG-lipids). These lipids can self-assemble into LNPs under controlled microfluidic mixing with an aqueous phase containing nucleic acids. PEG-lipids can prevent or reduce aggregation, degradation, and opsonization of LNPs, while structured lipids promote nanoparticle stability and integrity.

[0249] In some embodiments, the LNPs comprise the ionizable lipid DLin-MC3-DMA (MC3). In some embodiments, the LNPs comprise DLin-MC3-DMA / DSPC / cholesterol / PEG-lipid in a molar ratio of 50:10:38.5:1.5. In some embodiments, the delivery vector is not an LNP.

[0250] In some embodiments, the lipid-based delivery vector is or comprises a liposome. The liposome may comprise a cationic lipid, such as the cationic lipids disclosed herein. Exemplary liposomes include multilamellar vesicles (MLV), oligolamellar vesicles (OLV), unilamellar vesicles (UV), small unilamellar vesicles (SUV), medium unilamellar vesicles (MUV), large unilamellar vesicles (LUV), giant unilamellar vesicles (GUV), multivesicular vesicles (MW), unilamellar or oligolamellar vesicles produced by reverse phase evaporation (REV), multilamellar vesicles produced by reverse phase evaporation (MLV-REV), stable multilamellar vesicles (STV), and the like. These include frozen and thawed vesicles (SPLVs), frozen and thawed MLVs (FATMLVs), vesicles prepared by extrusion (VETs), vesicles prepared by French press (FPVs), vesicles prepared by fusion (FUVs), dehydrated-rehydrated vesicles (DRVs), and bubblesomes (BSVs).

[0251] In some examples, the liposomes provided herein also comprise a carrier lipid. In some embodiments, the carrier lipid is a phospholipid. The carrier lipid is optionally any non-phosphate polar lipid. In some examples, the liposomes provided herein comprise a carrier lipid selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC; lecithin), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylglycerol (DMPC), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylglycerol (DMPC), dipalmitoylphosphatidylglycerol (DPPG), dimyristo ... The liposomes may comprise glycerol (DMPG), dipalmitoyl phosphatidic acid (DPPA), dimyristoyl phosphatidic acid (DMPA), distearoyl phosphatidic acid (DSPA), dipalmitoyl phosphatidylserine (DPPS), dimyristoyl phosphatidylserine (DMPS), distearoyl phosphatidylserine (DSPS), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), or the like, or a combination thereof. In some embodiments, the liposomes further comprise a sterol (e.g., cholesterol) that regulates liposome formation. In some embodiments, the liposomes comprise an electron-neutral lipid.

[0252] In some embodiments, the liposome comprises a cationic lipid. The cationic lipid may have a head group with a positive charge (e.g., a permanent or substantially permanent positive charge). Non-limiting examples of cationic lipids for use in liposomes include 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium bromide (DDAB), and 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate (DOSPA). In some embodiments, the delivery vector is not a liposome.

[0253] In some embodiments, the lipid-based delivery vector is or comprises a vesicle, micelle, or microsphere. In some embodiments, the delivery vector is not a vesicle, micelle, or microsphere.

[0254] The lipid-based delivery vector may be or may include a micelle. In some instances, the micelle is a polymeric micelle characterized by a core-shell structure in which a hydrophobic core is surrounded by a hydrophilic shell. In some instances, the hydrophilic shell further comprises a hydrophilic polymer or copolymer and a pH-sensitive component.

[0255] Exemplary hydrophilic polymers or copolymers include, but are not limited to, poly(N-substituted acrylamide), poly(N-acryloylpyrrolidine), poly(N-acryloylpiperidine), poly(N-acryl-L-amino acid amide), poly(ethyloxazoline), methyl cellulose, hydroxypropyl acrylate, hydroxyalkyl cellulose derivatives and poly(vinyl alcohol), poly(N-isopropylacrylamide), poly(N-vinyl-2-pyrrolidone), polyethylene glycol derivatives, and combinations thereof.

[0256] The delivery vector may be or include a polymeric micelle that exhibits pH-sensitive properties, for example, formed by using pH-sensitive polymers including, but not limited to, methacrylic acid, copolymers of methacrylic acid esters and acrylic acid esters, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, cellulose acetate phthalate, or cellulose acetate trimellitate.

[0257] The delivery vector may include a pH-sensitive moiety, which may include, but is not limited to, an alkyl acrylate, such as methacrylate, ethyl acrylate, propyl acrylate, and butyl acrylate, or an amino acid, such as glutamic acid.

[0258] The delivery vectors disclosed herein may be non-viral vectors. In some embodiments, non-viral vectors allow for superior delivery of polynucleotide constructs or polynucleotides when administered repeatedly, compared to viral vectors, for example, due to reduced immunogenicity.

[0259] The delivery vector disclosed herein can be a non-viral lipid-based delivery vector. The non-viral lipid-based delivery vector can be, for example, the LDV disclosed herein, liposome, lipoplex, lipid nanoparticle, vesicle, or micelle.

[0260] In some embodiments, the delivery vector is or comprises a poloxamer, nanoparticle, polyplex, or dendrimer.

[0261] The delivery vector can be an inorganic nanoparticle, such as a nanoparticle, e.g., a gold, silica, iron oxide, titanium, calcium phosphate, PLGA, poly(s-amino ester) (PBAE, e.g., PBAE-447), or hydrogel nanoparticle. In some embodiments, the delivery vector is not a nanoparticle, e.g., is not an inorganic nanoparticle.

[0262] Nucleic acids can be encapsulated in particles through electrostatic association and physical entrapment. Polymeric conjugates with degradable disulfide bonds can be used to prevent or delay dissociation of the cargo nucleic acid from nanoparticles after systemic administration. Nanoparticles can be encapsulated with lipid coatings to improve oral bioavailability, minimize enzymatic degradation, and cross the blood-brain barrier. The nanoparticle surface can also be PEGylated to improve water solubility, in vivo circulation, and stealth properties.

[0263] The delivery vector can be a polyplex, for example, a complex of one or more polymers and nucleic acids. The polyplex can include a cationic polymer. The polyplex can be produced by self-assembly through ionic interactions. The polyplex can include polyethyleneimine, chitosan, poly(β-amino ester), and / or polyphosphoramidate. In some embodiments, the delivery vector is not a polyplex.

[0264] The delivery vector can be a dendrimer. Dendrimers can be spherical, highly branched macromolecules. The surface of dendrimer particles can be functionalized, e.g., with a positive surface charge (cationic dendrimers), and used to deliver nucleic acids. The dendrimer-nucleic acid complex is taken up into cells via endocytosis. In some embodiments, the delivery vector is not a dendrimer.

[0265] In some embodiments, the delivery vector is or comprises a viral vector, a gamma-retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector. In some embodiments, the delivery vector is not a viral vector. In some embodiments, the delivery vector is not a retroviral vector. In some embodiments, the delivery vector is not a lentiviral vector. In some embodiments, the delivery vector is not an adenoviral vector. In some embodiments, the delivery vector is not an adeno-associated viral vector.

[0266] In some embodiments, the delivery vector is untargeted or formulated for non-targeted delivery, which can facilitate delivery of the polynucleotide construct to a wide range of cell types, including, for example, target cells and non-target cells (e.g., adipocytes and non-adipocytes, or white adipocytes and control cells that are not white adipocytes). The delivery vector can be capable of non-targeted delivery or can be configured for non-targeted delivery. Specificity of expression in target cells during non-targeted delivery can be facilitated by an expression regulatory region, such as a cell type-specific promoter.

[0267] In some embodiments, the delivery vector can be targeted or formulated for targeted delivery, facilitating preferential delivery of the polynucleotide construct to a target cell type or population, such as, for example, adipocytes or white adipocytes. The delivery vector can target a receptor that is specifically or preferentially expressed on the target cells, such as an adipocyte-specific receptor or surface protein, or a receptor or surface protein that shows higher expression on adipocytes compared to control cells. The specificity of expression in the target cells during targeted delivery can be further enhanced by an expression regulatory region, such as a cell type-specific promoter.

[0268] The delivery vectors disclosed herein can exclude cells. For example, in some embodiments, a delivery vector administered to an organism does not contain cells, but rather contains a polynucleotide that is delivered to a cell after administration (e.g., parenteral administration) of the delivery vector to the organism.

[0269] IV. System In some embodiments, the present disclosure provides systems comprising delivery vectors and polynucleotide constructs for achieving a target cell-specific reduction in proliferation and / or survival of target cells. The systems disclosed herein may find utility in a wide range of therapeutic applications in which it is desirable to modulate the proliferation or survival characteristics of adipocytes, with minimal or no substantial reduction in the proliferation or survival characteristics of control cells, e.g., non-adipocytes.

[0270] The systems disclosed herein (e.g., systems for in vivo delivery of polynucleotide constructs encoding cytotoxic proteins) can include a delivery vector disclosed herein and a polynucleotide construct disclosed herein. The delivery vector can be, for example, a non-viral vector, a lipid-based delivery vector (LDV), or a non-viral LDV disclosed herein. The polynucleotide construct can include a transcriptional promoter that is selectively or preferentially active in target cells (e.g., adipocytes or white adipocytes) and drives expression of a transgene encoding a therapeutic protein, such as a cytotoxic protein. The production and / or activation of specific or preferential cytotoxic proteins in adipocytes can provide a strategy for selectively killing or reducing adipocytes without the use of exogenously administered toxins. In some embodiments, the production and / or activation of specific or preferential cytotoxic proteins in adipocytes can provide a strategy for selectively killing or reducing adipocytes without target cell-specific delivery of a polynucleotide construct. Selective or preferential expression can be achieved by utilizing combinations of the promoters disclosed herein in conjunction with transcriptional regulatory mechanisms provided by the target cell.

[0271] The system disclosed herein may further include an inducer. For example, the system may include (i) a delivery vector disclosed herein, (ii) a polynucleotide construct encoding an inducible cytotoxic protein (e.g., rapamycin-inducible caspase), and (iii) an inducer (e.g., rapamycin or a structural analog thereof). The inducer or rapamycin or a structural analog thereof may be or include FK506, C-20-methyltolylrapamycin (MaRap), C16(S)-butylsulfonamidorapamycin (C16-BS-Rap), C16-(S)-7-methylindolerapamycin (AP21976 / C16-AiRap), C16-(S)-3-mechlindolerapamycin (C16-iRap), sirolimus, tacrolimus, everolimus, temsirolimus, or deforolimus.

[0272] The system may include (i) an LDV disclosed herein, (ii) a polynucleotide encoding a rapamycin-inducible cytotoxic protein, and (iii) rapamycin or a structural analog thereof.

[0273] The systems disclosed herein may further include, for example, one or more safety elements disclosed herein.

[0274] The systems disclosed herein may include, for example, (i) a safety element, (ii) a polynucleotide construct encoding an inducible cytotoxic protein (e.g., a rapamycin-inducible caspase), (iii) an inducer (e.g., rapamycin or a structural analog thereof), and / or (iv) a delivery vector disclosed herein.

[0275] The systems disclosed herein may further include an extracellular matrix remodeling factor, for example, (i) an extracellular matrix remodeling factor, (ii) a polynucleotide construct encoding an inducible cytotoxic protein (e.g., rapamycin-inducible caspase), (iii) an inducer (e.g., rapamycin or a structural analog thereof), and / or (iv) a delivery vector disclosed herein.

[0276] V. Method Disclosed herein, in some embodiments, are methods for reducing the survival or persistence of target cells (e.g., adipocytes or white adipocytes), the methods comprising contacting the target cells with a polynucleotide construct, delivery vector, and / or system disclosed herein.

[0277] In some embodiments, disclosed herein are methods for reducing proliferation of target cells (e.g., adipocytes or white adipocytes), the methods comprising contacting the target cells with a polynucleotide construct, delivery vector, inducer, and / or system disclosed herein. The contacting step can be in vitro. The contacting step can be in vivo. The polynucleotide construct can encode an inducible cytotoxic protein (e.g., rapamycin-inducible caspase), and the method can comprise contacting the cells with an inducer (e.g., rapamycin or a structural analog thereof).

[0278] In some embodiments, target cells are irradiated with less than about 100 mM, less than about 10 mM, less than about 1 mM, less than about 500 μM, less than about 100 μM, less than about 50 μM, less than about 10 μM, less than about 5 μM, less than about 4 μM, less than about 3 μM, less than about 2 μM, less than about 1 μM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM In some embodiments, the polynucleotide construct, delivery vector, inducer, and / or system is contacted with the polynucleotide construct, delivery vector, inducer, and / or system at a concentration of less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 10 pM, or less than about 1 pM.

[0279] In some embodiments, target cells are irradiated with at least about 100 mM, at least about 10 mM, at least about 1 mM, at least about 500 μM, at least about 100 μM, at least about 50 μM, at least about 10 μM, at least about 5 μM, at least about 4 μM, at least about 3 μM, at least about 2 μM, at least about 1 μM, at least about 900 nM, at least about 800 nM, at least about 700 nM, at least about 600 nM, at least about 500 nM, at least about 400 nM, at least about 300 nM, at least about 200 nM, at least about 100 nM, at least about 90 nM, at least about 80 nM, at least about 70 nM, In some embodiments, the polynucleotide construct, delivery vector, inducing agent, and / or system is contacted with a concentration of at least about 60 nM, at least about 50 nM, at least about 40 nM, at least about 30 nM, at least about 20 nM, at least about 10 nM, at least about 5 nM, at least about 4 nM, at least about 3 nM, at least about 2 nM, at least about 1 nM, at least about 900 pM, at least about 800 pM, at least about 700 pM, at least about 600 pM, at least about 500 pM, at least about 400 pM, at least about 300 pM, at least about 200 pM, at least about 100 pM, at least about 10 pM, or at least about 1 pM.

[0280] In some embodiments, after the contacting step, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% of the adipocytes or white adipocytes are killed. Killing can be determined by cytotoxicity assays, such as LDH release assays, dye exclusion assays, flow cytometry assessment, or other suitable methods.

[0281] In some embodiments, after the contacting step, up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, or up to 90%, up to 95%, up to 97%, or up to 99% of the adipocytes or white adipocytes are dead.

[0282] In some embodiments, after the contacting step, about 1 to 90%, about 1 to 80%, about 1 to 70%, about 1 to 60%, about 1 to 50%, about 1 to 40%, about 1 to 30%, about 1 to 20%, about 1 to 10%, about 1 to 5%, about 5 to 90%, about 5 to 80%, about 5 to 70%, about 5 to 60%, about 5 to 50%, about 5 to 40%, about 5 to 30%, about 5 to 20%, about 5 to 10%, about 10 to 90%, about 10 to 80%, about 10 to 70%, about 10 to 60%, about 10 to 50%, about 10 to 40%, about 10 to 30%, about 10 to 20%, About 20-90%, about 20-80%, about 20-70%, about 20-60%, about 20-50%, about 20-40%, about 20-30%, about 30-90%, about 30-80%, about 30-70%, about 30-60%, about 30-50%, about 30-40%, about 40-90%, about 40-80%, about 40-70%, about 40-60%, about 40-50%, about 50-90%, about 50-80%, about 50-70%, about 50-60%, about 60-90%, about 60-80%, about 60-70%, about 70-90%, about 70-80%, or about 80-90% are killed.

[0283] The polynucleotide constructs or systems disclosed herein may exhibit preferential killing of adipocytes or white adipocytes over control cells, such as non-adipocytes or cells that are not white adipocytes. The control cells may be, for example, muscle cells, hepatocytes, bone cells, erythrocytes, neurons, leukocytes, lymphocytes, monocytes, or fibroblasts, epithelial cells, or a combination thereof. In some embodiments, the control cells are brown adipocytes. In some embodiments, contacting one or more populations of cells, including adipocytes and / or control cells, with the polynucleotide construct or system results in adipocyte death (e.g., apoptosis) that is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 250-fold, at least 500-fold, or at least 1000-fold greater than control cell death.

[0284] The disclosed methods may include administering to a subject a polynucleotide, system, delivery vector, inducer, and / or pharmaceutical composition disclosed herein. The subject may be a mammal. The subject may be human.

[0285] The polynucleotide construct, system, delivery vector, inducer, and / or pharmaceutical composition can be administered to an individual subject, for example, parenterally, orally, or by topical application. Administration can be local. Administration can be systemic. The polynucleotide construct, system, delivery vector, inducer, and / or pharmaceutical composition can be administered to an individual subject, for example, intravenously, intraperitoneally, intramuscularly, subcutaneously, intracerebrally, intraventricularly, intraarticularly, intraarterially, intrathecally, intracapsularly, subcapsularly, intraorbitally, intracardially, intradermally, subcutaneously, intrathecally, or intracranially, e.g., by injection or infusion. Administration can be by local injection or infusion. Administration can be via systemic injection or infusion. Administration can be via intravenous injection or infusion. Administration can be via subcutaneous injection or infusion. Administration can be via local injection into adipose tissue, e.g., a body cavity or anatomical region disclosed herein. Administration can be via intraperitoneal injection.

[0286] Various administration schedules can be used. In some embodiments, the polynucleotide construct, polynucleotide, system, delivery vector, inducer, and / or pharmaceutical composition is administered to a subject once. In some embodiments, the polynucleotide construct, polynucleotide, system, delivery vector, inducer, and / or pharmaceutical composition is administered to a subject two or more times. In some embodiments, the systems disclosed herein, including non-integrating polynucleotide constructs, can allow for repeated administration with reduced potential for toxicity, for example, based on a limited number of copies of the polynucleotide construct that are degraded and / or diluted over time as cells expressing the cytotoxic protein undergo cell death. The transgene can be expressed in target cells (e.g., adipocytes) without genomic integration. For example, the transgene can be expressed from an episomal vector, such as DNA, RNA, circular DNA, plasmid, circular RNA, or minicircle. The transgene can be transiently expressed. For example, expression of the transgene can decrease as the nucleic acid encoding it is degraded.

[0287] In some embodiments, the present disclosure provides a method for reducing adipose tissue volume in a subject, the method comprising administering to the subject an effective amount of a polynucleotide construct, delivery vector, inducer, system, and / or pharmaceutical composition disclosed herein. The polynucleotide construct can encode an inducible cytotoxic protein (e.g., rapamycin-inducible caspase), and the method can comprise administering to the subject an inducer (e.g., rapamycin or a structural analog thereof).

[0288] The inducing agent (e.g., rapamycin or a structural analog thereof) can be administered by any suitable route of administration, for example, via parenteral, oral, topical, local, systemic, subcutaneous, intravenous, intramuscular, intraperitoneal, subcutaneous, intracerebral, intraventricular, intraarticular, intraarterial, intrathecal, intracapsular, subcapsular, intraorbital, intracardiac, intradermal, subcutaneous, intrathecal, or intracranial routes. Administration can be via local injection or infusion. Administration can be via systemic injection or infusion. Administration can be via intravenous injection or infusion. Administration can be via subcutaneous injection or infusion. Administration can be via local injection into adipose tissue, for example, in a body cavity or anatomical region disclosed herein. Administration can be via intraperitoneal injection. The inducing agent can be administered by the same route as the polynucleotide construct or delivery vector. The inducing agent can be administered by a different route than the polynucleotide construct or delivery vector, for example, the polynucleotide construct or delivery vector can be administered by local parenteral administration and the inducing agent can be administered orally.

[0289] In some embodiments, the inducer is administered locally, for example, to adipose tissue. In some embodiments, the limited diffusion and / or limited half-life of the inducer can advantageously limit the activity of the cytotoxic proteins disclosed herein to outside of adipocytes, such as white adipocytes. In some embodiments, the inducer is administered systemically.

[0290] Adipose tissue can be reduced in a subject by the methods disclosed herein. Adipose tissue can be reduced, for example, in the visceral, subcutaneous, and / or abdominal regions of a subject. Adipose tissue can be reduced in a single anatomical region or body cavity of a subject. Adipose tissue can be reduced in one or more specific regions or body cavities disclosed herein. For example, in some embodiments, adipose tissue is reduced in subcutaneous fat. In some embodiments, adipose tissue is reduced in visceral fat. In some embodiments, a promoter, expression control region, or safety element disclosed herein contributes to the specific or preferential reduction of adipose tissue in one or more specific regions or body cavities. In some embodiments, an administration route disclosed herein contributes to the specific or preferential reduction of adipose tissue in one or more specific regions or body cavities. Adipose tissue can be reduced in multiple anatomical regions or body cavities of a subject. Adipose tissue loss can be determined, for example, before and after treatment, by imaging techniques such as skinfold calipers, circumference measurements, hydrostatic weighing, air displacement plethysmography, bioelectrical impedance analysis, bioimpedance spectroscopy, electrical impedance myography, multicompartmental models, DEXA scans, or combinations thereof.

[0291] In some embodiments, the methods disclosed herein reduce adipose tissue (e.g., white adipose tissue) in a subject (e.g., in a specific anatomical region or body cavity or systemically) by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%.

[0292] In some embodiments, partial reduction of adipose tissue is more advantageous than complete reduction of adipose tissue, for example, due to physiological processes mediated by adequate amounts of adipose tissue. In some embodiments, the methods disclosed herein reduce the adipose tissue (e.g., white adipose tissue) in a subject (e.g., in a specific anatomical region or body cavity, or systemically) by up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, or up to 90%.

[0293] In some embodiments, the methods disclosed herein provide for reducing adipose tissue (e.g., white adipose tissue) in a subject (e.g., in a specific anatomical region or body cavity, or systemically) by about 10-90%, about 10-80%, about 10-70%, about 10-60%, about 10-50%, about 10-40%, about 10-30%, about 10-20%, about 20-90%, about 20-80%, about 20-70%, about 20-60%, about 20-50%, about 20-60%, about 20-70%, about 20-80%, about 20-90%, about 20-80%, about 20-70%, about 20-60%, about 20-50%, about 20-90%, about 20-90%, about 20-90%, about 20-60%, about 20-60%, about 20-60%, about 20-70%, about 20-80%, about 20-90%, about 20-90%, about 20-90%, about 20-90%, about 20-90%, about 20-6 ... Decrease by 40%, about 20-30%, about 30-90%, about 30-80%, about 30-70%, about 30-60%, about 30-50%, about 30-40%, about 40-90%, about 40-80%, about 40-70%, about 40-60%, about 40-50%, about 50-90%, about 50-80%, about 50-70%, about 50-60%, about 60-90%, about 60-80%, about 60-70%, about 70-90%, about 70-80%, or about 80-90%.

[0294] In some embodiments, brown adipose tissue is reduced by up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, or up to 90%.

[0295] In some embodiments, the subject has a reduction in visceral adipose tissue (eg, white visceral adipose tissue).

[0296] In some embodiments, the methods disclosed herein reduce visceral adipose tissue (e.g., white visceral adipose tissue) in a subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%.

[0297] In some embodiments, the methods disclosed herein reduce visceral adipose tissue (e.g., white visceral adipose tissue) in a subject by up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, or up to 90%.

[0298] In some embodiments, the methods disclosed herein provide for reducing visceral adipose tissue (e.g., white visceral adipose tissue) in a subject by about 10-90%, about 10-80%, about 10-70%, about 10-60%, about 10-50%, about 10-40%, about 10-30%, about 10-20%, about 20-90%, about 20-80%, about 20-70%, about 20-60%, about 20-50%, about 20-40%, about 20-30%, about 30-40%, about 30-50%, about 30-50%, about 30-60%, about 30-60%, about 30-70%, about 30-80%, about 30-90%, about 30-90%, about 30-90%, about 30-90%, about 30-90%, about 30-90%, about 30-60%, about 30-60%, about 30-70%, about 30-60%, about 30-40%, about 30-50%, about 30-60%, about 30-50%, about 30-60%, about 30-70%, about 30-80%, about 30-9 ... Decrease by 0-90%, about 30-80%, about 30-70%, about 30-60%, about 30-50%, about 30-40%, about 40-90%, about 40-80%, about 40-70%, about 40-60%, about 40-50%, about 50-90%, about 50-80%, about 50-70%, about 50-60%, about 60-90%, about 60-80%, about 60-70%, about 70-90%, about 70-80%, or about 80-90%.

[0299] In some embodiments, brown visceral adipose tissue is reduced by up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, or up to 90%.

[0300] In some embodiments, subcutaneous adipose tissue (eg, white subcutaneous adipose tissue) is reduced in the subject.

[0301] In some embodiments, the methods disclosed herein reduce subcutaneous adipose tissue (e.g., white subcutaneous adipose tissue) in a subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%.

[0302] In some embodiments, the methods disclosed herein reduce subcutaneous adipose tissue (e.g., white subcutaneous adipose tissue) in a subject by up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, or up to 90%.

[0303] In some embodiments, the methods disclosed herein provide for reducing subcutaneous adipose tissue (e.g., white subcutaneous adipose tissue) in a subject by about 10-90%, about 10-80%, about 10-70%, about 10-60%, about 10-50%, about 10-40%, about 10-30%, about 10-20%, about 20-90%, about 20-80%, about 20-70%, about 20-60%, about 20-50%, about 20-40%, about 20-30%, about 30-40%, about 30-50%, about 30-60%, about 30-60%, about 30-70%, about 30-80%, about 30-90%, about 30-90%, about 30-90%, about 30-90%, about 30-90%, about 30-90%, about 30-60%, about 30-60%, about 30-70%, about 30-60%, about 30-40%, about 30-50%, about 30-60%, about 30-50%, about 30-60%, about 30-60%, about 30-70%, about 30-80%, about 30-9 ... Decrease by 0-90%, about 30-80%, about 30-70%, about 30-60%, about 30-50%, about 30-40%, about 40-90%, about 40-80%, about 40-70%, about 40-60%, about 40-50%, about 50-90%, about 50-80%, about 50-70%, about 50-60%, about 60-90%, about 60-80%, about 60-70%, about 70-90%, about 70-80%, or about 80-90%.

[0304] In some embodiments, brown subcutaneous adipose tissue is reduced by up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, or up to 90%.

[0305] In some embodiments, abdominal adipose tissue (eg, white abdominal adipose tissue) is reduced in the subject.

[0306] In some embodiments, the methods disclosed herein reduce abdominal adipose tissue (e.g., white abdominal adipose tissue) in a subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%.

[0307] In some embodiments, the methods disclosed herein reduce abdominal adipose tissue (e.g., white abdominal adipose tissue) in a subject by up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, or up to 90%.

[0308] In some embodiments, the methods disclosed herein provide for reducing abdominal adipose tissue (e.g., white abdominal adipose tissue) in a subject by about 10-90%, about 10-80%, about 10-70%, about 10-60%, about 10-50%, about 10-40%, about 10-30%, about 10-20%, about 20-90%, about 20-80%, about 20-70%, about 20-60%, about 20-50%, about 20-40%, about 20-30%, about 30-40%, about 30-50%, about 30-50%, about 30-60%, about 30-60%, about 30-70%, about 30-80%, about 30-90%, about 30-80%, about 30-90%, about 30-60%, about 30-60%, about 30-70%, about 30-80%, about 30-90%, about 30-90%, about 30-90%, about 30-90%, about 30-90%, about 30-60%, about 30-60%, about 30-40%, about 30-40%, about 30-50%, about 30-60%, about 30-40%, about 30-50%, about 30-60%, about 30-50%, about 30-60%, about 30-60%, about 30-70%, about 30-80%, about 30-90%, about 30-90%, about 30-90%, about 30-60%, about 30-60%, about 30-40%, about 30-50%, about 30-60%, about 30-40%, about 30-50%, about 30-60%, about 30-4 Decrease by 0-90%, about 30-80%, about 30-70%, about 30-60%, about 30-50%, about 30-40%, about 40-90%, about 40-80%, about 40-70%, about 40-60%, about 40-50%, about 50-90%, about 50-80%, about 50-70%, about 50-60%, about 60-90%, about 60-80%, about 60-70%, about 70-90%, about 70-80%, or about 80-90%.

[0309] In some embodiments, brown abdominal adipose tissue is reduced by up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, or up to 90%.

[0310] In some embodiments, disclosed herein are methods for treating a disease in a subject in need thereof, the methods comprising administering to the subject an effective amount of a polynucleotide construct, delivery vector, system, and / or pharmaceutical composition disclosed herein. The polynucleotide construct can encode an inducible cytotoxic protein (e.g., rapamycin-inducible caspase), and the method can comprise administering to the subject an inducer (e.g., rapamycin or a structural analog thereof).

[0311] The disease to be treated may be, include, or be associated with a metabolic disorder. The disease may be, include, or be associated with a lipidemia. The disease may be, include, or be associated with a rare lipidemia disorder. The disease may be, include, or be associated with a rare lipidemia disorder. The disease may be, include, or be associated with a lipoma. The disease may be, include, or be associated with a fat accumulation, e.g., excessive adiposity. The disease may be, include, or be associated with obesity. The disease may be, include, or be associated with morbid obesity. The disease may be, include, or be associated with morbid obesity. The disease may be, include, or be associated with overweight. The disease may be, include, or be associated with an enzyme deficiency or abnormality. The disease may be, include, or be associated with an enzyme deficiency or abnormality. The disease may be, include, or be associated with cancer.

[0312] In some embodiments, the compositions, systems, or methods disclosed herein are used to reduce a subject's body mass index (BMI). BMI can be described as a subject's weight (kilograms) divided by the square of their height (meters). In some embodiments, the treated subject has a BMI of at least 20, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40. In some embodiments, the methods reduce BMI by at least 1 unit, at least 2 units, at least 3 units, at least 4 units, at least 5 units, at least 5 units, at least 7 units, at least 8 units, at least 9 units, or at least 10 units.

[0313] In some embodiments, the disease is, includes, or is associated with lipedema. Lipedema is a disease characterized by abnormal fat deposition in the legs. Lipedema is a chronic medical condition that can be characterized by symmetrical accumulation of adipose tissue (fat), particularly in the legs. Lipedema can cause pain, swelling, and easy bruising, can make daily activities difficult, and may not respond as well to diet and exercise as other types of fat accumulation. The disease can be, for example, stage 1 lipedema, stage 2 lipedema, or stage 3 lipedema.

[0314] In some embodiments, the condition is, involves, or is associated with Dercum's disease (adiposis dolorosa). The skin disease is a rare disorder of unknown etiology characterized by painful subcutaneous fatty tissue deposits with variable localization throughout the body. The deposits appear histologically as lipomas and are associated with overweight or obese conditions and various psychiatric disorders (anxiety, depression, sleep disorders). The lipomas or fatty masses can be small and difficult to palpate or larger nodules and can cause severe chronic pain.

[0315] In some embodiments, the polynucleotide constructs, delivery vectors, systems, and / or pharmaceutical compositions disclosed herein are useful in methods for cosmetic fat removal.

[0316] Treatment may include pharmaceutical or other intervention regimens to achieve beneficial or desired results in a subject. Beneficial or desired results include, but are not limited to, therapeutic benefit and / or preventive benefit. Therapeutic benefit may refer to the eradication or amelioration of the signs or symptoms of the underlying disorder being treated. Therapeutic benefit may be achieved by reducing or improving one or more physiological signs or symptoms associated with the underlying disease, so that an improvement can be observed or detected in the subject. A preventive effect may include delaying, preventing, or reducing the appearance of a disease or condition, delaying, preventing, or reducing the onset of symptoms of a disease or condition, delaying, preventing, reducing, or reversing the progression of a disease or condition, or any combination thereof. For preventive benefit, a subject at risk of developing a particular disease or reporting one or more physiological symptoms of a disease can receive treatment even if the disease has not been diagnosed.

[0317] Exemplary methods of treatment can include administration of the polynucleotide constructs, polynucleotides, systems, inducers, and / or delivery vectors disclosed herein, including as part of a pharmaceutical composition. The expression constructs, polynucleotides, systems, and / or delivery vectors can be administered in an amount effective to treat or prevent the disease or condition.

[0318] "Treatment" (and grammatical variants thereof, such as "treat" or "treating") can refer to clinical intervention in an attempt to alter the natural course of the individual (subject) being treated, and can be performed prophylactically or during the course of clinical pathology. Desirable effects of treatment can include prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, reduction in the rate of disease progression, improvement or palliation of the disease state, and remission or improved prognosis.

[0319] The pharmaceutical composition of the present disclosure may comprise the composition disclosed herein and a pharmaceutically acceptable excipient. The pharmaceutical composition may, for example, comprise (i) the polynucleotide construct disclosed herein, the inducer disclosed herein, and / or the delivery vector disclosed herein, and (ii) a pharmaceutically acceptable excipient. The pharmaceutical composition may be formulated for, for example, systemic, local, parenteral, intratumoral, intravenous, intraperitoneal, subcutaneous, transdermal, or intramuscular administration. These compositions may take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, etc.

[0320] The systems, compositions (e.g., pharmaceutical compositions), and methods of the present disclosure can be tested in vitro and / or in vivo for desired therapeutic or prophylactic activity. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include the effect of the system on a cell line or patient tissue sample. The effect on the cell line and / or tissue sample can be determined using techniques including, but not limited to, proliferation and apoptosis assays. According to the present disclosure, in vitro assays that can be used to determine whether administration of a particular compound is indicated include in vitro cell culture assays in which a patient tissue sample is grown in culture, exposed to, or otherwise administered, a compound, and the effect of such compound on the tissue sample is observed.

[0321] In some embodiments, the polynucleotide construct is integrated into the genome of the host cell. The polynucleotide construct can be inserted into the genome of the cell in a targeted manner (e.g., at one or more specific sites) or a non-targeted manner (e.g., at one or more non-specific sites). For targeted integration, the polynucleotide sequence to be inserted can be flanked by homology arms containing sequences complementary to the genomic DNA sequence to be targeted for insertion (e.g., via homologous recombination and / or homology-directed repair). A double-strand break can be introduced at the target site in the genome, and the homology arms can facilitate the insertion of the polynucleotide. In some cases, the polynucleotide can be excised from the vector (e.g., via a nuclease) and inserted into the genome of the cell. The polynucleotide can be inserted into a safe harbor locus. Various enzymes can catalyze the insertion of foreign DNA into the host genome. Non-limiting examples of gene editing tools and technologies include CRISPR, TALEN, zinc finger nucleases (ZFNs), meganucleases, Mega-TALs, and transposon-based systems. [Example]

[0322] VI. Working Examples Example 1: Construction Design Polynucleotide constructs are designed for use in gene therapy approaches to induce adipocyte death and adipose tissue loss.

[0323] The polynucleotide construct comprises, from 5' to 3', (i) a promoter and / or expression regulatory region disclosed herein, such as the ADIPOQ, FABP4, PLIN1, PPARγ, PPARγ1, PPARγ2, CD36, LPL, LEP, CIDEC, TUSC5, CIDEA, or LIPE promoter, or a functional fragment thereof, and (ii) a transgene encoding a cytotoxic protein disclosed herein, such as a caspase (e.g., inducible caspase 9, such as rapamycin-inducible caspase 9). Expression of the transgene is driven by the promoter and / or expression regulatory region. Certain polynucleotide constructs further encode one or more extracellular matrix remodeling factors and / or safety elements (e.g., regulatory RNAs or regulatory RNA target sites).

[0324] The expression cassette is cloned into the NTC Nano plasmid. A control construct is generated in which the same promoter and regulatory region drives expression of a reporter gene (e.g., eGFP, luciferase, or E2). An additional control utilizes a constitutive or control promoter, such as the CMV promoter.

[0325] Example 2: Production of LDV formulations For in vitro purposes, LDVs containing FAST protein were hand-mixed with various lipid formulations (eg, as disclosed herein) and tested to assess transfection efficiency.

[0326] For in vivo purposes, FAST-LDVs are prepared to a final concentration of 2 mg / mL in PBS. LDVs are concentrated using a 100 kDa Ultra filter (Amicon, UFC810096) according to the manufacturer's instructions. LDVs are filter-sterilized through a 0.2 μm Acrodisc Supor filter (Amicon, UFC910008). Particle size, polydispersity index (PDI), and zeta potential are measured for the final sample using a Malvern Zetasizer Range and Universal 'Dip' Cell Kit (Malvern, ZEN1002) according to the manufacturer's instructions. Nucleic acid encapsulation efficiency is calculated using a modified Quant-IT PicoGreen dsDNA assay (Thermo Fisher Scientific).

[0327] Example 3: In vitro evaluation of adipocyte transfection, adipocyte-specific gene expression, and adipocyte-specific killing Human adipose-derived stromal / stem cells (Obatala Catalog No. OS-101) are cultured in Obatala Sciences StromaQual stromal medium until at least 80% confluence. Adipogenesis is induced using AdipoQual Differentiation Medium (Obatala Catalog No. OS-002).

[0328] Adipocytes were transfected in vitro using LDVs containing the FAST proteins disclosed herein with 500-2000 ng of pDNA encapsulated in LDVs for 96-well plates (300 μl cell culture medium final) and 250-1000 ng for 48-well plates (1000 μl cell culture medium final).

[0329] To test transfection efficiency, a reporter construct (e.g., with eGFP driven by adipocyte-specific (e.g., adiponectin) promoter / expression regulatory region or CMV-controlled promoter) is transfected using the LDV formulation disclosed herein. The transfected cells are imaged for eGFP expression over a 10-day period. The transfected cells are also analyzed by flow cytometry to determine the percentage of eGFP-positive cells. For adipocyte sample preparation, Obatala Sciences ObaFlow (catalog number OS-304) is used. Reporter gene expression is quantified.

[0330] The ability of polynucleotide constructs, transcription promoters, and / or expression regulatory regions to specifically or preferentially induce expression in target cells is tested. Adipocytes, control cells, and / or their co-cultures are transfected with a plasmid utilizing the transcription promoter or expression regulatory region disclosed herein to drive the expression of a reporter gene. The proportion of cells expressing the reporter gene is determined (e.g., via flow cytometry). A construct with a constitutive promoter is used as a control. Expression by target cells (e.g., adipocytes) is compared with expression by control cells.

[0331] The ability of polynucleotide constructs to induce specific target cell death (e.g., apoptosis of white adipocytes) is tested. Adipocytes, control cells, and / or their cocultures are transfected with plasmids utilizing transcriptional promoters or expression control regions preferentially or specifically active in human adipocytes to drive the expression of a cytotoxic protein (e.g., iCasp9). Constructs with constitutive promoters are used as controls. After treatment, incubation and addition of a chemical inducer of dimerization for inducible caspases are performed, and the viability of adipocytes and control cells is determined using assays such as flow cytometry (e.g., with annexin V or propidium iodide), LDH release, incucyte, or other viability / cytotoxicity assays. Target cell (e.g., adipocyte) death is compared to that of control cells.

[0332] Example 4: Fat volume reduction in human explants Human tissue explants were obtained from abdominoplasty ("abdominal crease") surgeries via a commercial vendor (e.g., HypoSkin® from GenoSkin, containing epidermis, dermis, and hypodermis with normal adipose / subcutaneous tissue architecture). The explants were maintained in GenoSkin culture medium and treated with 12 μg of LDV formulation containing a polynucleotide construct that expresses inducible caspase-9.

[0333] After 72 hours of treatment, a chemical inducer of dimerization (CID) was added to the culture medium to activate iCasp9. After 24 hours of treatment with CID, explants were fixed and processed for H&E staining (Figure 1). Cross sections were imaged and analyzed to determine the area of ​​fat-positive tissue (mm 2 The treatment resulted in a 20% reduction in fat compared to the control (Figure 2).

[0334] Example 5: Adipocyte-specific transgene expression in vivo In vivo studies were performed using adult female dB / dB mice (Jackson Lab). Mice received a subcutaneous injection of 100 μL of test agent (e.g., 200 μg of a plasmid expressing the reporter E2-Crimson driven by the adiponectin promoter "AQ-E2-Crimson") or PBS control. Seventy-two hours after treatment, mice were imaged (e.g., via IVIS), demonstrating that the compositions and methods disclosed herein can be used to induce adipocyte-specific expression in vivo (Figure 3).

[0335] Example 6: In vivo transgene expression In vivo studies were performed using adult female dB / dB mice (Jackson Lab). Five-month-old mice received a subcutaneous injection of 100 μL of the test agent (e.g., 200 μg of LDV containing a plasmid expressing a 1:1 combination of eGFP and luciferase, each driven by a CMV promoter) or PBS control. Twenty-four hours after injection, the animals were intraperitoneally injected with D-luciferin and imaged using an AMI HT in vivo imager (Figure 4). Skin and fat pad samples were then collected near the injection site (Figure 5). Samples were lysed, and sandwich ELISA was performed using Meso Scale Discovery (MSD), which is used to measure transgene expression, demonstrating that eGFP was expressed (Figure 6). The results demonstrate that administration of a polynucleotide construct can result in localized transgene expression.

[0336] Example 7: In vivo fat reduction In vivo studies were performed using adult dB / dB mice. Five-month-old mice received a subcutaneous injection of 100 μL of PBS control or 200 μg of LDV containing a plasmid carrying the expression of a cytotoxic protein (e.g., iCasp9) driven by a promoter / expression control region that induces specific or preferential expression in adipocytes. A chemical inducer of dimerization (e.g., rapamycin) was then administered to the animals via intraperitoneal injection.

[0337] The effect of the polynucleotide construct on adipose tissue volume is quantified or monitored over time via, for example, DEXA scans, body weight measurements, circumference measurements, hydrostatic weighing, air displacement plethysmography, bioelectrical impedance analysis, bioimpedance spectroscopy, electrical impedance myography, or a combination thereof.

[0338] VII. Additional Sequences

[0339] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5]

[0340] The degree of sequence identity between two sequences can be determined, for example, by comparing two sequences using a computer program designed for this purpose (e.g., global or local alignment algorithm).Non-limiting examples include BLASTp, BLASTn, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, GAP, BESTFIT, Needle (EMBOSS), Stretcher (EMBOSS), GGEARCH2SEQ, Water (EMBOSS), Matcher (EMBOSS), LALIGN, SSEARCH2SEQ, or another suitable method or algorithm.Global alignment algorithms such as the Needleman and Wunsch algorithm can be used to align the entire length of two sequences, maximizing the number of matches and minimizing the number of gaps.Default settings can be used.

[0341] To generate a similarity score for two amino acid sequences, a scoring matrix can be used that assigns positive scores to some non-identical amino acids (e.g., amino acids with similar physiochemical properties and / or amino acids that exhibit frequent substitutions in orthologs, homologs, or paralogs). Non-limiting examples of scoring matrices include PAM30, PAM70, PAM250, BLOSUM45, BLOSUM50, BLOUM62, BLOSUM80, and BLOSUM90.

[0342] Amino acids can include genetically encoded amino acids and non-genetically occurring amino acids. Amino acids can include naturally occurring amino acids and non-naturally occurring amino acids. Amino acids can be L- or D-form. The substitutions disclosed herein can include conservative and / or non-conservative amino acid substitutions. A conservative amino acid substitution can be the substitution of one amino acid for another amino acid with similar biochemical properties (e.g., charge, size, and / or hydrophobicity). A non-conservative amino acid substitution can be the substitution of one amino acid for another amino acid with different biochemical properties (e.g., charge, size, and / or hydrophobicity). A conservative amino acid change can be, for example, a substitution that has minimal effect on the secondary or tertiary structure of a polypeptide. A conservative amino acid change can be an amino acid change from one hydrophilic amino acid to another hydrophilic amino acid. Hydrophilic amino acids can include Thr (T), Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K), and Arg (R). A conservative amino acid change can be from one hydrophobic amino acid to another hydrophilic amino acid. Hydrophobic amino acids can include Ile (I), Phe (F), Val (V), Leu (L), Trp (W), Met (M), Ala (A), Gly (G), Tyr (Y), and Pro (P). A conservative amino acid change can be from one acidic amino acid to another acidic amino acid. Acidic amino acids can include Glu (E) and Asp (D). A conservative amino acid change can be from one basic amino acid to another basic amino acid. Basic amino acids can include His (H), Arg (R), and Lys (K). A conservative amino acid change can be from one polar amino acid to another polar amino acid. Polar amino acids can include Asn (N), Gln (Q), Ser (S), and Thr (T). A conservative amino acid change can be from one nonpolar amino acid to another, including Leu (L), Val (V), Ile (I), Met (M), Gly (G), and Ala (A).A conservative amino acid change can be from one aromatic amino acid to another. Aromatic amino acids can include Phe (F), Tyr (Y), and Trp (W). A conservative amino acid change can be from one aliphatic amino acid to another aliphatic amino acid. Aliphatic amino acids can include Ala (A), Val (V), Leu (L), and Ile (I). In some embodiments, a conservative amino acid substitution is from one amino acid to another within one of the following groups: Group I: Ala, Pro, Gly, Gln, Asn, Ser, Thr; Group II: Cys, Ser, Tyr, Thr; Group III: Val, Ile, Leu, Met, Ala, Phe; Group IV: Lys, Arg, His; Group V: Phe, Tyr, Trp, His; and Group VI: Asp, Glu.

[0343] The proteins or polypeptides disclosed herein can include an N-terminal methionine. The proteins or polypeptides disclosed herein can lack an N-terminal methionine.

Claims

1. A polynucleotide construct for selective killing of human adipocytes, comprising: (a) a transcription promoter that is preferentially or specifically active in human adipocytes; (b) a transgene encoding a cytotoxic protein, wherein expression of the cytotoxic protein is regulated by the transcription promoter.

2. The polynucleotide construct of claim 1 , wherein the polynucleotide construct comprises DNA.

3. The polynucleotide construct of claim 1 , wherein the polynucleotide construct comprises double-stranded DNA.

4. The polynucleotide construct of claim 1 , wherein the polynucleotide construct is a plasmid.

5. The polynucleotide construct of claim 1 , wherein the polynucleotide construct is a minicircle.

6. The polynucleotide construct of claim 1 , wherein the transcription promoter comprises an ADIPOQ promoter or a functional fragment thereof.

7. 2. The polynucleotide construct of claim 1, wherein the transcription promoter comprises a FABP4, PLIN1, PPARγ, PPARγ1, PPARγ2, CD36, LPL, LEP, CIDEC, TUSC5, CIDEA, or LIPE promoter, or a functional fragment thereof.

8. 2. The polynucleotide construct of claim 1, wherein the transcription promoter comprises a nucleic acid sequence having at least about 80% sequence identity to any one of SEQ ID NOs: 1 and 56-72.

9. 2. The polynucleotide construct of claim 1, wherein the promoter is at least 50% more active in human adipocytes than in control cells that are not human adipocytes.

10. 10. The polynucleotide construct of claim 9, wherein the control cell is a muscle cell, a hepatocyte, a bone cell, an erythrocyte, a neuron, a leukocyte, a lymphocyte, or a fibroblast.

11. The polynucleotide construct of claim 1, wherein the cytotoxic protein induces non-inflammatory cell death when expressed in the human adipocyte.

12. The polynucleotide construct of claim 11 , wherein the cytotoxic protein induces apoptosis when expressed in the human adipocyte.

13. The polynucleotide construct of claim 12 , wherein the cytotoxic protein comprises a caspase or its catalytic domain.

14. The polynucleotide construct of claim 13 , wherein the caspase comprises an inducible caspase or its catalytic domain.

15. The polynucleotide construct of claim 14 , wherein the caspase comprises a rapamycin-inducible caspase.

16. The polynucleotide construct of claim 15, wherein the rapamycin-inducible caspase comprises an FKBP-rapamycin binding (FRB) domain.

17. The polynucleotide construct of claim 15 , wherein the rapamycin-inducible caspase comprises an FK506 binding protein (FKBP) domain.

18. 18. The polynucleotide construct of claim 17, wherein the FKBP domain is an FKBP12 domain.

19. The polynucleotide construct of claim 15, wherein the rapamycin-inducible caspase comprises, from N-terminus to C-terminus, an FRB domain, an FKBP12 domain, and the caspase or a functional fragment thereof.

20. The polynucleotide construct of claim 13, wherein the caspase is a non-inducible caspase.

21. The polynucleotide construct of claim 13, wherein the caspase is an autoactivating caspase.

22. The polynucleotide construct of claim 13 , wherein the caspase comprises caspase 9 or its catalytic domain.

23. The polynucleotide construct of claim 13 , wherein the caspase comprises caspase 1 or its catalytic domain.

24. The polynucleotide construct of claim 13 , wherein the caspase comprises caspase 3 or its catalytic domain.

25. 2. The polynucleotide construct of claim 1, wherein the cytotoxic protein comprises caspase 8, BAX, DFF40, HSV-TK, cytosine deaminase, or a catalytic domain thereof.

26. 13. The polynucleotide construct of claim 12, wherein the cytotoxic protein comprises an amino acid sequence having at least 80% sequence identity or similarity to any one of SEQ ID NOs: 3-13.

27. The polynucleotide construct of claim 1 , wherein the adipocyte is a white adipocyte.

28. The polynucleotide construct of claim 1 , wherein the adipocyte is not a brown adipocyte.

29. The polynucleotide construct of claim 1 , further comprising a safety element that reduces expression of said cytotoxic protein in said control cells that are not human adipocytes.

30. 30. The polynucleotide construct of claim 29, wherein the control cell is a muscle cell, a hepatocyte, a bone cell, an erythrocyte, a neuron, a leukocyte, a lymphocyte, or a fibroblast.

31. 30. The polynucleotide construct of claim 29, wherein expression of the safety element is driven by a regulatory element that is active in the control cells but less active or substantially inactive in the human adipocytes.

32. 32. The polynucleotide construct of claim 31 , wherein the safety element comprises a regulatory RNA or a target site for a regulatory RNA that targets the transcript encoding the cytotoxic protein for degradation.

33. 33. The polynucleotide construct of claim 32, wherein the regulatory RNA is an siRNA or an miRNA.

34. 32. The polynucleotide construct of claim 31 , wherein the safety element comprises a transcriptional repressor that reduces expression mediated by the transcriptional promoter.

35. A lipid-based delivery vector (LDV) comprising the polynucleotide construct of any one of claims 1 to 34.

36. 36. The LDV of claim 35, wherein the LDV comprises a fusion-associated small transmembrane (FAST) protein.

37. 37. The LDV of claim 36, wherein the FAST protein comprises the ectodomain of a first reovirus FAST protein and the endodomain of a second reovirus FAST protein.

38. 37. The LDV of claim 36, wherein the FAST protein comprises p10, p13, p14, p15, p16, p22, or a functional domain thereof.

39. 37. The LDV of claim 36, wherein the FAST protein comprises a fusion of a first domain derived from a p14 FAST protein or a p10 FAST protein with a second domain derived from a p14 FAST protein or a p15 FAST protein.

40. 37. The LDV of claim 36, wherein the FAST protein comprises the ectodomain of p14 and the endodomain of p15.

41. 36. The LDV of claim 35, wherein the LDV comprises an ionizable lipid.

42. 42. The LDV of claim 41, wherein the molar ratio of said ionizable lipid to said polynucleotide construct is between about 2:1 and 25:

1.

43. 43. The LDV of claim 42, wherein the molar ratio is about 5:1, about 7.5:1, about 10:1, or about 15:

1.

44. 42. The LDV of claim 41, wherein the ionizable lipid comprises Dlin-KC2-DMA (KC2), DODMA, DODAP, DOBAQ, DOTMA, 18:1 EPC, DOTAP, DDAB, 18:0 EPC, 18:0 DAP, or 18:0 TAP.

45. 36. The LDV of claim 35, wherein the LDV is configured to deliver the polynucleotide construct to a human adipocyte upon contact of the human adipocyte with the LDV.

46. The LDV of claim 35, wherein the LDV is configured to deliver the polynucleotide construct to the human adipocyte upon administration of the LDV to a subject.

47. 36. The LDV of claim 35, wherein the LDV is formulated for non-targeted delivery to the human adipocytes and non-adipocytes.

48. A cell comprising the polynucleotide construct of any one of claims 1 to 34.

49. A method for reducing the viability of a population of adipocytes, comprising contacting the population of adipocytes with an LDV described in any one of claims 35 to 47 under conditions that promote uptake of the polynucleotide construct by the adipocytes.

50. 50. The method of claim 49, wherein the adipocytes comprise white adipocytes.

51. 50. The method of claim 49, wherein the adipocytes are human adipocytes.

52. 50. The method of claim 49, wherein during the contacting step, the LDV containing the polynucleotide construct is present at a concentration of at least 1 nM.

53. 50. The method of claim 49, wherein at least about 1% of the white adipocytes in the population are killed.

54. 50. The method of claim 49, wherein up to about 95% of the white adipocytes in the population are killed.

55. 50. The method of claim 49, wherein between about 5% and 80% of the white adipocytes are killed.

56. A method for reducing adipose tissue volume, comprising administering to a subject an effective amount of the LDV of any one of claims 35 to 47.

57. 57. The method of claim 56, wherein the LDV is administered systemically.

58. 57. The method of claim 56, wherein the LDV is administered topically.

59. 57. The method of claim 56, wherein the LDV is administered via injection into adipose tissue.

60. 57. The method of claim 56, wherein the LDV is administered into visceral fat.

61. 57. The method of claim 56, wherein the LDV is administered into subcutaneous fat.

62. 57. The method of claim 56, wherein the LDV is administered into abdominal fat.

63. 57. The method of claim 56, wherein the adipose tissue volume is reduced by at least about 5%.

64. 57. The method of claim 56, wherein the adipose tissue volume is reduced by up to about 95%.

65. 57. The method of claim 56, wherein the adipose tissue volume is reduced by about 5% to about 80%.

66. 57. The method of claim 56, wherein the adipose tissue is white adipose tissue.

67. 57. The method of claim 56, wherein the reduction in adipose tissue volume is determined by DEXA scanning to quantify adipose tissue before and after administering the LDV containing the polynucleotide construct.

68. 57. The method of claim 56, wherein the cytotoxic protein is an inducible caspase, and the method further comprises administering to the subject an inducer of the inducible caspase.

69. 57. The method of claim 56, wherein the cytotoxic protein is a rapamycin-inducible caspase, and the method further comprises administering rapamycin or a structural analog thereof to the subject.

70. 57. The method of claim 56, wherein the LDV is administered to the subject two or more times.

71. 70. The method of claim 69, wherein the rapamycin or structural analog thereof is administered to the subject two or more times.

72. 57. The method of claim 56, wherein the method treats cellulite in the subject.

73. 57. The method of claim 56, wherein the method treats a metabolic disorder in the subject.

74. 57. The method of claim 56, wherein the method treats Dercam's disease in the subject.

75. A system for selective killing of human adipocytes, comprising a polynucleotide construct according to any one of claims 1 to 34 or an LDV according to any one of claims 35 to 47, and rapamycin or a structural analogue thereof.

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