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WO2024197314A2 - Procédé de thérapie génique de précision de maladies provoquées par un dysfonctionnement endothélial - Google Patents

Procédé de thérapie génique de précision de maladies provoquées par un dysfonctionnement endothélial Download PDF

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WO2024197314A2
WO2024197314A2 PCT/US2024/021370 US2024021370W WO2024197314A2 WO 2024197314 A2 WO2024197314 A2 WO 2024197314A2 US 2024021370 W US2024021370 W US 2024021370W WO 2024197314 A2 WO2024197314 A2 WO 2024197314A2
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promoter
specific
selective
construct
recombinase
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WO2024197314A3 (fr
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Youyang ZHAO
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Northwestern University
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Northwestern University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links

Definitions

  • the vascular endothelium is a monolayer of endothelial cells (ECs) lining the luminal surface of blood and lymphatic vessels.
  • ECs endothelial cells
  • the endothelial monolayer plays a crucial role in vascular homeostasis and maintenance of tissue fluid balance. It helps to maintain an anti -thrombotic and anti-inflammatory state of the microvascular bed and control the tone and proliferative state of the underlying vascular smooth muscle cells. It regulates blood flow and deliver plasma-borne macromolecules.
  • ECs also mediate diverse biological functions such as endocytosis, metabolism, and directing organ regeneration and repair.
  • ECs Under adverse conditions (as for example, infection, tissue necrosis, immune reactions, or hypercholesterolemia), ECs are activated, leading to inflammation and endothelial barrier disruption (increased vascular permeability, edema formation, release of proinflammatory cytokines, and leukocyte extravasation). Endothelial dysfunction figures prominently in the etiologies of many diseases such as atherosclerosis, the pathological process underlying the major cardiovascular diseases (myocardial infarction, stroke, coronary artery disease, and peripheral artery disease), neurodegenerative disease, cancer and cancer metastasis, diabetic complications, hypertension, pulmonary hypertension, sepsis, and acute respiratory distress syndrome (ARDS).
  • endothelial barrier disruption increased vascular permeability, edema formation, release of proinflammatory cytokines, and leukocyte extravasation.
  • Endothelial dysfunction figures prominently in the etiologies of many diseases such as atherosclerosis, the pathological process
  • ECs exhibit structural, phenotypic, and functional heterogeneity in different tissues. Tissue-specific EC dysfunction contributes to different diseases. In the blood-brain barrier, ECs are bound by tight junctions to maintain a highly selective, low-permeability barrier. Endothelial dysfunction in the blood-brain barrier can lead to Alzheimer disease, epilepsy, and multiple sclerosis. The cardiac endothelium plays a crucial role in promoting cardiomyocyte proliferation and maturation via paracrine signaling whereas limited EC proliferative potential results in suboptimal repair of damaged heart tissue after ischemic injury. Thus, vascular endothelial dysfunction in the heart can contribute to heart attack and recovery, and heart failure.
  • Hypertension is attributable to dysfunction of the cardiovascular ECs but not the pulmonary vascular ECs.
  • the pulmonary circulation is different from systemic cardiovascular circulation.
  • Pulmonary vascular EC dysfunction uniquely contribute to pulmonary vascular diseases such as pulmonary arterial hypertension, and ARDS as well as pulmonary fibrosis and COPD.
  • Secretion of angiocrine factors from pulmonary ECs has been shown to improve lung alveolar regeneration, whereas angiocrine factors from liver sinusoidal ECs are critical in modulating hepatic regeneration.
  • Vascularization is critical for tumorigenesis. Due to tissue-specific EC dysfunction in different diseases, methods for selectively targeting ECs in specific tissues are of interest.
  • a construct for selectively expressing a therapeutic polynucleotide in a specific cell type, a specific organ, or both comprising: the therapeutic polynucleotide; a first selective promoter; and a second selective promoter; wherein each of the first selective promoter and the second selective promoter are selected from: a cell type-specific promoter; an organ- specific promoter; and a disease-specific promoter; and wherein the therapeutic polynucleotide is operably linked to at least one of the first selective promoter and the second selective promoter.
  • the construct further comprises: a sequence encoding a DNA recombinase; and a target sequence; wherein the target sequence comprises a stop signal flanked by two DNA recombinase binding sites; wherein the construct comprises from 5’ to 3’ the first selective promoter, the sequence encoding the DNA recombinase, the second selective promoter, the target sequence, and the therapeutic polynucleotide; wherein the first selective promoter is operably linked to the sequence encoding the DNA recombinase; wherein the second selective promoter is operably linked to the therapeutic polynucleotide; and wherein the DNA recombinase binding sites are oriented such that the stop signal is excised from the construct when the DNA recombinase is transcribed.
  • the DNA recombinase is selected from Cre recombinase, Dre recombinase, and Flp recombinase.
  • the cell type-specific promoter is a common endothelial cell promoter.
  • the common endothelial cell promoter is selected from: a CDH5 promoter; a PEC AMI promoter; an ERG promoter; a TEK promoter; a KDR promoter; a SOX17 promoter; a VWF promoter; an ESMI promoter; a PROXI promoter; a VEGFR3 promoter, a FLT4 promoter; a PDPN promoter; and a LYVE1 promoter.
  • the organ-specific promoter is selected from a lung-specific promoter, a heart-specific promoter, an aorta-specific promoter, a skeletal muscle-specific promoter, a brain-specific promoter, a liverspecific promoter, a kidney-specific promoter, a pancreas-specific promoter, an adipose tissuespecific promoter, a spleen-specific promoter, a bone marrow-specific promoter, an intestinespecific promoter, a mammary gland-specific promoter, and a tumor-specific promoter.
  • the lung-specific promoter is selected from a TMEM100 promoter, a HPGD promoter, GRPT1 promoter, a ADRB1 promoter, a SCN7a promoter, a FOXF1A promoter, a NCKAP5 promoter, a RASGEF1A promoter, a FENDRR promoter, and a PRX promoter;
  • the heart-specific promoter is selected from a DCN promoter, a CYTL1 promoter, a WT1 promoter, a SLC28A2 promoter, a EEPD1 promoter, a KCNA5 promoter, a CAR8 promoter, a FBLN1 promoter, a MEOX2 promoter, a RFTN1 promoter, a LAMB1 promoter, a BCL6 promoter, and a MYADM promoter;
  • the aorta-specific promoter is selected from a ACTA2 promoter, a BMX1 promoter,
  • the skeletal muscle-specific promoter is selected from a MLLT4 promoter, a CXCL10 promoter, a CXCL2 promoter, a ADAMTS4 promoter, a RND1 promoter, a CXCL1 promoter, a IL6 promoter, a SPHK1 promoter, a ICAM1 promoter, a PFKFB3 promoter, and a TUBB6 promoter;
  • the brain-specific promoter is selected from a SLC2A1 promoter, a PTN promoter, a LEF1 promoter, a CLDN5 promoter, a CLDN1 promoter, a CLDN3 promoter, a SLCO1C1 promoter, a SLCO1A4 promoter, a SLC22A8 promoter, a
  • the first selective promoter is a CDH5 promoter; and the second selective promoter is a TMEM100 promoter or a HPGD promoter.
  • the construct further comprises at least one additional selective promoter.
  • a method for selectively expressing a therapeutic polynucleotide in a specific cell type, a specific organ, or both, in a subject in need thereof comprising administering to the subject a construct comprising: the therapeutic polynucleotide; a first selective promoter; and a second selective promoter; wherein each of the first selective promoter and the second selective promoter are selected from: a cell type-specific promoter; an organ-specific promoter; and a disease-specific promoter.
  • the construct further comprises: a sequence encoding a DNA recombinase; and a target sequence; wherein the target sequence comprises a stop signal flanked by two DNA recombinase binding sites; wherein the construct comprises from 5’ to 3’ the first selective promoter, the sequence encoding the DNA recombinase, the second selective promoter, the target sequence, and the therapeutic polynucleotide; wherein the first selective promoter is operably linked to the sequence encoding the DNA recombinase; wherein the second selective promoter is operably linked to the therapeutic polynucleotide; and wherein the DNA recombinase binding sites are oriented such that the target sequence is excised from the construct when bound by the DNA recombinase.
  • the DNA recombinase is selected from Cre recombinase, Dre recombinase, and Flp recombinase.
  • the cell type-specific promoter is a common endothelial cell promoter.
  • the common endothelial cell promoter is selected from: a CDH5 promoter; a PEC AMI promoter; an ERG promoter; a TEK promoter; a KDR promoter; a SOX17 promoter; a VWF promoter; an ESMI promoter; a PROXI promoter; a VEGFR3 promoter; a FLT4 promoter; a PDPN promoter; and a LYVE1 promoter.
  • the organ-specific promoter is selected from a lung-specific promoter, a heart-specific promoter, an aorta-specific promoter, a skeletal muscle-specific promoter, a brainspecific promoter, a liver-specific promoter, a kidney-specific promoter, a pancreas-specific promoter, an adipose tissue-specific promoter, a spleen-specific promoter, a bone marrow-specific promoter, an intestine-specific promoter, a mammary gland-specific promoter, and a tumorspecific promoter.
  • the lung-specific promoter is selected from a TMEM100 promoter, a HPGD promoter, GRPT1 promoter, a ADRB1 promoter, a SCN7a promoter, a FOXF1A promoter, a NCKAP5 promoter, a RASGEF1A promoter, a FENDRR promoter, and a PRX promoter;
  • the heart-specific promoter is selected from a DCN promoter, a CYTL1 promoter, a WT1 promoter, a SLC28A2 promoter, a EEPD1 promoter, a KCNA5 promoter, a CAR8 promoter, a FBLN1 promoter, a MEOX2 promoter, a RFTN1 promoter, a LAMB1 promoter, a BCL6 promoter, and a MYADM promoter;
  • the aorta-specific promoter is selected from a ACTA2 promoter, a BMX1 promoter,
  • the first selective promoter is a CDH5 promoter; and the second selective promoter is a TMEM100 promoter or a HPGD promoter.
  • the construct further comprises at least one additional selective promoter.
  • the construct is complexed with a nanoparticle.
  • FIGS. 1A-1B Aging impairs lung vascular repair following LPS challenge.
  • FIG. 1A Persistent increase of lung vascular permeability in aged mice following LPS challenge (i.p.). Young adult mice (3-5 mos.) were challenged with 2.5 mg/kg of LPS while aged mice (19-21 mos.) with 1.0 mg/kg of LPS. Lungs were collected at various times for EBA flux assay.
  • FIG. IB Lung edema in aged mice at 72h post-LPS. *P ⁇ 0.05; ****p ⁇ 0.0001. Kruskal-Wallis (A); Oneway ANOVA (B).
  • FIGS. 2A-2C Sustained inflammatory lung injury in aged mice following LPS challenge.
  • FIG. 2A Sustained increase of MPO activity in aged lungs following LPS challenge.
  • FIGS. 2B and 2C Representative micrographs of H & E staining and quantification of lung injury. Scale bar 40 pm. ****p ⁇ 0.0001. One-way ANOVA.
  • FIGS. 3A-3B Defective endothelial proliferation in aged lungs following LPS challenge.
  • FIG. 3B Quantification of cell proliferation in mouse lungs at basal (Ctl) and 72h post-LPS. ****p ⁇ 0.0001. One-way ANOVA (Tukey).
  • FIGS. 4A-4B Failure of FoxMl induction in aged lungs following LPS challenge.
  • FIG. 4A QRT-PCR analysis of FoxMl expression in mouse lungs at indicated times post-LPS.
  • FIG. 4B QRT-PCR analysis showing marked increase of expression of genes at 72h post-LPS in lungs of young mice but not aged mice. **P ⁇ 0.01; ****p ⁇ 0.0001.
  • FIGS. 5A-5C Forced expression of FoxMl normalized resolution of inflammatory lung injury and promoted survival of aged mice.
  • FIG. 5B Marked decrease of lung vascular permeability in F0XM1 plasmid-administered mice at 72h post-LPS compared to vector mice.
  • FIG. 5C MPO activity assay demonstrating normal resolution of lung inflammation in F0XM1 plasmid-administered aged mice. ****P ⁇ 0.0001.
  • FIGS. 6A-6C Transient FoxMl expression in lung ECs of aged WT mice reactivated lung EC proliferation in F0XM1 plasmid DNA-administered mice.
  • FIG. 6A Representative micrographs of anti-BrdU staining showing reactivated EC proliferation in F0XM1 plasmid DNA- administered mice at 72h post-LPS. Arrows point to proliferating ECs. Scale bar, 50 pm.
  • FIG. 6B Quantification of cell proliferation.
  • FIG. 6C Quantitative RT-PCR analysis of FoxMl target genes. ****/’ ⁇ 0.0001.
  • One-way ANOVA (Tukey).
  • FIGS. 7A-7G Construction of a lung EC-specific promoter system to drive lung EC- specific expression of FoxMl in aged mice leading to normalized vascular repair.
  • FIG. 7A Diagram presentation of the plasmid DNA expressing FOXM1 under the control of the dual promoter system for lung EC-specific expression. CDH5 promoter-driven Dre expression will remove the STOP signal in TMFM100 expressing cells leading to lung EC-specific gene expression.
  • FIGS. 7B-7D Quantitative RT-PCR analysis of gene expression.
  • FIGS. 8A-8C Constructs comprising lung EC-specific dual promoter system with CDH5 and TMEM100 promoters and a Cre/LoxP recombinase system (FIG. 8A), a Flp/Frt recombinase system (FIG. 8B), or a Dre/Rox recombinase system (FIG. 8C).
  • FIGS. 9A-9C Constructs comprising lung EC-specific dual promoter system with PECAM1 and TMEM100 promoters and a Dre/Rox recombinase system (FIG. 9A), a Cre/Lox recombinase system (FIG. 9B), or a Flp/Frt recombinase system (FIG. 9C).
  • FIGS. 10A-10F Constructs comprising lung-EC specific dual promoter systems using CDH5 and HPGD promoters and a Dre/Rox recombinase system (FIG. 10A), a Cre/Lox recombinase system (FIG. 10B), or a Flp/Frt recombinase system (FIG. 10C).
  • FIGS. 11A-11C Constructs comprising lung-EC specific triple promoter systems using a common EC promoter of either CDH5 o PEC AMI and another 2 lung endothelial gene promoters (TMEM100 and HPGD .
  • the constructs comprise and Dre/Rox and Cre/Lox recombinase systems (FIG. 11 A), and Dre/Rox and Flp/Frt recombinase systems (FIG. 11B), Cre/Lox and Flp/Frt recombinase systems (FIG. 11C).
  • RSR Rox-STOP-Rox
  • LSL Lox-STOP-Lox
  • FSF Frt-STOP- Frt.
  • FIGS. 12A-12G Constructs illustrate generalization of organ-specific EC promoter system for gene modulation in an organ and EC-specific manner comprising a common EC promoter and an organ-specific EC promoter.
  • the constructs comprise Dre/Rox recombinase systems (FIG. 12A), a Cre/Lox recombinase system (FIG. 12B), and a Flp/Frt recombinase system (FIG. 12C), a Dre/Rox and Cre/Lox recombinase system (FIG. 12D), a Dre/Rox and Flp/Frt recombinase system (FIG.
  • FIG. 12E a Cre/Lox and Flp/Frt recombinase system
  • FIG. 12G a Dre/Rox, Cre/Lox, and Flp/Frt recombinase system
  • FIGS. 13A-13C Constructs illustrate generalization of organ-specific EC promoter system for gene modulation in an organ and EC-specific manner comprising a common EC promoter and an organ-specific promoter which is active in ECs but not limited in ECs.
  • the constructs comprise Dre/Rox recombinase systems (FIG. 13 A), a Cre/Lox recombinase system (FIG. 13B), and a Flp/Frt recombinase system (FIG. 13C).
  • FIGS. 14A-14C Constructs comprise two organ-specific EC promoters, and a Dre/Rox recombinase system (FIG. 14A), a Cre/Lox recombinase system (FIG. 14B), and a Flp/Frt recombinase system (FIG. 14C).
  • FIGS. 15A-15C Constructs comprise two organ-specific promoters, and a Dre/Rox recombinase system (FIG. 15 A), a Cre/Lox recombinase system (FIG. 15B), and a Flp/Frt recombinase system (FIG. 15C).
  • FIGS. 16A-16C Nanoparticle delivery of plasmid DNA expressing Cre under the control of CDH5 promoter into TdTomato mice resulted in TdTomato expression in ECs of various organs.
  • FIG. 16A Construct delivered.
  • FIG. 16B Graphic presentation of experiment protocol.
  • FIG. 16C Confocal microscopy analysis of TdTomato expression in ECs of various organs.
  • FIGS. 17A-17B CDH5p-Dre-Tmeml00p-Rox-STOP-Rox dual promoter system drove lung EC-specific expression of Cre leading to activation of TdTomato expression (red) in lung ECs of TdTomato mice but not in other vascular ECs (aorta, brain, heart, skeletal muscle).
  • the plasmid DNA including the construct (FIG. 17A) was delivered to TdTomato mice by nanoparticles.
  • various organ tissues were collected for immunostaining with anti- CD31 (white) to identify ECs and confocal microscopy (FIG. 17B). Nuclei were counterstained with DAPI (blue). Scale bar, 20 pm.
  • FIGS. 18A-18B CDH5-Dre-HPGD-Rox-STOP-Rox dual promoter system drove lung EC-specific activation of TdTomato expression (red) in TdTomato mice four days after plasmid DNA delivery.
  • the plasmid DNA including the construct (FIG. 18A) was delivered to TdTomato mice by nanoparticles.
  • Various organ tissues were collected for immunostaining with anti-CD31 (white) to identify ECs and confocal microscopy (FIG. 18). Nuclei were counterstained with DAPI (blue). Scale bar, 20 pm.
  • vascular endothelial cells exhibit structural, phenotypic, and functional heterogeneity in different tissues. Tissue-specific EC dysfunction contributes to different diseases. Although many efforts have been devoted to identifying organ-specific endothelial genes, these genes are typically also expressed in some other cell types to some extent. Thus, selective targeting of tissuespecific EC dysfunction, for example, by normalization of gene expression and/or signal transduction in disease-causing tissue-specific ECs is a beneficial precision gene therapy approach for treating a wide range of human diseases with minimal off-target effects.
  • the present disclosure uses an artificial promoter system for organ-specific vascular EC targeting.
  • a construct for selectively expressing a therapeutic polynucleotide in a specific cell type and/or a specific organ in a subject in need thereof comprising the therapeutic polynucleotide; a first selective promoter, and a second selective promoter, wherein each of the first selective promoter and the second selective promoter are selected from: a cell type-specific promoter, an organ-specific promoter, and/or a disease-specific promoter.
  • the cell type specific promoter is a common endothelial cell (EC) promoter.
  • the endothelium is a single layer of squamous endothelial cells (ECs) that line the interior surface of blood vessels and lymphatic vessels.
  • ECs squamous endothelial cells
  • the endothelium forms an interface between circulating blood or lymph in the lumen and the rest of the vessel wall.
  • ECs form the barrier between vessels and tissue and control the flow of substances and fluid into and out of a tissue.
  • ECs in direct contact with blood are called vascular endothelial cells whereas those in direct contact with lymph are known as lymphatic endothelial cells.
  • the vascular endothelium a monolayer of EC, constitutes the inner cellular lining of arteries, veins and capillaries and therefore is in direct contact with the components and cells of blood.
  • the endothelium is not merely a barrier between blood and tissues, but it is also an endocrine organ.
  • Vascular ECs have a key role in the development and maintenance of the functional
  • constructs are used herein to refer to a recombinant polynucleotide, i.e., a polynucleotide that was formed artificially by combining at least two polynucleotide components from different sources (natural or synthetic).
  • the constructs described herein comprise the coding region of a transgene of interest (a “therapeutic polynucleotide”) operably linked to a promoter that (1) is associated with another gene found within the same genome, (2) is from the genome of a different species, or (3) is synthetic.
  • constructs can be generated using conventional recombinant DNA methods.
  • a "therapeutic polynucleotide” as used herein refers to DNA sequence encoding a polypeptide or an RNA that induces a positive therapeutic effect when expressed.
  • a therapeutic polynucleotide may comprise several operably linked fragments, such as a promoter, a 5' leader sequence, a coding sequence and a 3' non-translated sequence, such as sequence encoding a polyadenylation site.
  • "Expression” of a polynucleotide refers to the process wherein a gene is transcribed into an RNA and/or translated into a protein.
  • polynucleotide refers to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof.
  • a polynucleotide may refer to a polydeoxyribonucleotide (containing 2-deoxy-D-ribose), a polyribonucleotide (containing D- ribose), and to any other type of polynucleotide that is an N glycoside of a purine or pyrimidine base.
  • nucleic acid refers only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA.
  • an oligonucleotide also can comprise nucleotide analogs in which the base, sugar, or phosphate backbone is modified as well as non-purine or non-pyrimidine nucleotide analogs.
  • DNA or RNA of genomic, natural, or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand).
  • protein or “polypeptide” or “peptide” may be used interchangeable to refer to a polymer of amino acids.
  • a “polypeptide” or “protein” is defined as a longer polymer of amino acids, of a length typically of greater than 50, 60, 70, 80, 90, or 100 amino acids.
  • a “peptide” is defined as a short polymer of amino acids, of a length typically of 50, 40, 30, 20 or less amino acids.
  • a protein typically comprises a polymer of naturally or non- naturally occurring amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine).
  • amino acids e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine).
  • a “promoter” or “transcription regulatory sequence” refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, such as a therapeutic polynucleotide sequence, and is typically located upstream with respect to the direction of transcription of the coding sequence.
  • a promoter is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter, including e g. attenuators or enhancers, but also silencers.
  • a selective promoter may be a promoter that is only active or is primarily active in a specific cell type (“cell type-specific promoter”), a specific organ (“organ-specific promoter”), a specific tissue (“tissue-specific promoter”), a specific disease (“disease-specific promoter”), a specific tumor type (“tumor-specific promoter”), etc.
  • the selective promoter may be at least 10-, 100-, 1000-fold more active in the selected cell type, tissue, organ, etc. relative to other cell types, tissues, organs, etc.
  • Some organ-specific promoters are expressed in endothelial cells and also other cell types.
  • activity of the cell-specific promoter limits the activity of the organspecific promoter only in the selected cell type.
  • the cell type-specific promoter is only active in the selected cell type.
  • the selective promoter is only active in the selected cell type, tissue, organ, disease condition, etc.
  • the selective promoters disclosed herein are identified as promoters that regulate a particular gene.
  • a CDH5 promoter is a promoter that regulates the gene CDH5, which encodes vascular endothelial cadherin (VE-cadherin).
  • the promoters described herein may be from human or other species including, but limited to mouse, rat, dog, pig, and monkey, and analogs thereof or synthetic analogs with key elements of selectivity.
  • a cell type-specific promoter may be a common endothelial cell.
  • a “common endothelial cell promoter” refers to a promoter that is expressed in endothelial cells, regardless of the tissue or organ that the endothelial cell is located in. Common endothelial promoters include, but are not limited to a CDH5 promoter; a PEC AMI promoter; an ERG promoter; a TEK promoter; a KDR promoter; a SOX17 promoter; a VWF promoter; and an ESMI promoter.
  • Common endothelial promoters can also be a lymphatic endothelial cell-specific promoters, including but not limited to a PROXI promoter, a VEGFR3 promoter, a FLT4 promoter, a PDPN promoter, and a LYVE1 promoter.
  • the PROXI, VEGFR3, FLT4, PDPN, and LYVE1 promoters are common lymphatic EC-specific promoters.
  • the CDH5 promoter may comprise a sequence having at least 85%, 90%, 95%, or 100% identity to SEQ ID NO: 3.
  • a selective promoter may be characterized by a combination of specificities.
  • a promoter may be both cell type-specific and organ-specific.
  • Exemplary constructs comprising dual specificity promoters are found throughout the specification and figures, particularly FIGS. 8-15.
  • An organ-specific promoter may be a lung-specific promoter.
  • lung specific promoters include, but are not limited to a GRPT1 promoter, a ADRB1 promoter, a SCN7a promoter, a TMEM100 promoter, a HPGD promoter, a FOXF1A promoter, aNCKAP5 promoter, a RASGEF1A promoter, a FENDRR promoter, and a PRX promoter.
  • Exemplary constructs comprising lung specific promoters include those described herein and in FIGS. 7-11, 17, and 18. These lung-specific promoters are expressed in endothelial cells.
  • the HPGD promoter may comprise a sequence having at least 85%, 90%, 95%, or 100% identity to SEQ ID NO: 10.
  • the TMEM100 promoter may comprise a sequence having at least 85%, 90%, 95%, or 100% identity to SEQ ID NO: 7.
  • An organ-specific promoters may be a heart-specific promoter.
  • exemplary heart specific promoters include, but are not limited to a DON promoter, a CYT11 promoter, a WT1 promoter, a SLC28A2 promoter, a EEPD1 promoter, a KCNA5 promoter, a CAR8 promoter, a FBLN1 promoter, a ME0X2 promoter, a RFTN1 promoter, a LAMB1 promoter, a BCL6 promoter, and a MY ADM promoter. These heart-specific promoters are expressed in endothelial cells.
  • An organ-specific promoter may be an aorta-specific promoter.
  • aorta specific promoters include, but are not limited to a ACTA2 promoter, a BMX1 promoter, a TMEM26 promoter, a IGFBP5 promoter, a IL33 promoter, a DRAM1 promoter, a EHD3 promoter, a ESMI promoter, a RARB promoter, a FKBP5 promoter, a F.NPF.P promoter, and a CDC14A promoter. These aorta-specific promoters are expressed in endothelial cells.
  • An organ-specific promoter may be a skeletal muscle-specific promoter.
  • skeletal muscle specific promoters include, but are not limited to a CXCL10 promoter, a MLLT4 promoter, a CXCL2 promoter, a ADAMTS4 promoter, a RND1 promoter, a CXCL1 promoter, a IL6 promoter, a SPHK1 promoter, a ICAM1 promoter, a PFKFB3 promoter, and a TUBB6 promoter. These skeletal muscle-specific promoters are expressed in endothelial cells.
  • An organ-specific promoter may be a brain -specific promoter.
  • exemplary brain specific promoters include, but are not limited to a SLC2A1 promoter, a PTN promoter, a LEF1 promoter, a CLDN5 promoter, a CLDN1 promoter, a CLDN3 promoter, SLCO1C1 promoter, a SLCO1A4 promoter, a SLC22A8 promoter, a MFSD2A promoter, a SLC38A3 promoter, a SPOCK2 promoter, a FOXF2 promoter, a EDN3 promoter, a STRA6 promoter, and a SLC38A5 promoter.
  • These brain-specific promoters are expressed in endothelial cells.
  • An organ-specific promoter may be a liver-specific promoter.
  • liver specific promoters include, but are not limited to a DNASE1L3 promoter, a CLEC4G promoter, a FCGR2B promoter, a STAB2 promoter, a OIT3 promoter, a BMP2 promoter, a AASS promoter, a MRC1 promoter, a PLXNC1 promoter, and a WNT2 promoter. These liver-specific promoters are expressed in endothelial cells.
  • An organ-specific promoter may be a kidney-specific promoter.
  • kidney specific promoters include, but are not limited to a DRAM1 promoter, a DKK2 promoter, a ESMI promoter, a IGFBP5 promoter, a PBX1 promoter, a BOC promoter, a IGFBP3 promoter, a IRX3 promoter, a TNFAIP2 promoter, and a PTPRU promoter. These kidney-specific promoters are expressed in endothelial cells.
  • An organ-specific promoter may be a pancreas-specific promoter.
  • pancreas specific promoters include, but are not limited to a CELE2A promoter, a PRSS2 promoter, a CELA1 promoter, a TRY4 promoter, a PNLIP promoter, a CTRB1 promoter, a CELA3B promoter, a CEL promoter, a CLPS promoter, and a CPA1 promoter. These pancreas-specific promoters are expressed in endothelial cells.
  • An organ-specific promoter may be an adipose-specific promoter.
  • adipose specific promoters include, but are not limited to a CAR3 promoter, a CSF2RB promoter, a ANGPTL4 promoter, a EGR2 promoter, a GPR160 promoter, a IFI27L2A promoter, a LM01 promoter, a CLDN15 promoter, a HEXIM 1 promoter, and a LGALS1 promoter. These adiposespecific promoters are expressed in endothelial cells.
  • An organ-specific promoter may be a spleen-specific promoter.
  • exemplary spleen specific promoters include, but are not limited to STAB2, LTC4S, ANKRD2, STAR. These spleenspecific promoters are expressed in endothelial cells.
  • An organ-specific promoter may be a bone marrow-specific promoter.
  • Exemplary bone marrow specific promoters include, but are not limited to SIGLECH, SELL, LAIR1. These bone marrow-specific promoters are expressed in endothelial cells.
  • An organ-specific promoter may be an intestine-specific promoter.
  • exemplary intestine specific promoters include, but are not limited to ACE, COL15A1, SCGB3A1. These intestinespecific promoters are expressed in endothelial cells.
  • An organ-specific promoter may be a mammary gland-specific promoter.
  • exemplary mammary gland specific promoters include, but are not limited to CCRN4L. These mammary gland-specific promoters are expressed in endothelial cells.
  • An organ-specific promoter may be a tumor-specific promoter.
  • Tumor-specific genes or specific variants may be specifically expressed, differentially expressed in tumors or cancerous lesions.
  • Exemplarily tumor-specific promoters include, but are not limited to VWA1, an alphafetoprotein (AFP) promoter, a Cholecystokinin-A receptor (CCKAR) promoter, a carcinoembryonic antigen (CEA) promoter, a C-erbB2/neu promoter, a cyclooxygenase (COX-2) promoter, a CXCR4 promoter, a E2F promoter, a human epididymis protein 4 (HE4), a L-plastin (LP) promoter, a MUC1 promoter, a Prostate-specific antigen (PSA) promoter, a Survivin promoter, a tyrosinase-related protein 1 (TRP1) promoter, a EGFR promoter, and a Tyrosin
  • An organ-specific promoter may be a disease-specific promoters, wherein the promoter is activated by specific pathological condition(s).
  • pathological specific promoters include, but are not limited to a ICAM1 promoter, a VCAM1 promoter, a RAB5A promoter, a CTTN promoter, a ITGB1 promoter, a MMP9 promoter, a TNFA promoter, a IL6 promoter, a TGFB promoter, a PDGF promoter, a CXCL12 promoter, and a BCL6 promoter.
  • Suitable organ-specific promoters include promoters that are expressed in endothelial cells and any other cell type specific to that organ.
  • the term "operably linked” refers to a linkage of polynucleotide (or polypeptide) elements in a functional relationship.
  • a nucleic acid is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame.
  • the construct may further include a DNA recombinase system.
  • the construct may further comprise: a sequence encoding a DNA recombinase; and a target sequence; wherein the target sequence comprises a stop signal flanked by two DNA recombinase binding sites; wherein the construct comprises from 5’ to 3’ the first selective promoter, the sequence encoding the DNA recombinase, the second selective promoter, the target sequence, and the therapeutic polynucleotide; wherein the first selective promoter is operably linked to the sequence encoding the DNA recombinase; wherein the second selective promoter is operably linked to the therapeutic polynucleotide; and wherein the DNA recombinase binding sites are oriented such that the target sequence is excised from the construct when bound by the DNA recombinase.
  • DNA recombinases e.g. Cre recombinase, Dre recombinase, Flp recombinase
  • Cre recombinase e.g. Cre recombinase, Dre recombinase, Flp recombinase
  • DNA recombinases are enzymes that catalyze directionally sensitive DNA exchange reactions between short DNA recombinase binding sites (e.g. lox, rox, frt) that are specific to each recombinase. These reactions enable four basic functional modules: excision/insertion, inversion, translocation and cassette exchange, which may be used individually or combined in a range of configurations to control gene expression.
  • the “stop signal” or “stop cassette” prevents translation of a downstream gene.
  • the DNA recombinase binding sites may be oriented in the same orientation, and therefore configured to excise the stop signal when bound by the DNA recombinase.
  • the sequence encoding the DNA recombinase is operably linked to the first selective promoter.
  • the DNA recombinase is transcribed.
  • the second selective promoter is operably linked to the therapeutic polynucleotide, but is separated by the target sequence. The stop signal within the target sequence prevents transcription of the therapeutic polynucleotide, even when in the presence of the transcription factors and regulatory proteins necessary to activate the second selective promoter.
  • the DNA recombinase protein can bind the cognate recombining sites (e.g., recombining LoxP sites for Cre, recombining RoxP sites for Dre, recombining Frt sites for Flp) and excise the stop signal, thereby allowing transcription of the therapeutic polynucleotide under control of the second selective promoter.
  • cognate recombining sites e.g., recombining LoxP sites for Cre, recombining RoxP sites for Dre, recombining Frt sites for Flp
  • DNA recombinase systems that may be used in the constructs disclosed herein include, but are not limited to, a Cre/Lox system, a Dre/Rox system, and a Flp/Frt system.
  • the construct comprises the configuration shown in FIG. 7A, wherein CDH5 is the first selective promoter, TMEM100 is the second selective promoter, FOXM1 is the therapeutic polynucleotide, and the construct comprises a Dre/Rox recombinase system.
  • the construct may comprise a sequence having at least 85%, 90%, 95%, or 100% identity to SEQ ID NO: 1.
  • the construct may comprise a sequence having at least 85%, 90%, 95%, or 100% identity to SEQ ID NO: 1, except the sequence encoding FOXM1 is replaced with another therapeutic polynucleotide.
  • the construct comprises the configuration shown in FIG. 10A, wherein CDH5 is the first selective promoter, HPGD is the second selective promoter, and FOXM1 is the therapeutic polynucleotide, and the construct comprises a Dre/Rox recombinase system.
  • the construct may comprise a sequence having at least 85%, 90%, 95%, or 100% identity to SEQ ID NO: 2.
  • the construct may comprise a sequence having at least 85%, 90%, 95%, or 100% identity to SEQ ID NO: 2, except the sequence encoding FOXM1 is replaced with another therapeutic polynucleotide.
  • Substantial identity of amino acid sequences means that a polynucleotide or polypeptide comprises a sequence that has at least 85% sequence identity to a reference sequence (SEQ ID NO) using a sequence alignment program; preferably BLAST using standard parameters.
  • a preferred percent identity of polynucleotides and polypeptides can be any integer from 85% to 100%.
  • a preferred percent identity may be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to a reference sequence.
  • the construct may be part of a vector.
  • a “vector” is a nucleic acid molecule capable of transporting another nucleic acid to which it is linked.
  • the four maj or types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced.
  • Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e g., lentiviral vectors).
  • certain vectors are capable of directing the expression of exogenous genes to which they are operatively linked.
  • Suitable vectors are known in the art and contain the necessary elements in order for the gene encoded within the vector to be expressed as a protein in the host cell.
  • plasmid and also “minicircle DNA” as well as “nanoplasmid” refers to a circular double stranded DNA loop into which additional DNA segments may be ligated, specifically exogenous DNA segments encoding the mutant a-gal protein.
  • viral vector is used to describe a virus particle that is used to deliver genetic material (e.g., the constructs of the present invention) into cells, wherein additional DNA segments may be ligated into the viral genome.
  • Viral vectors include replication defective retroviruses (including lentiviruses), adenoviruses, and adeno-associated viruses (AAV)), which serve equivalent functions.
  • a viral vector may be used to the extent it can tolerate the size of the inserted nucleic acid.
  • the construct is in a plasmid.
  • a "transgene” refers to a gene that has been introduced into a host cell.
  • the transgene may comprise sequences that are native to the cell, sequences that do not occur naturally in the cell, or combinations thereof.
  • a transgene may contain sequences coding for one or more proteins that may be operably linked to appropriate regulatory sequences for expression of the coding sequences in the cell.
  • Transduction refers to the delivery of a nucleic acid molecule into a recipient host cell, such as by non-viral carriers such as polymer nanoparticles or lipid nanoparticles, by a viral vector, such as rAdv or rAAV.
  • transduction of a target cell by a rAAV virion leads to transfer of the rAAV vector contained in that virion into the transduced cell.
  • "Host cell” or “target cell” refers to the cell into which the nucleic acid delivery takes place.
  • the constructs and plasmids described herein may be complexed with a nanoparticle.
  • Suitable nanoparticles for carrying the constructs and plasmids include, but are not limited to, nanoparticles that comprise a polyethylene glycol (PEG)-Z>-poly (D,L-lactide) (PLA) (PEG-/>- PLA, PLA-PEG)) copolymer or a PEG-Z>-poly(lactic acid-co-glycolic acid) (PLGA) (PEG-Z>- PLGA, PLGA-PEG) copolymer, as described in International Publication Nos. W02020/077178A1 and WO2022/182745A1.
  • Nanoparticles include lipid nanoparticles and PEI600-MA5/PEG-OA/Cho nanoparticles, and other nanoparticles with positive charges.
  • an endothelium-targeting nanoparticle, EndoNPl (Mountview Therapeutics LLC), is used.
  • a method for selectively expressing a therapeutic polynucleotide in at least one of a specific cell type and a specific organ in a subject in need thereof comprising administering to the subject a construct comprising the therapeutic polynucleotide; a first selective promoter; and a second selective promoter; wherein each of the first selective promoter and the second selective promoter are selected from: a cell type-specific promoter; an organ-specific promoter; and a disease-specific promoter.
  • the method may comprise administering any of the constructs described herein.
  • the construct comprises the configuration shown in FIG. 7A, wherein CDH5 is the first selective promoter, TMEM100 is the second selective promoter, FOXM1 is the therapeutic polynucleotide, and the construct comprises a Dre/Rox recombinase system.
  • the construct may comprise a sequence having at least 85%, 90%, 95%, or 100% identity to SEQ ID NO: 1.
  • the construct comprises the configuration shown in FIG.
  • CDH5 is the first selective promoter
  • HPGD is the second selective promoter
  • FOXM1 is the therapeutic polynucleotide
  • the construct comprises a Dre/Rox recombinase system.
  • the construct may comprise a sequence having at least 85%, 90%, 95%, or 100% identity to SEQ ID NO: 2.
  • administering refers to dispensing, delivering or applying the substance to the intended subject by any suitable route for delivery, including delivery by either the parenteral/oral route, intramuscular injection, subcutaneous/intradermal injection, intravenous injection, retro-orbital injection, intrathecal administration, buccal administration, transdermal delivery, topical administration, and administration by the intranasal or respiratory tract route.
  • a “subject in need thereof’ as utilized herein may refer to a subject in need of treatment for a disease or disorder.
  • the disease or disorder may be associated with dysregulated gene expression or function.
  • the subject in need thereof may include a subject having a disease or condition associated with tissue-specific EC dysfunction, for example changes in gene expression and or signal transduction in disease-causing tissue-specific EC.
  • the subject in need thereof may include a subject having a disease or condition associated with vascular EC dysfunction.
  • Diseases associated with EC dysfunction include, but are not limited to hypertension, peripheral vascular disease, stroke, atherosclerosis, diabetes, chronic kidney failure, pulmonary hypertension, ARDS, cancer, cancer metastasis, and infectious diseases.
  • the term “subject” may be used interchangeably with the terms “individual” and “patient” and includes human and non-human mammalian subjects.
  • the therapeutic polynucleotide delivered may be used to treat a disease or a means of therapy in response to a disease.
  • treatment refers to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible.
  • the aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder or condition.
  • the terms “a”, “an”, and “the” mean “one or more.”
  • a molecule should be interpreted to mean “one or more molecules.”
  • “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus ⁇ 10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.
  • the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.”
  • the terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims.
  • the terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims.
  • the term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.
  • Example 1 A Method for Precision Gene Therapy of Diseases Caused by Endothelial Dysfunction
  • Acute respiratory distress syndrome is a form of acute-onset hypoxemic respiratory failure with bilateral pulmonary infiltrates that is caused by acute inflammatory edema of the lungs not attributable to left heart failure (32-34).
  • Common causes of ARDS include sepsis, pneumonia, inhalation of harmful substances, burns, major trauma with shock and massive transfusion. Sepsis, with annual U.S. incidence of over 750,000, is the most common cause.
  • Endothelial injury characterized by persistently increased lung microvascular permeability resulting in protein-rich lung edema, is a hallmark of acute lung injury (ALI) and ARDS (35-38).
  • the forkhead box (Fox) transcriptional factors share homology in the winged helix or forkhead DNA-binding domains (49, 50).
  • FoxMl was the first to be identified as a proliferation-specific transcriptional factor and is expressed during cellular proliferation to control the transcription of many cell cycle genes (51-53).
  • FoxMl is expressed in many types of cells, such as cardiomyocytes, endothelial cells (ECs), hepatocytes, lung epithelium cells, and smooth muscle cells (22-24).
  • ECs endothelial cells
  • hepatocytes hepatocytes
  • lung epithelium cells hepatocytes
  • smooth muscle cells 22-24.
  • FoxMl expression is restricted to intestinal crypts, thymus and testes (49, 50).
  • FoxMl is silenced in terminally differentiated cells, it can be induced after organ injury: We have previously reported that FoxMl is induced in lung ECs in the repair phase but not in the injury phase in young adult mice following sepsis challenge (24). In EC-restricted Foxml knockout mice, pulmonary vascular EC proliferation and endothelial barrier recovery are defective following inflammatory injury (24, 44). FoxMl also promotes re-annealing of the endothelial adherens junctional complex to restore endothelial barrier function after vascular injury (45). These results demonstrated the importance of FoxMl in vascular repair.
  • EBA Evans blue-conjugated albumin
  • MPO myeloperoxidase
  • MPO activity was similarly increased versus basal controls at 24h post-LPS in young adult and aged mice (FIG. 2A). Although MPO activity returned to basal levels in lungs of young adult mice at 72h post-LPS, it remained elevated in aged lungs (FIG. 2A). H & E staining showed similar degrees of injury at 24h post-LPS in young and aged mouse lungs (FIGS. 2B and 2C). At 96h, lung structure and cellular infiltrates were largely normalized in young mice whereas they remained abnormal in aged mice (FIGS. 2B and 2C). Together, these data demonstrated impaired resolution of inflammation in aged lungs following LPS challenge.
  • FoxMl is a critical reparative transcriptional factor (43-46)
  • FoxMl was markedly induced in the lungs of young adult mice during the recovery phase (at 48 and 72h but not in the injury phase, e.g., 24h post-LPS) but not in aged lungs following LPS challenge (FIG. 4A).
  • FoxMl target genes essential for cell cycle progression such as Cdc25c, and Ccna2 were not induced in aged lungs (FIG. 4B).
  • FoxMl expression and its transcriptional target genes were markedly induced at 72h post-LPS challenge (FIG. 4).
  • Nanoparticle-directed FoxMl expression in endothelium normalizes endothelial regeneration and resolution of inflammatory lung injury in aged WT mice
  • nanoparticle delivery of F0XM1 plasmid DNA resulted in a marked increase of FoxMl expression in aged WT mice at 72h post-LPS compared to vector DNA-administered mice, which was comparable to FoxMl expression in young adult mice.
  • EBA flux was drastically decreased in F0XM1 plasmid DNA- administered mice compared to vector DNA-administered mice (FIG. 5B).
  • Lung MPO activity returned to a level close to basal level in F0XM1 plasmid DNA-administered mice (FIG. 5C).
  • TMEM100 vascular endothelial (VE)- cadherin (CDH5) and platelet and endothelial adhesion molecule 1 (PECAM1, also known as CD31).
  • VE vascular endothelial
  • PECAM1 platelet and endothelial adhesion molecule 1
  • TMEM100 is considered as a lung-specific endothelium gene (48), however it is also expressed in the right atrium in mice (48) and other cell types in human including pericytes and smooth muscle cells (48).
  • Dre/Rox system to control TmemlOO promoter by human CDH5 promoter (FIG. 7A).
  • the Cre/LoxP system can al so be employed to construct this dual promoter system.
  • mice Male and female mice were studied. Anti-BrdU and anti-CD31 immunostaining were employed to quantify EC proliferation in mouse lungs, indicative of endothelial regeneration. Lung vascular permeability was determined by EBA assay whereas lung edema was assessed by lung wet/dry weight ratio. Lung inflammation was determined by MPO activity and expression of proinflammatory genes. Lung injury was also assessed by H & E staining.
  • mice received a single dose of LPS (Escherichia coli O55:B5, Santa Cruz Biotechnology) by i.p. injection.
  • LPS Erysia coli O55:B5, Santa Cruz Biotechnology
  • the LPS dose was dependent on the age of the mice (3-9 mos. old, 2.5-3.5 mg/kg; 19-20 mos. old, 1.0 mg/kg; 21-22 mos. 0.5 mg/kg; 24 mos., 0.3 mg/kg). All mice were anesthetized with ketamine/xylazine (100/5 mg/kg BW, i.p.) prior to tissue collection.
  • mice were treated with a single dose of LPS and monitored for 4-7 days.
  • the sections were then incubated with Alexa Fluor 594-conjugated secondary antibodies (1 :200, Thermal Fisher Scientific).
  • the nuclei were counterstained with DAPI (Thermal Fisher Scientific).
  • BrdU+ nuclei were quantified in 12-15 random field for each section and the average number of BrdU+ nuclei in every 1000 nuclei was used.
  • GTCGTTTCTGCTGTGATTCC-3' (SEQ ID NO: 12); mouse Cdh5 primers, 5’- AGACAAGGATGTGGTGCCAG-3’ (SEQ ID NO: 13) and 5’-
  • mice cyclophilin primers 5'- CTTGTCCATGGCAAATGCTG-3 (SEQ ID NO: 15) and 5 -TGATCTTCTTGCTGGTCTTGC- 3' (SEQ ID NO: 16).
  • Primers for mouse Cdc25c, Ccna2 Ccnbl, Cerf Tnfax ⁇ AIl6 were purchased from Qiagen. The mouse gene expression was normalized to cyclophilin.
  • Nanoparticle delivery of plasmid DNA for EC-specific overexpression of Foxml in lungs of aged mice 20pg plasmid DNA expressing human F0XM1 under the control of either CDH5 promoter (EC-specific) or the dual CDH5 and TmemlOO promoter system (lung EC-specific) and 60 pl endothelium-targeted nanoparticles (EndoNPl, Mountview Therapeutics LLC) were mixed and incubated at room temperature for 10 min administered to aged (20-21 mos. old) C57BL/6 mice retro-orbitally at 24h post-LPS challenge.
  • CDH5 promoter EC-specific
  • TmemlOO promoter system lung EC-specific
  • 60 pl endothelium-targeted nanoparticles 60 pl endothelium-targeted nanoparticles
  • Example 2 Lung EC-specific gene expression driven by lung EC-specific dual promoter systems
  • plasmid DNA expressing Cre under the control of the CDH5p-DvQ-Tmem K)()p- o - STOP-Rox-Cre dual promoter system was delivered i.v. to adult TdTomato reporter mice.
  • TdTomato mice carry the fluorescent reporter expressing membrane-targeted tandem dimer Tomato (TdTomato) after Cre-mediated excision of the Lox-STOP -Lox cassette.
  • CDH5p-Cre plasmid DNA were administered as positive control while plasmid DNA without Cre was used as a negative control (FIG. 16).
  • FIG. 16 shows that the nanoparticles could deliver the CDH5-Cre plasmid to all the organs leading to activation of TdTomato expression in ECs, demonstrating that the failed activation of TdTomato in vascular ECs of aorta, brain, heart, skeletal muscle of the C I)H5p- vQ-Tmem 1 OOp- Rox-STOP-Rox-Cre dual promoter plasmid DNA-administered reporter mice (FIG. 17) was not ascribed to failure of plasmid DNA delivery.
  • FIG. 17 shows that the failed activation of TdTomato in vascular ECs of aorta, brain, heart, skeletal muscle of the C I)H5p- vQ-Tmem 1 OOp- Rox-STOP-Rox-Cre dual promoter plasmid DNA-administered reporter mice
  • FIG. 17 demonstrated that the lung EC-specific dual promoter system activated Cre expression only in lung ECs, leading to TdTomato expression, not in other organ ECs.
  • FIG. 18 showed the CDHSp-D e-Hpgd /vw;/ Ze/-Rox-STOP-Rox- Cre dual promoter system was also lung EC-specific which activated Cre expression and resultant TdTomato expression selectively in lung vascular ECs.
  • Aird WC The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome. Blood. 101, 3765-77 (2003).
  • Hepatocyte nuclear factor 3/fork head homolog 11 is expressed in proliferating epithelial and mesenchymal cells of embryonic and adult tissues. Mol Cell Biol. 17, 1626-41 (1997).

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Abstract

La présente invention concerne des constructions comprenant des promoteurs spécifiques d'une cellule et/ou spécifiques d'un organe qui régulent l'expression sélective d'un gène dans un type de tissu et de cellule particulier. L'invention concerne également des procédés d'administration de gènes à des types de cellules et de tissus spécifiques à l'aide des constructions décrites ici.
PCT/US2024/021370 2023-03-23 2024-03-25 Procédé de thérapie génique de précision de maladies provoquées par un dysfonctionnement endothélial Pending WO2024197314A2 (fr)

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