[go: up one dir, main page]

WO2025027060A1 - Facteur de transcription runx3 codé par un acide nucléique - Google Patents

Facteur de transcription runx3 codé par un acide nucléique Download PDF

Info

Publication number
WO2025027060A1
WO2025027060A1 PCT/EP2024/071649 EP2024071649W WO2025027060A1 WO 2025027060 A1 WO2025027060 A1 WO 2025027060A1 EP 2024071649 W EP2024071649 W EP 2024071649W WO 2025027060 A1 WO2025027060 A1 WO 2025027060A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
runx3
artificial nucleic
pharmaceutical composition
rna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2024/071649
Other languages
English (en)
Inventor
Joanna Rejman
Joseph ARBOLEDA-VELASQUEZ
Leo A. KIM
Michael N. O´HARE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Curevac SE
Schepens Eye Research Institute Inc
Original Assignee
Curevac SE
Schepens Eye Research Institute Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Curevac SE, Schepens Eye Research Institute Inc filed Critical Curevac SE
Publication of WO2025027060A1 publication Critical patent/WO2025027060A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • Nucleic add such as RNA has the potential to provide highly specific and individual treatment options for the therapy of a large variety diseases, disorders, or conditions, e.g. ophthalmic diseases, disorders, or conditions.
  • nucleic acid-based treatments such as RNA for clinical applications has mainly focused on immunotherapeutics for multiple clinical applications. Pathologies caused by increased or decreased function or activity of a gene, such as transcription factors, are more difficult to address with nucleic acid-based therapeutics.
  • Transcription factors include a wide number of proteins that initiate and regulate the transcription of genes, protein synthesis, and subsequent altered cellular function. Transcription factor malfunctions play a crucial role in the development and progression of various diseases and conditions such as diseases and conditions. For example, increased RUNX1 function, a member of the Runt-related transcription factor family, is a hallmark of pathological epithelial to mesenchymal transition (EMT), aberrant angiogenesis, degeneration, and fibrosis; processes underlying proliferative vitreoretinopathy (PVR) and other multiple prevalent conditions in the eye and elsewhere.
  • EMT epithelial to mesenchymal transition
  • PVR proliferative vitreoretinopathy
  • transcription factors may represent powerful therapeutic targets for treating or preventing numerous diseases
  • nucleic add sequences for example RNA
  • RNA may represent a promising class of molecules to provide the information for expressing intracellular proteins such as transcription factors.
  • the underlying object of the invention is to provide nucleic acid-based therapeutics for produdng transcription factors suitable for treating or preventing diseases in a cell or a subject, in particular diseases and conditions associated with pathological epithelial to mesenchymal transition (EMT), pathological cell proliferation, aberrant angiogenesis, aberrant neovascularization, degeneration, fibrosis, and cancer.
  • EMT epithelial to mesenchymal transition
  • the present invention is inter alia directed to a nucleic add, preferably RNA, comprising at least one coding sequence encoding a RUNX3 transcription factor or a fragment or variant thereof, wherein the nucleic acid preferably comprises at least one heterologous untranslated region (UTR).
  • a nucleic add preferably RNA
  • the nucleic acid preferably comprises at least one heterologous untranslated region (UTR).
  • pharmaceutical compositions comprising the nucleic add, preferably formulated in polyethylene glycol/peptide polymers, polymeric carriers, or lipid-based carriers.
  • PVR proliferative vitreoretinopathy
  • EPR epiretinal membranes
  • ERM epiretinal membrane
  • a fibrocellular tissue found on the inner surface of the retina. It is semi-translucent and proliferates on the surface of the internal limiting membrane.
  • ERMs also commonly known as cellophane maculopathy or macular puckers, are avascular (having few or no blood vessels), semitranslucent, fibrocellular membranes that form on the inner surface of the retina.
  • Most patients with ERMs have no symptoms. In such cases, patients typically have normal or near-normal vision. However, ERMs can slowly progress, leading to a vague visual distortion that can be perceived better by closing the non- or less-affected eye.
  • a surgical procedure called vitrectomy is the only option so far in eyes that require treatment.
  • PVR is a blinding, relatively common complication of retinal detachment often associated with eye trauma driven by RUNX1 -mediated epithelial-mesenchymal transition (EMT) that currently lacks medical treatment.
  • EMT epithelial-mesenchymal transition
  • PVR is characterized by the development of membranous intraocular scar tissue (membranes that consist of proliferating cells and extracellular matrix) and is the most common cause of failure after retinal detachment surgery.
  • Increased RUNX1 function a member of the Runt-related transcription factor family, is a hallmark of pathological EMT, aberrant angiogenesis, degeneration, and fibrosis; processes underlying PVR and other multiple prevalent conditions in the eye and elsewhere.
  • the invention is inter alia based on the surprising finding that nucleic acid, e.g. RNA, that encode a RUNX3 transcription factor can be used as specific inhibitors or antagonists of the activity of cellular target transcription factors, e.g. RUNX1 , in particular transcription factors that have a pathologic transcription factor activity, e.g. transcription factors that are overexpressed or overactive in a disease, disorder, or condition.
  • RUNX1 e.g. RUNX1
  • transcription factors that have a pathologic transcription factor activity e.g. transcription factors that are overexpressed or overactive in a disease, disorder, or condition.
  • RUNX3 was effective in inter alia reducing EMT and PVR in the eye. That is even more surprising as RUNX3 is essentially not expressed in human ocular cells or tissues.
  • RUNX3 may inhibit RUNX1 e.g. by preventing its nuclear translocation and/or by reducing the interaction with the cellular transcription co-factor CBFbeta and/or by binding to the RUNX1 promotor itself.
  • RUNX3 strongly reduced proliferation in human microvascular retinal endothelial cells (HMREC) and primary human cell cultures derived from surgically excised membranes from eyes of patients with PVR ( Figure 1 and 2).
  • HMREC human microvascular retinal endothelial cells
  • Figure 1 and 2 primary human cell cultures derived from surgically excised membranes from eyes of patients with PVR
  • RUNX3 reduced cell clusters identified as fibroblasts, as well as lead to a reduction of proliferation marker expression (Figure 3).
  • RNA-encoded RUNX3 strongly reduced proliferation and ocular pathology triggered by injection of human PVR cells in a rabbit eye ( Figure 4).
  • formulated RUNX3 mRNA reduced lesion size in a laser-CNV mouse model, 7 days after treatment ( Figure 5).
  • LNP formulated mRNA encoding RUNX3 was effectively expressed in various and reduced proliferation in HUVEC cells ( Figures 6-7).
  • nuclei acid in particular RNA
  • RNA can be leveraged to provide a RUNX3 transcription factor for reducing or inhibiting various pathological conditions including EMT, pathological cell proliferation, aberrant angiogenesis, aberrant neovascularization, degeneration, fibrosis, and cancer.
  • nuclei acid such as RNA providing RUNX3 may be used in treating or preventing ocular diseases, e.g. PVR and/or epiretinal membranes (ERM).
  • ocular diseases e.g. PVR and/or epiretinal membranes (ERM).
  • the present invention provides a nucleic acid comprising at least one coding sequence (cds) encoding at least one RUNX3 transcription factor, or a fragment or variant thereof.
  • the nucleic add is an RNA, more preferably an mRNA.
  • the RUNX3 transcription factor is human.
  • the present invention provides a pharmaceutical composition comprising at least one nucleic acid comprising at least one cds encoding at least one RUNX3 transcription factor, or a fragment or variant thereof.
  • the nucleic add is formulated in polyethylene glycol/peptide polymers, polymeric carriers, or lipid-based carriers (e.g. LNPs).
  • the formulation is selected from LNPs.
  • the present invention provides a kit or kit of parts comprising at least one nucleic acid of the first aspect or at least one pharmaceutical composition of the second aspect.
  • the disease, disorder or condition is an ocular disease, disorder, or condition, preferably PVR.
  • a further aspect relates to a method of reducing the activity of RUNX1 in a cell or a subject.
  • Figure 1 Formulated RUNX3 mRNA induces the expression of RUNX3 and inhibits the proliferation and migration of C-PVR.
  • 1A Shows the RUNX3 protein expression in C-PVR cells 24 hours after transfection of formulated RUNX3 mRNA (RUNX3_A and RUNX3_B) which was assessed via western blot.
  • Formulated RUNX3 mRNA (RUNX3_A and RUNX3_B) is non-toxic (1B) and reduces proliferation and migration of C-PVR cells (1C and 1D) in C-PVR. Further information is provided in the Example section, Example 3.
  • Figure 2 Formulated RUNX3 mRNA induces the expression of RUNX3 and inhibit the proliferation and migration of HMREC.
  • 2A shows the RUNX3 protein expression in HMREC cells 24 hours after transfection of formulated RUNX3 mRNA (RUNX3_B) which was assessed via western blot.
  • Formulated RUNX3 mRNA (RUNX3_B) reduces proliferation and migration of HMREC cells after treatment with formulated RUNX3 mRNA ( Figure 2B and 2C and 2 D). Further information is provided in the Example section, Example 3.
  • FIG. 3 Single-cell RNA sequencing analysis of C-PVR cells treated with formulated RUNX3 mRNA (RUNX3_A), Luciferase mRNA and control (non-treated cells).
  • 3A and 3B show locations within the UMAPs plot of nontreated cells (control) and formulated RUNX3 mRNA (RUNX3_A) treated cells. Treatment with RUNX3_A completely abolished the cluster that identifies as fibroblast (black arrow).
  • 3C shows unsupervised clustering and a reduction of RUNX1 expression in formulated RUNX3 mRNA treated cells.
  • 3D and 3E show the distribution within the UMAPs plot of RUNX3 comparing control and formulated RUNX3 mRNA therapy.
  • Proliferation markers such as Cyclin D1 and D2 (CCND1 , CCND2), and PCNA and CDK4 were also reduced, indicating that the RUNX3 treatment inhibits proliferation of C-PVR.
  • fibrotic markers from the collagen family were downregulated with the RUNX3 therapy (3I). Further information is provided in Example 4.
  • Figure 4 Formulated RUNX3 mRNA (RUNX3_A) reduces pathology severity in a PVR rabbit model.
  • 4A OCT (optical coherence tomography) images shows the formation of aberrant membranes (pointed with black and white arrows) over the optic nerve in the luciferase group
  • 4B PVR score calculated from OCT images shows a reduction of formulated RUNX3 mRNA in comparison to formulated Luciferase mRNA
  • 4C H&E staining reveals the formation of the membrane formation over the retina and the effect of formulated RUNX3 on the reduction of the pathology progression.
  • Control (Luciferase) is shown in left, RUNX3 treatment on the right. Further information is provided in the Example section, Example 5.
  • FIG. 5 CVCM-formulated RUNX3 mRNA reduces lesion size in a laser-induced choroidal neovascularization (CNV) mice model after 7 days of injection.
  • 5A shows the photographic imaging and 5B shows the quantification of the lesions. Further information is provided in the Example section, Example 6.
  • Angiogenesis means the physiological process through which new blood vessels form from pre-existing vessels. Angiogenesis is particularly relevant to aberrant vessel growth in infants, children, adults, such as during tumor growth, and tumor-like growth, and e.g. in wet age-related macular degeneration, and proliferative diabetic retinopathy. Blood vessel growth may occur via the process of angiogenesis and/or vasculogenesis.
  • the processes are distinct, and the involvement of a protein or pathway in vasculogenesis (e.g., during embryonic development) does not necessarily indicate that the protein or pathway is relevant to angiogenesis, much less aberrant angiogenesis.
  • the involvement of a protein or pathway in embryonic angiogenesis does not indicate that targeting the protein or pathway would be capable of reducing the aberrant angiogenesis, much less sufficient for inhibiting aberrant angiogenesis or safe for targeting in an infant, child, or adult.
  • Vasculogenesis means the process of blood vessel formation occurring by a de novo production of endothelial cells. Vasculogenesis is particularly relevant to embryonic blood vessel formation. Vasculogenesis and angiogenesis are distinct from each other in that angiogenesis relates to the development of new blood vessels from (e.g., sprouting or extending from) pre-existing blood vessels, whereas vasculogenesis relates to the formation of new blood vessels that have not extended/sprouted from pre-existing blood vessels (e.g., where there are no pre-existing vessels). E.g., if a monolayer of endothelial cells begins sprouting to form capillaries, angiogenesis is occurring.
  • Vasculogenesis in contrast, is when endothelial precursor cells (angioblasts) migrate and differentiate in response to local cues (such as growth factors and extracellular matrices) to form new blood vessels. These new blood vessels formed by vasculogenesis are then pruned and extended through angiogenesis.
  • endothelial precursor cells angioblasts
  • local cues such as growth factors and extracellular matrices
  • Cationic, cationizable The term “cationic” means that the respective structure bears a positive charge, either permanently or not permanently, e.g. in response to certain conditions such as pH. Thus, the term “cationic” covers both “permanently cationic” and “cationisable”.
  • the term “permanently cationic” means, e.g., that the respective compound, group, or atom, is positively charged at any pH value or hydrogen ion activity of its environment. Typically, the positive charge results from the presence of a quaternary nitrogen atom.
  • the terms “cationic”, “cationisable”, and “permanently cationic” as used herein must be understood as defined in WO2023/031394 [p.12, line 32 to p.13, line 16].
  • Coding sequence cds: The term “coding sequence” and the corresponding abbreviation “cds” as used herein refers to a sequence of several nucleotide triplets, which may be translated into a peptide or protein.
  • a cds in the context of the present invention may be a DNA or RNA sequence consisting of a number of nucleotides that may be divided by three, which starts with a start codon, and which preferably terminates with a stop codon.
  • the cds encodes at least one RUNX3 transcription factor, or a fragment or variant thereof.
  • Core binding factors are heterodimeric transcription factors consisting of a DNA-binding CBFalpha subunit and non-DNA-binding CBFbeta subunit. DNA binding and heterodimerization is mediated by a single domain in the CBFalpha subunit called the Runt domain, while sequences flanking the Runt domain confer other biochemical activities such as transactivation.
  • the heterodimerization domain in CBFbeta is the only functional domain that has been identified in this subunit.
  • nucleic acid i.e. for a nucleic acid “derived from” (another) nucleic acid
  • nucleic acid which is derived from (another) nucleic acid, shares at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or are identical with the nucleic acid from which it is derived.
  • the term “derived from” means that the amino acid sequence, which is derived from (another) amino acid sequence, shares at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or are identical with the amino acid sequence from which it is derived.
  • Epithelial-mesenchymal transition The term epithelial-mesenchymal transition and the corresponding abbreviation “EMT” as used herein is e.g. characterized by a loss of cell adhesion, which leads to constriction and extrusion of new mesenchymal cells. EMT is a process by which epithelial cells lose their cell polarity, which leads to cellcell adhesion loss, and gain of migratory and invasive properties to become mesenchymal stem cells (which are multipotent stromal cells that can differentiate into a variety of cell types). EMT is essential for numerous developmental processes including mesoderm formation and neural tube formation.
  • EMT has also been shown to occur in wound healing, in organ fibrosis and in the initiation of metastasis in cancer progression.
  • EMT, and its reverse process, MET (mesenchymal-epithelial transition) are critical for development of many tissues and organs in the developing embryo, and numerous embryonic events such as gastrulation, neural crest formation, heart valve formation, palatogenesis and myogenesis.
  • Epithelial cells are closely connected to each other by tight junctions, gap junctions and adherens junctions, have an apico-basal polarity, polarization of the actin cytoskeleton and are bound by a basal lamina at their basal surface.
  • EMT Mesenchymal cells, on the other hand, lack this polarization, have a spindle-shaped morphology and interact with each other only through focal points.
  • Epithelial cells express high levels of E-cadherin, whereas mesenchymal cells express those of N-cadherin, fibronectin and vimentin.
  • EMT entails profound morphological and phenotypic changes to a cell. Based on the biological context, EMT has been categorized into 3 types: developmental (Type I), fibrosis and wound healing (Type II), and cancer (Type III). Loss of E-cadherin is a fundamental event in EMT.
  • EMT-TF EMT inducing TFs
  • SNAI l/Snail 1 , SNAI2/Snail 2 also known as Slug or Zinc finger protein
  • Zinc finger E-box binding homeobox 1 and 2 ZEB1 and ZEB2
  • transcription factor 3 TCF3
  • KLF8 krueppel-like factor 8
  • Twist also referred to as class A basic helix-loop-helix protein 38; bHLHa38
  • TCF4 homeobox protein Sineoculis homeobox homolog 1
  • FOXC2 fork-head box protein C2
  • TGFbeta transforming growth factor beta
  • FGF fibroblast growth factor
  • EGF epidermal growth factor
  • HGF hepatocyte growth factor
  • Wnt/beta-catenin and Notch hypoxia may induce EMT.
  • Ras-MAPK mitogen- activated protein kinases
  • Slug triggers the steps of desmosomal disruption, cell spreading, and partial separation at cell-cell borders, which comprise the first and necessary phase of the EMT process.
  • Wnt signaling pathway regulates EMT in gastrulation, cardiac valve formation and cancer.
  • Wnt pathway Activation of Wnt pathway in breast cancer cells induces the EMT regulator SNAIL and upregulates the mesenchymal marker, vimentin. Also, active Wnt/beta- catenin pathway correlates with poor prognosis in breast cancer patients in the clinic. Similarly, TGFbeta activates the expression of SNAIL and ZEB to regulate EMT in heart development, palatogenesis, and cancer. The breast cancer bone metastasis has activated TGFbeta signaling, which contributes to the formation of these lesions.
  • tumor protein 53 a well-known tumor suppressor
  • p53 tumor protein 53
  • p53 a well-known tumor suppressor
  • p53 represses EMT by activating the expression of various microRNAs - miR-200 and miR-34 that inhibit the production of protein ZEB and SNAIL, and thus maintain the epithelial phenotype.
  • the implantation of the embryo and the initiation of placenta formation are associated with EMT.
  • the trophoectoderm cells undergo EMT to facilitate the invasion of endometrium and appropriate placenta placement, thus enabling nutrient and gas exchange to the embryo.
  • EMT allows the cells to ingress in a specific area of the embryo - the primitive streak in amniotes, and the ventral furrow in Drosophila.
  • the cells in this tissue express E-cadherin and apical-basal polarity.
  • keratinocytes at the border of the wound undergo EMT and undergo re-epithelialization or MET when the wound is closed.
  • Snail2 expression at the migratory front influences this state, as its overexpression accelerates wound healing.
  • the ovarian surface epithelium undergoes EMT during post-ovulatory wound healing. Initiation of metastasis requires invasion, which is enabled by EMT.
  • Carcinoma cells in a primary tumor lose cellcell adhesion mediated by E-cadherin repression and breakthrough the basement membrane with increased invasive properties and enter the bloodstream through intravasation. Later, when these circulating tumor cells (CTCs) exit the bloodstream to form micro-metastases, they undergo MET for clonal outgrowth at these metastatic sites. Thus, EMT and MET form the initiation and completion of the invasion-metastasis cascade. At this new metastatic site, the tumor may undergo other processes to optimize growth. For example, EMT has been associated with programmed death ligand 1 (PD-L1) expression, particularly in lung cancer. Increased levels of PD-L1 suppresses the immune system which allows the cancer to spread more easily.
  • PD-L1 programmed death ligand 1
  • EMT has been shown to be induced by androgen deprivation therapy in metastatic prostate cancer.
  • Activation of EMT programs via inhibition of the androgen axis provides a mechanism by which tumor cells can adapt to promote disease recurrence and progression.
  • Brachyury, Axl (tyrosine protein kinase receptor UFO), MEK, and Aurora kinase A are molecular drivers of these programs, and inhibitors are currently in clinical trials to determine therapeutic applications.
  • Oncogenic protein kinase C iota type (PKC-iota) can promote melanoma cell invasion by activating Vimentin during EMT.
  • PKC-iota inhibition or knockdown resulted an increase E-cadherin and ras homolog gene family, member A (RhoA) levels while decreasing total Vimentin, phophorylated Vimentin (S39) and partitioning defective 6 homolog alpha (Par6) in metastatic melanoma cells.
  • CSCs cancer stem cells
  • Fibrosis inter alia relates to pathological wound healing in which e.g. connective tissue replaces normal parenchymal tissue to the extent that it goes unchecked, leading to considerable tissue remodelling and the formation of permanent scar tissue.
  • connective tissue replaces normal parenchymal tissue to the extent that it goes unchecked, leading to considerable tissue remodelling and the formation of permanent scar tissue.
  • Chronic inflammation and repair are typically susceptible to fibrosis where an accidental excessive accumulation of extracellular matrix components, such as the collagen is produced by fibroblasts, leading to the formation of a permanent fibrotic scar. In response to injury, this is called scarring, and if fibrosis arises from a single cell line, this is called a fibroma.
  • fibrosis acts to deposit connective tissue, which can interfere with or totally inhibit the normal architecture and function of the underlying organ or tissue.
  • Fibrosis can be used to describe the pathological state of excess deposition of fibrous tissue, as well as the process of connective tissue deposition in healing. Defined by the pathological accumulation of extracellular matrix (ECM) proteins, fibrosis results in scarring and thickening of the affected tissue. It is in essence an exaggerated wound healing response which interferes with normal organ function. From the physiological perspective, fibrosis is similar to the process of scarring, in that both involve stimulated fibroblasts laying down connective tissue, including collagen and glycosaminoglycans. The process is initiated when immune cells such as macrophages release soluble factors that stimulate fibroblasts.
  • ECM extracellular matrix
  • pro-fibrotic mediator is TGFbeta, which is released by macrophages as well as any damaged tissue between surfaces called interstitium.
  • Other soluble mediators of fibrosis include CTGF, platelet-derived growth factor (PDGF), and interleukin 10 (IL-10). These initiate signal transduction pathways such as the AKT/mTOR and SMAD pathways that ultimately lead to the proliferation and activation of fibroblasts, which deposit extracellular matrix into the surrounding connective tissue. This process of tissue repair is a complex one, with tight regulation of extracellular matrix (ECM) synthesis and degradation ensuring maintenance of normal tissue architecture.
  • ECM extracellular matrix
  • Fibrosis can occur in many tissues within the body, typically as a result of inflammation or damage, and examples include pathologies in the lung (e.g. cystic fibrosis, idiopathic pulmonary fibrosis), pathologies in the liver (e.g. cirrhosis), or pathologies in the heart (e.g. myocardial fibrosis). Additionally, fibrosis is a key component of PVR pathology. Intraretinal fibrosis leads to stiffness of the retina and can prevent the retina from flattening after surgical membrane removal
  • fragment as used herein in the context of a nucleic acid sequence (e.g. RNA or DNA) or an amino acid sequence may typically be a shorter portion of a reference sequence of e.g. a nucleic acid sequence or an amino acid sequence. Accordingly, a fragment typically consists of a sequence that is identical to the corresponding stretch within the reference sequence.
  • a preferred fragment of a sequence in the context of the present invention consists of a continuous stretch of entities, such as nucleotides or amino acids corresponding to a continuous stretch of entities (i.e., nucleotides or amino adds) in the molecule the fragment is derived from, which represents at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the total length of the reference molecule from which the fragment is derived.
  • entities such as nucleotides or amino acids corresponding to a continuous stretch of entities (i.e., nucleotides or amino adds) in the molecule the fragment is derived from, which represents at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the total length of the reference molecule from which the fragment is derived.
  • Identity refers to the percentage to which two sequences are identical. To determine the percentage to which two sequences are identical, the sequences can be aligned (by also introducing gaps, if necessary) to be subsequently compared to one another. In the context of the invention, the substitution of a nucleotide by a modified nucleotide (e.g. U substituted by with N1 -methylpseudouridine (ml i )) shall not be considered for calculating percent identity.
  • the percentage to which two sequences are identical can e.g. be determined using an algorithm, e.g. an algorithm integrated in the BLAST program.
  • Neovascularization has to be understood as the (natural) process of formation of new blood vessels.
  • neovascularization is in the form of functional microvascular networks, capable of perfusion by red blood cells, which form to serve as collateral circulation in response to local poor perfusion or ischemia.
  • Growth factors that inhibit neovascularization include those that affect endothelial cell division and differentiation. These growth factors often act in a paracrine or autocrine fashion; they include fibroblast growth factor, placental growth factor, insulinlike growth factor, hepatocyte growth factor, and platelet-derived endothelial growth factor.
  • vasculogenesis angiogenesis
  • arteriogenesis arteriogenesis
  • pathologies and diseases can be associated with aberrant neovascularization, including ocular pathologies such as corneal neovascularization, retinopathy of prematurity, diabetic retinopathy, age-related macular degeneration, and choroidal neovascularization.
  • Aberrant neovascularization can also be associated with cardiovascular diseases e.g. Ischemic heart disease.
  • Nucleic add, nucleic acid molecule The terms “nucleic acid” or “nucleic acid molecule” as used herein preferably refers to DNA (molecules) or RNA (molecules). It is preferably used synonymously with the term polynucleotide.
  • a nucleic acid or a nucleic add molecule is a polymer comprising or consisting of nucleotide monomers, which are covalently linked to each other by phosphodiester-bonds of a sugar/phosphate-backbone.
  • Nucleic acid sequence, DNA sequence, RNA sequence The terms “nucleic acid sequence”, “DNA sequence”, “RNA sequence” refer to a particular and individual order of the succession of its nucleotides.
  • RNA is the usual abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e. a polymer consisting of nucleotide monomers. These nucleotides are usually adenosine-monophosphate (AMP), uridine-monophosphate (UMP), guanosine-monophosphate (GMP) and cytidine-monophosphate (CMP) monomers or analogs thereof, which are connected to each other along a so-called backbone. The backbone is typically formed by phosphodiester bonds between the sugar, i.e. ribose, of a first and a phosphate moiety of a second, adjacent monomer.
  • AMP adenosine-monophosphate
  • UMP uridine-monophosphate
  • GMP guanosine-monophosphate
  • CMP cytidine-monophosphate
  • RNA sequence The specific order of the monomers, i.e. the order of the bases linked to the sugar/phosphate-backbone, is called the RNA sequence.
  • RNA can be obtained by transcription of a DNA sequence, e.g., inside a cell or in vitro. In the context of the invention, the RNA may be obtained by RNA in vitro transcription or chemical synthesis.
  • RNA in vitro transcription relates to a process wherein RNA is synthesized in a cell-free system in vitro.
  • the RNA is obtained by transcribing a DNA template in the presence of a DNA-dependent RNA polymerase (e.g. T7, SP6), ribonucleotide triphosphates (NTPs, and optionally modified NTPs) and optionally, a cap analog, in an appropriate buffer (e.g. comprising MgCI2).
  • a DNA-dependent RNA polymerase e.g. T7, SP6
  • NTPs ribonucleotide triphosphates
  • cap analog optionally, a cap analog
  • Variant of a sequence:
  • the term “variant'’ as used herein in the context of a nucleic acid sequence refers to a variant of a nucleic acid sequence derived from another nucleic add sequence.
  • a variant of a nucleic add sequence may exhibit one or more nucleotide deletions, insertions, additions and/or substitutions compared to the nucleic acid sequence from which the variant is derived.
  • a variant of a nucleic acid sequence may at least 50%, 60%, 70%, 80%, 90%, or 95% identical to the nucleic acid sequence the variant is derived from.
  • a variant may be a functional variant in the sense that the variant has retained at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the function of the sequence where it is derived from.
  • a “variant” of a nucleic acid sequence may have at least 70%, 75%, 80%, 85%, 90%, 95% , or 99% nucleotide identity over a stretch of at least 30, 50, 75 or 100 nucleotides.
  • variants refers to a protein or peptide variant having an amino add sequence which differs from the original sequence in one or more mutation(s)/substitution(s), such as one or more substituted, inserted and/or deleted amino add(s).
  • these fragments and/or variants have the same, or a comparable specific property. Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three-dimensional structure or do not affect the binding region.
  • a variant of a protein may be a functional variant of the protein, which means that the variant exerts essentially the same, or at least 40%, 50%, 60%, 70%, 80%, 90% of the function of the protein it is derived from.
  • a “variant” of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch ofat least 30, 50, 75 or 100 amino acids of such protein or peptide.
  • the invention provides a nucleic acid comprising least one coding sequence (cds) encoding a RUNX3 (also known as AML2, CBFA3, PEBP2A3) transcription factor, or a fragment or variant thereof.
  • cds coding sequence
  • RUNX3 also known as AML2, CBFA3, PEBP2A3
  • specific features and embodiments that are described in the context of the first aspect, that is the nucleic add of the invention are likewise applicable to the second aspect (pharmaceutical composition), the third aspect (kit or kit of parts), or further aspects including medical uses and method of treatments.
  • Runt-related transcription factor (RUNX) proteins belong to a transcription factors family known to have key roles in diverse cellular processes such as cell proliferation, differentiation, senescence, apoptosis, epithelial-mesenchymal transition, inflammation, epigenetic memory and DNA repair.
  • RUNX family members share the evolutionarily conserved Runt domain, which binds to core-binding factor-P (CBFbeta) and mediates DNA binding.
  • CBFbeta core-binding factor-P
  • RUNX1 is essential for generation of hematopoietic stem cells and is involved in human leukemia.
  • RUNX2 is essential for skeletal development and has an oncogenic potential.
  • RUNX3 is a major tumor suppressor of gastric and many other solid tumors. In humans, RUNX3 is located on 1 p.13-p36.11 , a region of chromosome 1 that contains a tumor suppressor gene, where heterozygous deletion or mutation of one copy of the allele predisposes to cancer. It mediates binding of RUNX proteins to DNA as well as protein-protein interaction with the partner subunit CBFbeta. RUNX3 forms the heterodimeric complex core-binding factor (CBF) with CBFbeta.
  • CBF complex core-binding factor
  • RUNX members modulate the transcription of their target genes through recognizing a core consensus binding sequence within their regulatory regions via their Runt domain, while CBFbeta is a non-DNA-binding regulatory subunit that allosterically enhances the sequence-specific DNA-binding capacity of RUNX.
  • RUNX3 is mostly expressed in bone marrow and lymphoid tissues, skin, female tissues, liver, and gastrointestinal tract tissue. RUNX3 is essentially not expressed in the eye.
  • the nucleic acid comprising the at least one cds encoding the at least one RUNX3 transcription factor, or a fragment or variant thereof is an artificial nucleic acid.
  • an artificial nucleic acid refers to a nucleic acid that does not occur naturally.
  • an artificial nucleic acid may be understood as a non-natural nucleic add molecule.
  • Such nucleic acid molecules may be non-natural due to their sequence (e.g. G/C content modified cds, UTRs) and/or due to other modifications, e.g. structural modifications of nucleotides.
  • an artificial nucleic acid may be designed and/or generated by genetic engineering to correspond to a desired artificial sequence of nucleotides.
  • an artificial nucleic acid is a sequence that may not occur naturally, i.e.
  • artificial nucleic acid is not restricted to mean “one single molecule” but is understood to comprise an ensemble of essentially identical nucleic acid molecules.
  • artificial nucleic acid as used herein may relate to artificial DNA or, preferably, to artificial RNA.
  • the at least one RUNX3 transcription factor that is encoded by the nucleic acid is selected from a full length RUNX3 protein, or a N-terminally and/or a C-terminally truncated RUNX3 protein fragment.
  • the full length RUNX3 protein (e.g. isoform 1 having a length of 429 amino adds; isoform 2 having a length of 415 amino acids) comprises several domains, each of which being capable of interacting with various proteins to regulate RUNX activity in a spatio-temporal manner.
  • the N-terminal part comprises a conserved DNA binding domain, the Runt domain (RD), as well as the nuclear localization signal (NLS), wherein the C-terminal part comprises the transcription activation domain (AD) and the transcription inhibition domain (ID).
  • the full length RUNX3 protein is RUNX3 protein variant or protein isoform 1 .
  • the nucleic acid encoding the at least one RUNX3 transcription factor, or a fragment or variant thereof comprises a Runt domain (RD).
  • the Runt domain is a conserved DNA binding domain (typically comprising 129aa) which is located in the N-terminal part of RUNX proteins with more than 90% identity among the three RUNX genes. This domain is considered as the main part of RUNX proteins since, only this part binds to a specific motif in DNA. Furthermore, the Runt domain also contributes to nuclear localization and is able to translocate to the nucleus and bind to DNA with stronger affinity compared to the full protein.
  • N-terminally an/or a C-terminally truncated RUNX3 protein fragments as defined herein comprise at least the Runt domain (RD).
  • RD Runt domain
  • the Runt domain recognizes and binds the DNA motif or DNA core consensus binding sequence 5’- TGTGGT-3’ or 5-TGCGGT-3’.
  • Runt domain mediates binding of RUNX proteins to DNA as well as interaction with the core-binding factor subunit beta (CBFbeta).
  • the transcription co-factor CBFbeta is a subunit of a heterodimeric core-binding (transcription) factor (CBF) belonging to the PEBP2/CBF transcription factor family.
  • CBF regulates transcription via formation of a heterodimeric complex between RUNX, the CBFalpha-DNA-binding subunit, and CBFbeta.
  • CBFbeta is a non-DNA binding regulatory subunit; it allosterically enhances DNA binding by the alpha subunit (of e.g. RUNX) as the complex binds to the core site of various enhancers and promoters.
  • CBFbeta is imported to the nucleus by associating with RUNX factors, as CBF lacks a nuclear localization signal.
  • RUNX can bind DNA as a monomer in vitro
  • heterodimerization with the non-DNA binding transcription co-factor CBFbeta triggers flexible DNA-recognition loops, thus stabilizing the complex and increasing RUNX binding to DNA.
  • Binding of transcription co-factor CBFbeta enhances DNA binding affinity of RUNX by approximately 10-fold.
  • the nucleic acid encoding the at least one RUNX3 transcription factor, or a fragment or variant thereof comprises a transactivation domain (AD) and/or an inhibition domain (ID).
  • the transactivation domain (AD) and inhibition domain (ID) are important regions at the C-terminal of the RUNX3 transcription factor.
  • the transactivation domain binds to different cofactors and makes various combinations of transcription factors for activation of specific promoters.
  • the Inhibition domain inhibits RUNX3 activity by masking the activation domain or binding to some inhibitory proteins.
  • the nucleic acid encoding the at least one RUNX3 transcription factor, or a fragment or variant thereof activates or represses transcription regulation of genes, preferably genes involved in pathological EMT, induction of epithelial cell differentiation, pathological cell proliferation, aberrant angiogenesis, aberrant neovascularization, degeneration, osteoarthritis, cancer or metastasis, inflammation, and/or fibrosis.
  • the nucleic acid encoding the at least one RUNX3 transcription factor, or a fragment or variant thereof binds to or interacts with other transcription factors and/or inhibitory proteins, preferably transcription factors and/or inhibitory proteins involved in pathological EMT, induction of epithelial cell differentiation, pathological cell proliferation, aberrant angiogenesis, aberrant neovascularization, degeneration, osteoarthritis, cancer or metastasis, inflammation, and/or fibrosis.
  • transcription factors and/or inhibitory proteins preferably transcription factors and/or inhibitory proteins involved in pathological EMT, induction of epithelial cell differentiation, pathological cell proliferation, aberrant angiogenesis, aberrant neovascularization, degeneration, osteoarthritis, cancer or metastasis, inflammation, and/or fibrosis.
  • the activation domain is responsible for modulating RUNX function through interaction with certain cofactors
  • the exposed Runt domain by itself can interact with many cofactors, including Ets, C/EBP, and CBFbeta which may consequently regulate a number of RUNX functions.
  • the RUNX3 transcription factor, or a fragment or variant thereof comprises or consists of an amino acid sequence selected from or derived from the GenBank accession number NM_004350.3, NM_001031680.2, NM_001320672.1 , XM_005246024.5, XM_011542351 .2, XM_047433131 .1 , XM_054339349.1 orXM_054339350.1 .
  • Table A provides exemplary human cellular RUNX3 transcript and protein IDs.
  • Table A Exemplary sequences of cellular transcripts and proteins of the RUNX3 transcription factor
  • Preferred amino add and nucleic acid sequences in the context of the invention are provided in Table 1. Therein, each row corresponds to suitable RUNX3 proteins or protein fragments encoded by the nucleic acid.
  • Column A provides a short description of the respective RUNX3 protein or protein fragment.
  • Column B provides the amino acid SEQ ID NO of respective RUNX3 protein or protein fragment.
  • Column C provides SEQ ID NOs of wild type or reference cds encoding the respective RUNX3 protein or protein fragment.
  • Column D provides SEQ ID NOs of G/C optimized (opt1) cds encoding the respective RUNX3 protein or protein fragment.
  • Column E provides SEQ ID NOs of human codon usage adapted (opt3) cds encoding the respective RUNX3 protein or protein fragment.
  • Table 1 Preferred amino acid sequences and coding sequences
  • the nucleic acid encoding the at least one RUNX3 transcription factor comprises or consists of an amino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 129-163, e.g., to the full length of the sequence or fragments or variants of any of these.
  • the nucleic acid encoding the at least one RUNX3 transcription factor comprises or consists of an amino acid sequence being identical or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 129- 133, e.g., the full length or a fragment or a variant thereof.
  • the nucleic acid encoding the at least one RUNX3 transcription factor comprises or consists of an amino acid sequence being identical or at least 90% identical to SEQ ID NO: 129, e.g., the full length or a fragment or a variant thereof.
  • the RUNX3, or a fragment or variant thereof (that is provided by the nucleic acid) is produced in the cytosol upon administration of the nucleic acid to a cell, tissue, or subject.
  • the administration of the nucleic add e.g. RNA
  • the nucleic add leads to a translation of the at least one cds into at least one transcription factor protein.
  • the term relates to the protein product that is generated from the nucleic acid of the invention by translating the cds of the nucleic add into a protein.
  • the amino add sequence comprises or consists of an amino acid sequence selected or derived from
  • RUNX3 or a fragment or variant thereof, wherein the sequence comprises at least one, two, or more amino acid substitutions, deletions or insertions selected from K162R, K200R, K206R, K162Q, K200Q, K206Q, P323R, P323del, P324del, P325del, Y326del or 430insKKK, or any functionally equivalent amino acid substitution at position K162, K200, K206, K162, K200, K206, P323, P324, P325, Y326 or 430 (positions according to the RUNX3 sequence according to SEQ ID NO: 129). Suitable amino add sequences are also provided in Table 1.
  • RUNX3 delta 201 which comprises deletion of amino adds beyond 201 and only expresses the Runt domain for competitive binding to CBFbeta.
  • Other preferred variants comprise at least one, two, or more amino acid substitutions, deletions or insertions selected from K162R, K200R, 430 LLL and/or K206R which increase protein stability by preventing ubiquitin mediated degradation.
  • Other preferred variants comprise at least one, two, or more amino acid substitutions, deletions or insertions selected from K162Q, K200Q, and/or K206Q to mimic protein acetylation.
  • Another particularly preferred variant is RUNX3 delta 323-326, wherein the deletion of the PPXY motif necessary for Smurf mediated degradation.
  • Another preferred variant is RUNX3 with the P323R mutation, which disrupts the PPXY motif (positions according to the RUNX3 sequence SEQ ID NO: 129).
  • the amino add sequence comprises or consists of an amino add sequence selected or derived from isoform 2 RUNX3 or a fragment or variant thereof, wherein said amino acid sequence comprises at least one, two, or more amino acid substitutions, deletions or insertions selected from K148R, K186R and/or K192R which increase protein stability by preventing ubiquitin mediated degradation.
  • Another variant is RUNX3 delta 187 which only expresses the Runt domain for competitive binding to CBFbeta.
  • Other variants comprise at least one, two, or more amino acid substitutions, deletions or insertions selected from K148Q, K186Q, and/or K193Q to mimic protein acetylation.
  • a preferred variant is RUNX3 delta 309-312, wherein the deletion of the PPXY motif necessary for Smurf mediated degradation.
  • Another particularly preferred variant is RUNX3 with the P309R mutation, which disrupts the PPXY motif. (Positions according to the RUNX3 sequence according to SEQ ID NO: 131).
  • the RUNX3 transcription factor, or a fragment or variant thereof comprises or consists of an amino acid sequence being identical or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 129, 134, 136, 138, 140 e.g., the full length or a fragment thereof.
  • the amino acid sequence of RUNX3 comprises residues 68 to 196 selected or derived from RUNX3, residues 76 to 197 selected or derived from RUNX3, residues 68 to 201 selected or derived from RUNX3, or residues 1 to 201 selected or derived from RUNX3 (positions according to SEQ ID NO: 129).
  • the RUNX3 that comprises residues 68 to 196 may comprise an amino acid sequence according to SEQ ID NO: 134.
  • the RUNX3 that comprises residues 76 to 197 may comprise an amino acid sequence according to SEQ ID NO: 136.
  • the RUNX3 that comprises residues 68 to 201 may comprise an amino acid sequence according to SEQ ID NO: 138.
  • the RUNX3 that comprises residues 1 to 201 may comprise an amino acid sequence according to SEQ ID NO: 140.
  • Suitable amino add sequences are also provided in Table 1 .
  • the amino add sequence of RUNX3 comprises the full-length isoform 1 amino add sequence according to SEQ ID NO: 129.
  • the preferred RUNX3 comprises the Runt domain (RD) sequence according to SEQ ID NO: 134.
  • the preferred RUNX3 comprises the Runt domain (RD) sequence according to SEQ ID NO: 136.
  • the preferred RUNX3 comprises the Runt domain (RD) sequence according to SEQ ID NO: 138.
  • the RUNX3 transcription factor comprises a N-terminal region sequence according to SEQ ID NO: 140. Suitable amino add sequences are also provided in Table 1.
  • the RUNX3 transcription factor, or a fragment or variant thereof comprises or consists of an amino add sequence being identical or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 134, 136, 138, 140, e.g., the full length or a fragment thereof.
  • the RUNX3 transcription factor, or a fragment or variant thereof comprises or consists of an amino acid sequence being identical or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 140 or, e.g., the full length or a fragment thereof.
  • the RUNX3 transcription factor reduces EMT in a subject.
  • EMT is a process by which epithelial cells lose their cell polarity and cell-cell adhesion and gain migratory and invasive properties to become mesenchymal stem cells; these are multipotent stromal cells that can differentiate into a variety of cell types.
  • EMT is a dynamic and continuous biological process that contributes to organogenesis and disease. EMT has also been shown to occur in wound healing, in organ fibrosis and in the initiation of metastasis in cancer progression. Examples of EMT-assodated diseases include pathologic ocular fibrosis and proliferation, e.g.
  • conjunctival fibrosis e.g. ocular dcatricial pemphigoid
  • corneal scarring e.g., corneal epithelial down growth, and/or aberrant fibrosis
  • diseases in the anterior segment of the eye e.g., comeal opadfication and glaucoma
  • corneal dystrophies e.g., comeal opadfication and glaucoma
  • inflammation e.g., pterygium
  • macula edema e.g., retinal and vitreous hemorrhage
  • fibrovascular scarring neovascular glaucoma
  • age-related macular degeneration (ARMD) geographic atrophy
  • ROP retinopathy of prematurity
  • subretinal fibrosis epireti nal fibrosis
  • epireti nal fibrosis e.g. ocular dcatricial pe
  • EMT epithelial graft-versus-host disease
  • corneal scarring corneal epithelial downgrowth
  • conjunctival scarring eye tumors such as melanoma and metastatic tumors, or fibrosis.
  • the nucleic acid comprising at least one cds encoding the at least one RUNX3 reduces EMT of at least 5% , 10% , 25% , 50% , 60% , 70% , 80% or 90% upon administration to a cell or subject.
  • the RUNX3 reduces proliferation and/or migration of retinal pigment epithelial (RPE) cells in a subject.
  • RPE retinal pigment epithelial
  • the RPE is a pigmented cell layer just outside the neurosensory retina that nourishes retinal visual cells and is firmly attached to the underlying choroid and overlying retinal visual cells which functions both as a selective barrier to and a vegetative regulator of the overlying photoreceptor layer, thereby playing a key role in its maintenance. Dysfunction of the RPE is found in age-related macular degeneration and retinitis pigmentosa. RPE are also involved in diabetic retinopathy.
  • the RUNX3 (provided by the nucleic acid) reduces EMT in a subject.
  • the RUNX3 (provided by the nucleic acid) displays an anti-angiogenic effect in a subject.
  • the RUNX3 transcription factor functions as an angiogenesis inhibitor to reduce/or inhibit aberrant growth of new blood vessels (angiogenesis), e.g. within the eye of a subject.
  • the nucleic acid encoding the at least one RUNX3 transcription factor, or a fragment or variant thereof reduces the activity of a target transcription factor in a cell.
  • target transcription factori as used herein is intended to refer to the cellular transcription factor that is intended to be inhibited by the at least one RUNX3 transcription factor (encoded by the nucleic acid). In various embodiments, inhibiting the “target transcription factor'’ is associated with advantageous cellular or physiological effects as further outlined herein.
  • the target transcription factor is selected from a transcription factor that has an aberrant transcription factor activity or pathologic transcription factor activity.
  • the aberrant or pathologic target transcription factor activity is an overexpression and/or an overactivation.
  • the target transcription factor is selected from a transcription factor that has an aberrant or pathologic transcription factor activity (e.g. overexpression and/or an overactivation) associated with EMT, pathological cell proliferation, aberrant angiogenesis, aberrant neovascularization, degeneration, and/or fibrosis.
  • the target transcription factor is selected from a transcription factor that is overexpressed and/or overactive in a disease, disorder, or condition.
  • the target transcription factor is selected from a transcription factor that is overexpressed or overactive in an ocular disease, disorder, or condition.
  • the target transcription factor that shows pathologic transcription factor activity e.g. overexpression and/or an overactivation associated with EMT, pathological cell proliferation, aberrant angiogenesis, aberrant neovascularization, degeneration, and/or fibrosis.
  • the target transcription factor is RUNX1 .
  • the RUNX3 transcription factor (provided by the nucleic acid of the invention) is for reducing or inhibiting the activity of a RUNX1 in a cell.
  • the nucleic acid encoding the RUNX3 transcription factor as defined herein is a RUNX1 antagonist or a RUNX1 inhibitor.
  • Runt-related transcription factor 1 also known as acute myeloid leukaemia 1 protein (AML1) or core-binding factor subunit alpha-2 (CBFA2), is a protein that in humans is encoded by the RUNX1 gene.
  • RUNX proteins form a heterodimeric complex with core binding factor b (CBFbeta) which confers increased deoxyribonucleic add (DNA) binding and stability to the complex. That complex comprising RUNX (CBFalpha) proteins and CBFbeta is often referred to as heterodimeric CBF transcription factor.
  • RUNX1 can bind DNA as a monomer, but its DNA binding affinity is enhanced by 10-fold if it heterodimerizes with its co-transcription factor CBFbeta, also via the Runt domain.
  • An amino acid sequence for human RUNX1 is available in the UniProt database under accession No Q01196- 1 .
  • Amino acid sequences of additional isoforms are available in the UniProt database under accession No Q01196-2; Q01196-3; Q0119&4; Q01196-5; Q01196-6; Q01196-7; Q01196-8; Q01196-9; Q01196-10; and Q01196-11 .
  • the produced RUNX3 upon administration of the nucleic add to a cell or subject, reduces or prevents the interaction of cellular RUNX1 with cellular CBFbeta. Accordingly, the formation of a cellular RUNX1- CBFbeta heterodimeric complex is inhibited. Accordingly, in preferred embodiments, upon administration of the nucleic acid to a cell or subject, the produced RUNX3 reduces cellular RUNX1 -CBFbeta complex formation and/or activity. Suitably, as result of redudng cellular RUNX1 -CBFbeta complex formation and/or activity, the transcription activity of RUNX1 is reduced in the cell or subject.
  • the produced RUNX3 upon administration of the nucleic acid to a cell or subject, reduces the cellular expression of RUNX1 controlled genes or gene products.
  • the produced RUNX3 upon administration of the nucleic acid to a cell or subject, reduces the cellular expression of TGFbeta2 (TGF
  • MARVELD2 is a tight junction associated epithelial marker, as a predictor of the future state of the cell.
  • the produced RUNX3 transcription factor upon administration of the nucleic acid to a cell or subject, reduces the cellular expression of the RUNX1 target transcription factor.
  • the expression of transcription factors is often regulated by self-regulatory feedback loops. That means that e.g. transcription factors proteins can activate their own expression (self-activation).
  • the RUNX3 transcription factor of the present invention may reduce or inhibit the activity of the RUNX1 target transcription factor in a cell, that can also lead to a reduced expression of the RUNX1 target transcription factor as such. Accordingly, a further reduction of the cellular expression of the RUNX1 target transcription factor may increase or enhance advantageous cellular or physiological effects of the RUNX3 transcription factor that is provided by the nucleic acid.
  • the nucleic acid comprises or consists of at least one cds encoding at least one RUNX3 transcription factor as defined herein, preferably encoding any one ofSEQ ID NOs: 129-163, or fragments of variants thereof. It has to be understood that, on nucleic acid level, any sequence which encodes an amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 129-163, or fragments or variants thereof, may be selected and may accordingly be understood as suitable cds of the invention
  • the nucleic add comprises at least one cds that comprises at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequences according to any one of SEQ ID NOs: 164-268, or a fragment or a fragment or variant of any of these sequences.
  • the amino add sequence comprises or consists of an amino acid sequence selected or derived from RUNX3 or a fragment or variant thereof, wherein the sequence comprises at least one, two, or more amino acid substitutions, deletions as defined herein.
  • the nucleic acid comprises at least one cds that comprises at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 177-198, 212-233, 247-268, e.g. the full length or a fragment thereof.
  • the nucleic acid is a modified and/or stabilized nucleic acid.
  • the nucleic acid may thus be provided as a “stabilized nucleic acid” that is to say a nucleic acid showing improved resistance to in vivo degradation and/or showing improved stability in vivo, and/or showing improved translatability in vivo. This is particularly important in embodiments where the nucleic acid is an RNA.
  • the nucleic of the invention may be provided as a “stabilized nucleic acid”, preferably a “stabilized RNA”.
  • the nucleic add comprises at least one codon modified cds.
  • the amino acid sequence encoded by the at least one codon modified cds is not modified compared to the amino add sequence encoded by the corresponding wild type or reference cds.
  • codon modified cds relates to a cds that differs in at least one codon (triplets of nucleotides coding for one amino acid) compared to the corresponding wild type or reference cds.
  • a codon modified cds in the context of the invention may show improved resistance to in vivo degradation and/or improved stability in vivo, and/or improved translatability in vivo. Codon modifications in the broadest sense make use of the degeneracy of the genetic code wherein multiple codons may encode the same amino acid and may be used interchangeably to optimize/modify the cds for in vivo applications.
  • the at least one cds is a codon modified cds, wherein the codon modified cds is selected from a C maximized cds (as further defined in WO2021239880 [p.122, lines 33 to 39] which is hereby incorporated by reference); a CAI maximized cds (as further defined in WO2021239880 [p.123, lines 33 to 44] which is hereby incorporated by reference); a human codon usage adapted cds (as further defined in WO2021239880 [p.123, lines 7 to 17] which is hereby incorporated by reference); a G/C content modified cds (as further defined in WO2021239880 [p.123, lines 19 to 31] which is hereby incorporated by reference); and G/C optimized cds (“opt1 ”), or any combination thereof.
  • a C maximized cds as further defined in WO2021239880 [p.122, lines 33 to 39] which is hereby
  • At least one cds comprises a human optimized cds, wherein the nucleic acid sequence is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 234-268, or a fragment or a variant of any of these.
  • the at least one cds is G/C optimized cds as defined herein. Accordingly, in preferred embodiments, at least one cds comprises a G/C optimized cds, wherein the nucleic add sequence is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 199-233, or a fragment or a variant of any of these, preferably a nucleic acid sequence identical or at least 90% identical to SEQ ID NO: 199, or a fragment or a variant thereof.
  • the nucleic acid of the invention comprises at least one heterologous nucleic acid sequence element.
  • a preferred heterologous nucleic add sequence may be selected from at least one heterologous UTR.
  • heterologous sequence refers to a nucleic acid sequence that is not from the same gene. Accordingly, heterologous sequences may be derivable from a different gene in the same organism (e.g. human) or from a different organism. Heterologous sequences do naturally (in nature) not occur in the same nucleic acid molecule. In the context of the invention, a heterologous sequence is not selected or derived from a RUNX3 gene, e.g. a heterologous UTR is not selected from a UTR from a RUNX3 gene.
  • nucleic acid comprises at least one heterologous UTR, for example selected from at least one heterologous 5-UTR and/or at least one heterologous 3-UTR.
  • UTR untranslated region
  • UTR element refers to a part of a nucleic acid molecule typically located 5’ or 3’ of a cds.
  • a UTR is not translated into protein.
  • a UTR may be part of the nucleic add, e.g. an RNA.
  • a UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites, promotor elements etc.
  • the nucleic acid comprises a cds, and a 5-UTR and/or 3-UTR.
  • UTRs may harbour regulatory sequence elements that determine RNA turnover, stability, and localization.
  • UTRs may harbour sequence elements that enhance translation.
  • translation of the nucleic add into at least one peptide or protein is of paramount importance to therapeutic efficacy.
  • Certain combinations of 3’-UTRs and/or 5’-UTRs may enhance the expression of operably linked cds encoding peptides or proteins as defined herein.
  • Nucleic acid molecules harbouring said UTR combinations advantageously enable rapid and transient expression of encoded RUNX3 transcription factor after administration to a subject, preferably after ocular administration. Accordingly, the nucleic acid comprising certain combinations of 3'-UTRs and/or 5'-UTRs is particularly suitable for ocular administration.
  • the nucleic acid comprises at least one heterologous 5-UTR and/or at least one heterologous 3 -UTR.
  • Said heterologous 5’-UTRs or 3'-UTRs may be derived from naturally occurring genes or may be synthetically engineered.
  • the nucleic add comprises at least one cds as defined herein operably linked to at least one (heterologous) 3 -UTR and/or at least one (heterologous) 5-UTR.
  • the nucleic acid of the invention comprises at least one 3-UTR.
  • a 3-UTR is typically located between a cds and an (optional) poly(A) sequence.
  • a 3-UTR may comprise elements for controlling expression, also called regulatory elements. Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites.
  • the 3 -UTR comprises one or more of a polyadenylation signal, a binding site for proteins that affect a nucleic acid stability of location in a cell, or one or more miRNA or binding sites for miRNAs.
  • the at least one 3’-UTR comprises or consists of a nucleic add sequence derived or selected from a 3’-UTR of a gene selected from PSMB3, ALB7, alpha-globin, beta-globin, ANXA4, CASP1 , COX6B1 , FIG4, GNAS, NDUFA1 , RPS9, SLC7A3, TUBB4B, or from a homolog, a fragment, or variant of any one of these genes.
  • the at least one 3’-UTR that is derived or selected from PSMB3, ALB7, alpha-globin, betaglobin, ANXA4, CASP1 , COX6B1 , FIG4, GNAS, NDUFA1 , RPS9, SLC7A3, TUBB4B, comprises or consist of a nucleic add sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one ofSEQ ID NOs: 66-95, 112-123, or a fragment or a variant of any of these.
  • the nucleic acid of the invention comprises at least one 5’-UTR.
  • a 5’-UTR is typically located 5’ of the cds.
  • a 5’-UTR may start with the transcriptional start site and ends before the start codon of the cds.
  • a 5’-UTR may comprise elements for controlling gene expression, called regulatory elements.
  • regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites.
  • the nucleic acid comprises at least one 5’-UTR, which may be derivable from a gene that relates to an RNA with enhanced half-life (i.e. that provides a stable RNA).
  • the 5’-UTR comprises one or more of a binding site for proteins that affect a nucleic acid stability or nucleic acid location in a cell, or one or more miRNA or binding sites for miRNAs (as defined above).
  • the at least one 5’-UTR comprises or consist of a nucleic add sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 12-63, or a fragment or a variant of any of these.
  • the at least one 5 -UTR comprises a nucleic acid sequence derived or selected from a 5'- UTR of gene selected from HSD17B4, RPL32, AIG1 , alpha-globin, ASAH1 , ATP5A1 , COX6C, DPYSL2, MDR, MP68, NDUFA4, NOSIP, RPL31 , RPL35A, SLC7A3, TUBB4B, UBQLN2, or from a homolog, a fragment or variant of any one of these genes.
  • the at least one 5 -UTR derived or selected from HSD17B4, RPL32, AIG1 , alpha-globin, ASAH1 , ATP5A1 , COX6C, DPYSL2, MDR, MP68, NDUFA4, NOSIP, RPL31 , RPL35A, SLC7A3, TUBB4B, UBQLN2 comprises or consist of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 12- 45, 64, 65, or a fragment or a variant of any of these.
  • the nucleic acid comprises a 5 -UTR derived or selected from a HSD17B4 gene.
  • the at least one heterologous 5-UTR derived or selected from HSD17B4 comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 12, 13, 64, 65, or a fragment or a variant thereof, preferably SEQ ID NO: 13, or a fragment or a variant thereof.
  • the nucleic acid preferably the RNA, comprises at least one cds as defined herein operably linked to a 3 -UTR and/or a 5-UTR selected from the 5’-UTR/3’-UTR combinations (5’UTR/3’UTR) provided in WO2021239880 [p.127, line 35 to p.128, line 2], which is hereby incorporated by reference.
  • the at least one heterologous 5-UTR is selected from HSD17B4 and the at least one heterologous 3-UTR is selected from
  • PSMB3 (a-1 (HSD17B4/PSMB3)).
  • the nucleic acid preferably the RNA, comprises at least one cds as defined herein encoding at least one RUNX3 transcription factor as defined herein, wherein said cds is operably linked to a HSD17B45’-UTR and a PSMB33’-UTR (HSD17B4/PSMB3 (a-1)).
  • HSD17B45’-UTR a PSMB33’-UTR
  • PSMB33 PSMB33’-UTR
  • this embodiment is particularly beneficial for expressing the RUNX3 transcription factor in human cells of the eye e.g. retinal pigment epithelium (RPE) cells.
  • RPE retinal pigment epithelium
  • the nucleic acid is monocistronic, bidstronic, or multicistronic, preferably monocistronic.
  • the nucleic acid comprises a ribosome binding site, also referred to as “Kozak sequence” identical to or at least 80%, 85%, 90%, 95% identical to any one ofSEQ ID NOs: 1, 2, or sequences GCCGCCACC (DNA), GCCGCCACC (RNA), GCCACC (DNA), GCCACC (RNA), ACC (DNA) or ACC (RNA), or fragments or variants of any of these.
  • the “Kozak sequence” comprises or consists of RNA sequence ACC.
  • the nucleic acid comprises at least one poly(N) sequence, e.g. at least one poly(A) sequence, at least one poly(U) sequence, at least one poly(C) sequence, or combinations thereof.
  • the nucleic add e.g. the RNA, comprises at least one poly(A) sequence.
  • the nucleic acid comprises least two, three, or more poly(A) sequences.
  • poly(A) sequence refers to a sequence of up to about 1000 adenosine nucleotides, typically located at the 3’- end of a linear RNA. Typically, said poly(A) sequence is homopolymeric. Alternatively, a poly(A) sequence may be interrupted by at least one nucleotide different from an adenosine.
  • the at least one poly(A) sequence comprises about 20 to about 500 adenosines, about 40 to about 250 adenosines, about 60 to about 250 adenosines, preferably about 60 to about 150 adenosines. In embodiments, the at least one poly(A) sequence comprises about or more than 10, 50, 64, 75, 100, 200, 300, 400, or 500 adenosines.
  • the at least one poly(A) sequence comprises about 100 adenosine nucleotides (A100), preferably about 100 consecutive adenosine nucleotides.
  • the at least one poly(A) sequence as defined herein is located directly at the 3’ terminus of the nucleic acid, preferably the RNA.
  • the 3’-terminal nucleotide (that is the last 3’-terminal nucleotide in the polynucleotide chain) is the 3'-terminal A nucleotide of the at least one poly(A) sequence.
  • the 3’ terminus of the nucleic add consists of a poly(A) sequence (e.g. A100 or A30-N10-A70) and therefore terminates with an A.
  • ending on an adenosine nucleotide decreases the induction of interferons, e.g. IFNalpha, by the RNA of the invention if for example administered as a medicament into the eye. This is particularly important as the induction of interferons, e.g. IFNalpha, is thought to be one main factor for induction of side effects.
  • the at least one poly(A) sequence comprises about 100 adenosine nucleotides (A100), preferably about 100 consecutive adenosine nucleotides.
  • the at least one poly(A) sequence is obtained from a DNA template during RNA in vitro transcription.
  • the at least one poly(A) sequence is obtained in vitro by common methods of chemical synthesis.
  • the at least one poly(A) sequence is generated by enzymatic polyadenylation of the RNA (after RNA in vitro transcription).
  • the nucleic acid preferably the RNA, comprises at least one histone stem-loop (hSL).
  • hSL may be located in the 3’ region such as in the 3’-UTR.
  • the term refers to nucleic add sequences that forms a stem-loop structure.
  • a hSL may be derived from formulae (I) or (II) of W02012019780.
  • the nucleic acid may comprise at least one hSL sequence derived from the specific formulae (la) or (Ila) of W02012019780.
  • the at least one hSL sequence comprises or consists of a nucleic add sequence identical or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one ofSEQ ID NOs: 3, 4, or a fragment or variant of any of these, preferably SEQ ID NO: 4, or a fragment or variant thereof.
  • the nucleic acid comprises a 3’-terminal sequence element.
  • the 3’-terminal sequence element represents the 3’ terminus of the RNA.
  • a 3’-terminal sequence element may comprise at least one poly(A) sequence as defined herein and, optionally, at least one hSL as defined herein.
  • the at least one 3’- terminal sequence element comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one ofSEQ ID NOs: 5-11 , or a fragment or variant of these sequences, preferably SEQ ID NO: 5 or 6, or a fragment or variant of these sequences.
  • the nucleic acid is an isolated nucleic acid.
  • isolated nucleic acid does not comprise a cell or a subject that comprises said nucleic acid but relates to the nucleic add as an isolated molecule or ensemble of isolated molecules.
  • isolated nucleic acid can e.g. be a nucleic add isolated or purified from a cell (e.g. cell culture, bacterial culture), or can be a nucleic acid (e.g. RNA) isolated from an RNA in vitro transcription.
  • the nucleic acid of the invention is a therapeutic nucleic acid. Accordingly, the nucleic acid, preferably the RNA, is suitably used in a therapeutic context to provide a therapeutic modality for providing the RUNX3 transcription factor according to the invention.
  • the nucleic acid of the invention is selected from a DNA or an RNA.
  • the nucleic acid is a DNA.
  • the DNA may be any type of DNA that comprises a cds as defined herein including any type of single stranded, double stranded, linear, and circular DNA.
  • a suitable DNA in the context of the invention may be selected from bacterial plasmid, an adenovirus, a poxvirus, a parapoxivirus (orf virus), a vaccinia virus, a fowlpox virus, a herpes virus, an adeno-assodated virus (AAV), an alphavirus, a lentivirus, a lambda phage, a lymphocytic choriomeningitis virus and a Listeria sp, Salmonella sp.
  • the DNA a viral DNA, preferably an adeno-assodated virus DNA.
  • the nucleic is an RNA.
  • the RNA may be any type of RNA that comprises a cds as defined herein including any type of single stranded, double stranded, linear, and circular RNA.
  • the RNA is selected from mRNA, circular RNA, replicon RNA or self-replicating RNA, or viral RNA.
  • the RNA is a circular RNA.
  • circular RNA (circRNA) is an RNA connected to form a circle and therefore does not comprise a 3' or 5' terminus. Said circRNA comprises at least one cds as defined herein.
  • CircRNA construct designs can be taken from WO2023073228, claims 1 to 51 , hereby incorporated by reference.
  • the RNA is a replicon RNA or self-replicating RNA.
  • Such constructs may encode replicase elements derived from e.g. alphaviruses (e.g. SFV, SIN, VEE, or RRV) and a cds as defined herein.
  • the nucleic acid of the invention is an mRNA.
  • mRNA is preferred because mRNA allows for regulated dosage, transient expression, complete degradation of the mRNA after protein synthesis, and do not pose the risk of insertional mutations.
  • the mRNA of the invention is non-replicative.
  • the nucleic add preferably the RNA, comprises about 50 to about 20000 nucleotides, or about 500 to about 10000 nucleotides, or about 1000 to about 10000 nucleotides, or preferably about 1000 to about 5000 nucleotides, or even more preferably about 1000 to about 2000 nucleotides.
  • a modified nucleic acid or RNA may comprise nucleotide analogues/modifications, e.g. backbone modifications, sugar modifications and/or base modifications.
  • a backbone modification is a modification in which phosphates of the backbone of the nucleotides of the RNA are chemically modified.
  • a sugar modification is a chemical modification of the sugar of the nucleotides of the RNA.
  • a base modification is a chemical modification of the base moiety of the nucleotides of the RNA. Nucleotide analogues/modifications may be selected from those applicable fortranscription and/or translation.
  • the nucleic acid is an RNA that comprises at least one modified nucleotide.
  • the RNA comprises at least one modified nucleotide selected from WO2021239880 [p.136, line 17 to p.137, line 19], which is hereby incorporated by reference.
  • the RNA may comprise modified uridine nucleotides that preferably comprise a chemical modification in the 5-position of the uracil. Suitable modified uridine nucleotides may be selected from WO2021239880 [p.137, lines 15 to 19], which is hereby incorporated by reference.
  • 100% of the uracil in the frill nucleic acid sequence preferably the RNA sequence
  • 100% of the uracil in the frill nucleic add sequence preferably the RNA sequence are substituted with pseudouridine (ip).
  • the nucleic acid does not comprise chemically modified nucleotides.
  • a 5'-cap structure as defined herein is not considered to be a modified nucleotide in that spedfic context.
  • the nucleic acid is an RNA comprising a sequence consisting of G, C, A and U, and optionally, comprises a 5’-cap structure.
  • the nucleic acid preferably the RNA, comprises a 5’-cap structure.
  • the nucleic acid preferably the RNA
  • the RNA comprises a cap1 structure or a modified cap1 structure.
  • the term “5’-cap structure” refers to a 5’ modified nucleotide, particularly a guanine nucleotide, positioned at the 5’-end of an RNA.
  • the 5’-cap is typically connected via a 5’-5’-tri phosphate linkage to the RNA.
  • a 5’-cap may stabilize the RNA and/or enhance expression of RUNX3 and/or reduce the stimulation of the innate immune system after administration.
  • the 5’-cap (capO or cap1) structure may be formed in RNA in vitro transcription using a cap analog.
  • a cap1 or modified cap1 structure is generated using a cap analog, preferably a tri-nucleotide cap analog.
  • a cap analog preferably a tri-nucleotide cap analog.
  • Any cap analog derivable from the structures defined in claims 1-13 of WO2017053297 (hereby incorporated by reference) or, alternatively, any cap analog derivable from the structures defined in claim 1-37 ofW02023007019 (hereby incorporated by reference) may be suitably used to co-transcriptionally generate a cap1 or modified cap1 .
  • the cap1 structure is formed via co-transcriptional capping using tri-nucleotide cap analogues m7G(5’)ppp(5’)(2’OMeA)pG or m7G(5’)ppp(5’)(2’OMeG)pG or m7(3’OMeG)(5’)ppp(‘5)m6(2’OMeA)pG.
  • a particularly preferred cap1 analog in that context is m7G(5’)ppp(5’)(2’OMeA)pG.
  • the cap1 structure is a modified cap1 structure and is formed using co-transcriptional capping using tri-nucleotide cap analogue 3'OMe- m7G(5')ppp(5')(2'OMeA)pG .
  • the 5’-cap structure is formed via enzymatic capping using capping enzymes (e.g. vaccinia virus capping enzymes and/or cap-dependent 2’-0 methyltransferases) to generate capO, cap1 or cap2 structures.
  • capping enzymes e.g. vaccinia virus capping enzymes and/or cap-dependent 2’-0 methyltransferases
  • RNA comprises a cap structure, preferably a cap1 structure as determined by a capping assay (e.g. via an assay as described in cl. 27 to 46 of W02015101416).
  • the nucleic acid comprises a 5’-terminal sequence element comprising or consisting of a nucleic add sequence being identical or at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of sequences GGGAGA, AGGAGA, GGGAAA, AGAAUA, AGAUUA, GAUGGG or GGGCG, or a fragment or variant of these sequences, preferably AGGAGA, or a fragment or variant.
  • a 5’-terminal sequence element may comprise e.g. a binding site for T7 RNA polymerase.
  • the first nucleotide of said 5'-terminal start sequence may preferably comprise a 2’0 methylation, e.g. 2’0 methylated guanosine or a 2’0 methylated adenosine.
  • the nucleic add is an in vitro transcribed RNA, preferably an in vitro transcribed mRNA.
  • the nucleic add is a purified RNA, preferably a purified mRNA.
  • the RNA has been purified by (RP)HPLC, AEX, size exclusion chromatography (SEC), hydroxyapatite chromatography, tangential flow filtration (TFF), filtration, precipitation, core-bead flowthrough chromatography, oligo(dT) purification, and/or cellulose-based purification.
  • the RNA has been purified using RP-HPLC (preferably as described in W02008077592) and/or TFF (preferably as described in WO2016193206) and/or oligo d(T) purification (preferably as described in WO2016180430) to e.g. to remove dsRNA, non-capped RNA and/or RNA fragments.
  • the RNA has an integrity of at least 60%, 70%, 80%, 90%.
  • RNA integrity describes whether the complete RNA sequence is present. RNA integrity can be determined by RP-HPLC and may be based on determining the area under the peak of the expected full-length RNA in a chromatogram.
  • the nucleic acid is an RNA comprising the following elements preferably in 5’- to 3’-direction:
  • A) a 5’-cap structure preferably a cap1 structure or a modified cap1 structure as spedfied herein;
  • a 5’-UTR preferably selected or derived from a 5’-UTR of a HSD17B4 gene, or a fragment thereof;
  • a 3’-UTR preferably selected or derived from a 3’-UTR of a PSMB3 gene, or a fragment thereof;
  • G optionally, chemically modified nucleotides, suitably selected from ip or ml ip, preferably ml ip.
  • RNA sequences are provided in Table 2.
  • Table 2 Therein, the corresponding SEQ ID NOs of RNA constructs are provided in columns D-l, wherein columns D-F relates to RNA sequences comprising mRNA design HSD17B4/PSMB3 hSL-A100 and columns G-l relates to RNA sequences comprising mRNA design HSD17B4/PSMB3 A100.
  • Column A provides a short description of the respective RUNX3.
  • Column C provides the amino acid SEQ ID NO of respective amino acid sequence.
  • RNA sequences comprising a wild type or reference cds
  • columns E and H RNA sequences comprising a G/C optimized (opt1) cds
  • columns F and I RNA sequences comprising a human codon usage adapted (opt3) cds.
  • 'feature key i.e. “source” (for nucleic acids or proteins) or “misc eature” (for nucleic acids) or “REGION” (for proteins) of the respective SEQ ID NOs in the ST.26 sequence listing.
  • source for nucleic acids or proteins
  • mimisc eature for nucleic acids
  • REGION for proteins
  • the nucleic acid preferably the RNA comprises or consists of a nucleic acid sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 269-458, 461-462, or a fragment or variant of any of these sequences, optionally wherein at least one, preferably all uracil nucleotides in said
  • RNA sequences are replaced by pseudouridine (ip) nucleotides and/or N1 -methylpseudouridine (m1ip) nucleotides.
  • ip pseudouridine
  • m1ip N1 -methylpseudouridine
  • the nucleic acid preferably the RNA comprises or consists of a nucleic acid sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic add sequence selected from SEQ ID NOs: 269, 300, 331 , 362, 393, 424, 455-458, 461 -462, or a fragment or variant of any of these sequences, optionally wherein at least one, preferably all uradl nucleotides in said RNA sequences are replaced by pseudouridine (ip) and/or N1 -methylpseudouridine (m1ip), preferably m1ip.
  • pseudouridine ip
  • m1ip N1 -methylpseudouridine
  • the nucleic add of the invention is a N1 -methylpseudouridine (m1ip) modified RNA which is identical or at least 90% or 95% identical to a nucleic sequence according to SEQ ID NOs: 456 or 462, or a fragment or variant of any of these sequences.
  • the nucleic acid of the invention is a N1- methylpseudouridine (ml ip) modified 5’-cap1 mRNA that comprises or consists of an RNA sequence which is identical or at least 90% identical to a nucleic sequence according to SEQ ID NOs: 456 or 462, or a fragment or variant thereof.
  • the invention provides a pharmaceutical composition comprising at least one nucleic acid encoding at least one RUNX3 transcription factor or a fragment or variant thereof.
  • nucleic acid encoding RUNX3 the nucleic acid encoding RUNX3
  • compositions refers to any type of composition in which the specified ingredients (e.g. nucleic acid encoding RUNX3) may be incorporated, optionally along with any further constituents, usually with at least one pharmaceutically acceptable carrier or excipient.
  • the composition may be a dry composition such as a powder, a granule, or a solid lyophilized form.
  • the composition may be in liquid form, and each constituent may be independently incorporated in dissolved or dispersed (e.g. suspended or emulsified) form.
  • Compositions of the present invention are suitably sterile and/or pyrogen-free.
  • the at least one nucleic add of the pharmaceutical composition is selected from an RNA as further defined in the first aspect.
  • the at least one nucleic acid of the pharmaceutical composition is selected from an mRNA as further defined in first aspect.
  • the nucleic acid preferably the RNA as comprised in the pharmaceutical composition is provided in an amount of about 10ng to about 500pg, in an amount of about 1 pg to about 500pg, in an amount of about 1 pg to about 100pg, in an amount of about 1 pg to about 20pg.
  • the at least one nucleic add preferably the at least one RNA of the pharmaceutical composition, is formulated with a pharmaceutically acceptable carrier or excipient.
  • the at least one nucleic acid preferably the RNA
  • a formulation in that context may have the function of a transfection agent and/or may protect the nucleic add from degradation.
  • a compound for formulation is selected from peptides, proteins, lipids, polysaccharides, and/or polymers.
  • the at least one nucleic acid preferably the RNA
  • the at least one nucleic add preferably the at least one RNA of the pharmaceutical composition is complexed or associated with or at least partially complexed or partially associated with one or more cationic (cationic or preferably ionizable) or polycationic compound.
  • cationic means that the respective structure, compound, group, or atom bears a positive charge, either permanently or not permanently, e.g. in response to certain conditions such as pH.
  • cationic means that the respective structure, compound, group, or atom bears a positive charge, either permanently or not permanently, e.g. in response to certain conditions such as pH.
  • cationic means that the respective structure, compound, group, or atom bears a positive charge, either permanently or not permanently, e.g. in response to certain conditions such as pH.
  • cationic cationic
  • cationisable and “permanently cationic” must be understood as defined in WO2023/031394 [p.12, line 32 to p.13, line 16].
  • polycationic means that the respective structure, compound, or group, or atom bears a plurality of positive charges.
  • the term as used herein must be understood as defined in WO2021/156267 [p.88, line 12 to p.89, line 22].
  • the at least one cationic or polycationic compound is selected from a cationic or polycationic polymer, a cationic or polycationic polysaccharide, a cationic or polycationic lipid, a cationic or polycationic protein, a cationic or polycationic peptide, or any combinations thereof.
  • cationic or polycationic compounds being suitable in the context of the invention may be selected from p.88, line 24 to p.89, line 10 in WO2021/156267, the respective disclosure herewith incorporated by reference.
  • the at least one cationic or polycationic compound is a cationic or polycationic peptide or protein.
  • Preferred cationic or polycationic proteins or peptides that may be used for complexation can be derived from formula (Arg)l;(Lys)m;(His)n;(Orn)o;(Xaa)x of the patent application W02009030481 or WO2011026641 , the disclosure of W02009030481 or WO2011026641 relating thereto incorporated herewith by reference.
  • the at least one cationic or polycationic proteins or peptides preferably selected from SEQ ID NOs: 124-128, or any combinations thereof.
  • the pharmaceutical composition comprises at least one nucleic acid and a polymeric carrier.
  • polymeric carried refers to a compound that facilitates transport and/or complexation of another compound.
  • a polymeric carrier is typically a carrier that is formed of a polymer.
  • a polymeric carrier may be associated to its cargo (e.g. RNA) by covalent or non-covalent interaction.
  • a polymer may be based on different subunits, such as a copolymer.
  • the polymeric carrier used according to the present invention may comprise mixtures of cationic peptides, proteins or polymers and optionally further components as defined herein, which are cross-linked by disulfide bonds (via -SH groups).
  • the at least one nucleic acid is complexed or associated with a polyethylene glycol/peptide polymer, preferably comprising HO-PEG5000-S-(S-CHHHHHHRRRRHHHHHHC-S-)7-S-PEG5000-OH (SEQ ID NO: 127 as peptide monomer), HG-PEG5000-S-(S-CHHHHHHRRRRHHHHHHC-S-)4-S-PEG5000-GH (SEQ ID NO: 127 as peptide monomer), HG-PEG5000-S-(S-CGHHHHHRRRRHHHHHGC-S-)7-S-PEG5000-GH (SEQ ID NO: 128 as peptide monomer) and/or a polyethylene glycol/peptide polymer comprising H0-PEG5000-S-(S- CGHHHHHRRRRHHHHHGC-S-)4-S-PEG5000-GH (SEQ ID NO: 128 as peptide monomer) and/or a polyethylene glyco
  • the polymeric carrier is a peptide polymer, preferably a polyethylene glycol/peptide polymer as defined above, and a lipid component, preferably a lipidoid component.
  • a lipidoid is a lipid-like compound, i.e. an amphiphilic compound with lipid-like physical properties.
  • the lipidoid is a compound that comprises two or more cationic nitrogen atoms and at least two lipophilic tails.
  • the lipidoid may be free of a hydrolysable linking group, in particular linking groups comprising hydrolysable ester, amide or carbamate groups.
  • the cationic nitrogen atoms of the lipidoid may be cationisable or permanently cationic, or both types of cationic nitrogens may be present in the compound.
  • the lipidoid component may be anyone selected from the lipidoids provided in the table of page 50-54 ofW02017212009, the specific lipidoids of said table, and the specific disclosure relating thereto herewith incorporated by reference.
  • Particularly preferred lipidoid components in that context are 3-C12- OH, 3-C12-OH-cat, 3-C12-C3-OH.
  • the formulation comprises polyethylene glycol/peptide polymers (HO-PEG 5000-S-(S-CGH5R4H5GC-S-)7-S-PEG 5000-GH) and RNA complexed at the 1 :2 ratio (W/W), and, optionally, a 3-C12-OH lipidoid.
  • said formulations are particularly suitable for ocular administration.
  • the at least one nucleic acid preferably the RNA, is formulated in lipid-based carriers.
  • lipid-based carriers encompasses lipid-based delivery systems for nucleic acid, preferably RNA, which comprise a lipid component.
  • a lipid-based carrier may additionally comprise other components suitable for formulating a nucleic acid including a cationic or polycationic polymer, polysaccharide, protein, and/or peptide.
  • the nucleic add, preferably the RNA may completely or partially be incorporated or encapsulated in a lipid-based carrier, wherein the nucleic acid may be located in the interior space of the lipid-based carrier, within the lipid layer/membrane of the lipid-based carrier, or associated with the exterior surface of the lipid-based carrier.
  • the incorporation of nucleic acid into lipid-based carriers may be referred to as "encapsulation".
  • encapsulation refers to the essentially stable combination of nucleic acid such as RNA with one or more lipids into larger complexes or assemblies such as lipid-based carriers, preferably without covalent binding of the nucleic add.
  • the encapsulated nucleic acid preferably the RNA, may be completely or partially located in the interior of the lipid-based carrier (e.g. the lipid portion and/or an interior space) and/or within the lipid layer/membrane of the lipid-based carriers.
  • the lipid-based carrier is selected from a lipid nanoparticle (LNP), a liposome, a lipoplex, a solid lipid nanoparticle, a lipo-polyplex, and/or a nanoliposome.
  • the lipid-based carrier is a lipid nanoparticle (LNP).
  • LNPs are microscopic lipid particles having a solid or partially solid core. Typically, an LNP does not comprise an interior aqua space sequestered from an outer medium by a bilayer.
  • the nucleic acid may be encapsulated in the lipid portion of the LNP, enveloped by some or the entire lipid portion of the LNP.
  • An LNP may comprise any lipid capable of forming a particle to which the nucleic add such as the RNA may be attached, or in which the nucleic add such as the RNA may be encapsulated.
  • the lipid-based carrier preferably the LNP, comprise at least one or more lipids selected from at least one aggregation-reducing lipid, at least one cationic lipid or ionizable lipid, at least one neutral lipid or phospholipid, or at least one steroid or steroid analog, or any combinations thereof.
  • the lipid-based carriers, preferably the LNPs comprise (i) at least one aggregation-reducing lipid, (ii) at least one cationic lipid or ionizable lipid, (iii) at least one neutral lipid or phospholipid, (iv) and at least one steroid or steroid analog.
  • the lipid-based carrier comprises at least one cationic or ionizable lipid.
  • the at least one cationic or ionizable lipid may be cationisable or ionizable, i.e. it becomes protonated as the pH is lowered below the pK of the ionizable group of the lipid but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids.
  • the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.
  • the at least one cationic or ionizable lipid may carry a net positive charge at physiological pH.
  • the cationic or ionizable lipid comprises a tertiary nitrogen group or quaternary nitrogen group, most preferably a tertiary nitrogen group.
  • the at least one cationic or ionizable lipid may be selected from an amino lipid.
  • the at least one cationic lipid or ionizable lipid is selected from an amino lipid, preferably wherein the amino lipid comprises a tertiary amine group.
  • the at least one cationic or ionizable lipid selected or derived from formula HI-3 of WO2018078053 has the chemical term ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl) bis(2- hexyldecanoate), also referred to as ALC-0315, i.e. CAS Number 2036272-55-4.
  • suitable cationic or ionizable lipids may be selected or derived from lipids according to PCT claims 1 -14 of WO2021123332, or Table 1 of WO2021123332, the disclosure relating thereto herewith incorporated by reference. Accordingly, suitable cationic or ionizable lipids may be selected or derived from lipids according to Compound 1 to Compound 27 (C1-C27) of Table 1 of WO2021123332. In embodiments, the at least one cationic or ionizable lipid is selected or derived from SS-33/4PE-15 (see C23 in Table 1 of WO2021123332).
  • the at least one cationic or ionizable lipid is selected or derived from HEXA-C5DE-PipSS (see C2 in Table 1 of WO2021123332). In other embodiments, the at least one cationic or ionizable lipid is selected or derived from compound C26 (VitE-C4DE- Pip-thioether) as disclosed in Table 1 of WO2021123332.
  • the lipid-based carrier comprises at least one neutral lipid or phospholipid.
  • the lipid-based carrier comprises a steroid, steroid analog or sterol.
  • the steroid or steroid analog is selected or derived from cholesterol, cholesteryl hemisucdnate (CHEMS), or any derivate of these.
  • the steroid, steroid analog or sterol is cholesterol.
  • the lipid-based carriers comprise at least one aggregation reducing lipid or moiety.
  • aggregation reducing moiety refers to a molecule comprising a moiety suitable of reducing or preventing aggregation of the lipid-based carrier.
  • aggregation reducing lipid refers to a molecule comprising both a lipid portion and a moiety suitable of reducing or preventing aggregation of the lipid-based carriers.
  • the lipid-based carriers may undergo charge-induced aggregation, a condition which can be undesirable for the stability of the lipid-based carriers. Therefore, it can be desirable to include a compound or moiety which can reduce aggregation, e.g. by sterically stabilizing the lipid-based carriers.
  • Such a steric stabilization may occur when a compound having a sterically bulky but uncharged moiety that shields or screens the charged portions of a lipid- based carriers from close approach to other lipid-based carriers in the composition.
  • stabilization of the lipid-based carriers is achieved by including lipids which may comprise a lipid bearing a sterically bulky group which, after formation of the lipid-based carrier, is preferably located on the exterior of the lipid-based carrier.
  • Suitable aggregation reducing groups include hydrophilic groups, e.g. monosialoganglioside GM1 , polyamide oligomers (PAO), or certain polymers, such as poly(oxyalkylenes), e.g., polyethylene glycol) or polypropylene glycol).
  • hydrophilic groups e.g. monosialoganglioside GM1 , polyamide oligomers (PAO), or certain polymers, such as poly(oxyalkylenes), e.g., polyethylene glycol) or polypropylene glycol).
  • the aggregation reducing lipid is a polymer conjugated lipid.
  • polymer conjugated lipid refers to a molecule comprising both a lipid portion and a polymer portion, wherein the polymer is suitable of reducing or preventing aggregation of lipid-based carriers comprising the nucleic acid.
  • a polymer has to be understood as a substance or material consisting of very large molecules, or macromolecules, composed of many repeating subunits.
  • a suitable polymer in the context of the invention may be a hydrophilic polymer.
  • An example of a polymer conjugated lipid is a PEGylated or PEG-conjugated lipid.
  • the polymer conjugated lipid is selected from a PEG-conjugated lipid or a PEG-free lipid.
  • the polymer conjugated lipid is a PEG-conjugated lipid.
  • the average molecular weight of the PEG moiety in the PEG-conjugated lipid may ranges from 500 to 8,000 Daltons (e.g., from 1 ,000 to 4,000 Daltons). In one preferred embodiment, the average molecular weight of the PEG moiety is about 2,000 Daltons.
  • the PEG-conjugated lipid is selected or derived from 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000 DMG or DMG-PEG 2000), C10-PEG2K, or Cer8-PEG2K.
  • the polymer conjugated lipid is selected or derived from formula (IV) of WO2018078053, preferably from formula (IVa) of WO2018078053.
  • a preferred polymer-conjugated lipid is selected from ALC-0159 (2[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide).
  • the aggregation reducing lipid is selected from a PEG-free lipid, e.g. a PEG-free polymer conjugated lipid.
  • the aggregation reducing lipid is a PEG-free lipid that comprises a polymer different from PEG.
  • a PEG-free polymer conjugated lipid may be selected or derived from a “POZ-lipid”.
  • POZ lipid or respectively preferred polymer conjugated lipids are described in W02023031394, the full disclosure herewith incorporated by reference.
  • disclosure relating to polymer conjugated lipids as defined in any one of claims 1 to 8 of WO2023031394 is herewith incorporated by reference.
  • the polymer conjugated lipid is a PEG-free lipid selected from a POZ-lipid.
  • the aggregation-reducing lipid is selected or derived from PMOZ 1 , PMOZ 2, PMOZ 3, PMOZ 4, or PMOZ 5 of W02023031394.
  • the polymer conjugated lipid is selected or derived from PMOZ 4 according to formula “PMOZ4” of W02023031394, herewith incorporated by reference.
  • Lipid-based carrier compositions Lipid-based carrier compositions
  • the lipid-based carrier comprises at least one nucleic acid, preferably at least one RNA encoding RUNX3 as defined herein, a cationic or ionizable lipid as defined herein, an aggregation reducing lipid as defined herein, a neutral lipid as defined herein, and a steroid or steroid analog as defined herein.
  • the lipid-based carrier preferably the LNP, comprising the nucleic acid, preferably the RNA, comprise
  • the lipid-based carrier preferably the LNP, comprising the nucleic acid, preferably the RNA, comprise
  • At least one cationic lipid selected or derived from ALC-0315, SM-102, SS-33/4PE-15, HEXA-C5DE-PipSS or C26;
  • the nucleic acid e.g. the RNA
  • the lipid-based carrier preferably the LNP, comprises (i) to (iv) in a molar ratio of about 20-60% cationic lipid or ionizable lipid, about 5-25% neutral lipid, about 25-55% steroid or steroid analogue, and about 0.5-15% aggregation reducing lipid e.g. polymer conjugated lipid.
  • the lipid-based carrier preferably the LNP, comprise (i) to (iv) in a molar ratio of about 45- 60% cationic lipid or ionizable lipid, about 5-15% neutral lipid, about 25-45% steroid or steroid analog, and about 0.5- 2.5% aggregation reducing lipid e.g. polymer conjugated lipid.
  • the lipid-based carrier preferably the LNP, comprising the nucleic add, preferably the RNA, comprise
  • lipid-based carrier encapsulates the nucleic acid, preferably the RNA.
  • the amount of lipid comprised in the lipid-based carrier such as LNP may be selected taking the amount of the nucleic acid cargo into account. These amounts are suitably selected such as to result in an N/P ratio of the lipid-based carriers encapsulating the nucleic acid in the range of about 0.1 to about 20.
  • the N/P ratio is defined as the mole ratio of the nitrogen atoms (“N”) of the basic nitrogen-containing groups of the lipid to the phosphate groups (“P”) of the nucleic acid which is used as cargo.
  • the N/P ratio may be calculated on the basis that, for example, 1 pg nucleic acid typically contains about 3nmol phosphate residues, provided that the nucleic acid exhibits a statistical distribution of bases.
  • the “N”-value of the lipid or lipidoid may be calculated on the basis of its molecular weight and the relative content of permanently cationic and - if present - cationisable groups.
  • the N/P ratio can be in the range of about 1 to about 50. In other embodiments, the range is about 5 to about 20. In some embodiments, the N/P ratio is at about 17. In some embodiments, the N/P ratio is at about 14. In some embodiments, the N/P ratio is at about 6.
  • the lipid-based carrier as defined herein such as the LNP as defined herein have a defined size (particle size, homogeneous size distribution).
  • the size of a lipid-based carrier such as an LNP is typically described as Z-average size.
  • Z-average size refers to the mean diameter of particles as measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z- average with the dimension of a length, and the polydispersity index (PDI), which is dimensionless.
  • DLS dynamic light scattering
  • PDI polydispersity index
  • DLS dynamic light scattering
  • the lipid-based carrier preferably the LNP, has a Z-average size ranging from about 50nm to about 200nm, preferably from about 50nm to about 150nm, more preferably from about 50nm to about 120nm.
  • the polydispersity index (PDI) of the lipid-based carriers is typically in the range of 0.1 to 0.5. In a particular embodiment, a PDI is below 0.2. Typically, the PDI is determined by dynamic light scattering.
  • At least 70%, 80%, 90%, 95% of the nucleic acid molecules are encapsulated in a lipid-based carrier such as an LNP.
  • the percentage of encapsulation may be determined by a RiboGreen assay as known in the art.
  • the plurality of lipid-based carriers have a lamellar morphology and/or a bilayer morphology. In embodiments, at least 80%, 85%, 90%, 95% of the lipid-based carriers have a spherical morphology.
  • the surface of the lipid-based carrier preferably the LNP, is uncharged at pH 7.
  • the pharmaceutical composition additionally comprises a RUNX1 inhibitor selected from a small molecule inhibitor of RUNX1 , an inhibitory nucleic acid (siRNA) of RUNX1 , or a nucleic add encoding a RUNX1 inhibitor.
  • a RUNX1 inhibitor selected from a small molecule inhibitor of RUNX1 , an inhibitory nucleic acid (siRNA) of RUNX1 , or a nucleic add encoding a RUNX1 inhibitor.
  • RUNX1 inhibitors may be used that are provided in WO2019099560, WO2018093797, WO2019099595, and WO2021216378, the frill disclosure herewith incorporated by reference.
  • a suitable RUNX1 inhibitors is the small molecule ro5-3335 (see e.g. WO2018093797).
  • the CAS Registry Number for Ro5-3335 is 30195-30-3.
  • the composition additionally comprises a RUNX1 transcription factor inhibitor that is an RNA encoding a CBFbeta-SMMHC fusion protein according to WO2023144330.
  • the composition may additionally comprise an RNA encoding an RUNX1 -trap, wherein the RNA comprises or consists of a nucleic acid sequence which is identical or at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 820, 1579, 1581 , 910, 1580 or 1582 of WO2023144330 (referred as CBFB(1- 165)-SMMHC(1527-1877) or CBFbeta-SMMHCAC95), or a fragment or variant of that sequence.
  • the composition comprises an anti- inflammatory agent that suitably comprises a steroid or a nonsteroidal anti-inflammatory drug (NSAID).
  • NSAID nonsteroidal anti-inflammatory drug
  • the encoded RUNX3 transcription factor as defined herein is produced in said cell, tissue, or subject.
  • the RUNX3 transcription factor or a fragment or variant thereof is produced, preferably in an amount sufficient for reducing and/or inhibiting the activity of RUNX1 in said cell, tissue, or subject.
  • the RUNX3 transcription factor or a fragment or variant thereof is produced, preferably in an amount sufficient for reducing and/or inhibiting the activity of RUNX1 in said cell, tissue, or subject.
  • the pharmaceutical composition or nucleic acid is administered via ocular administration, wherein the ocular administration is selected from topical, intravitreal, intracameral, subconjunctival, into the ciliary body, subretinal, subtenon, retrobulbar, retronasa, orbital, topical, suprachoroidal, posterior juxtascleral, or intraoperative administration (e.g. during an ocular surgery).
  • ocular administration is selected from topical, intravitreal, intracameral, subconjunctival, into the ciliary body, subretinal, subtenon, retrobulbar, retronasa, orbital, topical, suprachoroidal, posterior juxtascleral, or intraoperative administration (e.g. during an ocular surgery).
  • ocular administration is selected from intravitreal or intraoperative administration.
  • Intravitreal administration e.g. via injection is one of the most common ways of administering a medicament into an eye. Accordingly, administration of the pharmaceutical composition or nucleic acid (e.g. an RNA encoding a RUNX3 transcription factor) via intravitreal administration is preferred in many medical applications in the context of eye diseases.
  • a preferred injection volume of the pharmaceutical composition is ranging from about 25pl to about 150pl, preferably from about 25pl to about 10OpI, more preferably from about 50pl to about 10Opl. In a particularly preferred embodiment, the injection volume is about 50pl.
  • the ocular administration is intraoperative administration.
  • Some disease, disorders or conditions in the eye occur after an ocular surgery or operation (e.g. PVR).
  • administration of the pharmaceutical composition or nucleic add e.g. an RNA encoding a RUNX3 transcription factor
  • intraoperative administration is preferred in medical applications where a disease, disorders or condition occurs after an ocular surgery or operation (e.g. PVR).
  • ocular administration of the pharmaceutical composition orthe nucleic acid leads to a production of the RUNX3 transcription factor in cells and/or tissues of the eye, preferably in cells and/or tissues selected from cornea, lens, ciliary body, vitreous, sclera, choroid, retina, optic nerve, macula, scleral cells, choroid cells, retinal cells, inflammatory cells, retinal pigment epithelium (RPE), Muller cells, microglia, photoreceptors, amacrine cells, choroidal melanocytes retinal ganglion cells, horizontal cells, bipolar cells, astrocytes, vitreous, trabecular mesh, conjunctiva, corneal endothelium, Bruch’s membrane, conjunctiva, and retinal or choroidal blood vessels or hyaloid vessels; or in cells of the brain comprising choroid plexus epithelial cells.
  • cells and/or tissues selected from cornea, lens, ciliary body, vitreous, scler
  • ocular administration of the pharmaceutical composition or the nucleic acid leads to a production of the RUNX3 transcription factor in retinal pigment epithelium (RPE) cells or cells derived from RPE cells.
  • RPE retinal pigment epithelium
  • the retinal pigment epithelium is the pigmented cell layer just outside the neurosensory retina that nourishes retinal visual cells and is firmly attached to the underlying choroid and overlying retinal visual cells.
  • the RPE forms a monolayer of cells beneath the sensory retina that is normally mitotically inactive except when it is participating in retinal wound repair, where it plays a central role.
  • the RPE usually stops proliferating; failure to do so can result in blinding disorders such as e.g. PVR or Epiretinal Membranes (ERM)and disciform scarring. For instance, after detachment of the sensory retina, the RPE changes in morphology and begins to proliferate.
  • Multi-layered colonies of dedifferentiated and transdifferentiated RPE cells are formed.
  • cells migrate onto the surface of the retina and form epiretinal membranes. These events have been implicated in the pathogenesis of proliferative vitreoretinopathy, severe scarring occurring in association with exudative macular degeneration, and poor or delayed recovery of vision after retinal reattachment.
  • ocular administration of the pharmaceutical composition or the nucleic acid reduces or prevents pathological EMT, pathological cell proliferation, aberrant angiogenesis, aberrant neovascularization, degeneration, and/or fibrosis. Accordingly, the administration leads to a production of the RUNX3 transcription factor which reduces or prevents pathological EMT, pathological cell proliferation, aberrant angiogenesis, aberrant neovascularization, degeneration, and/or fibrosis.
  • the ocular administration of the pharmaceutical composition or the nucleic acid is performed into a tamponade agent-filled human eye, a silicone-fi lied human eye, or a gas-filled human eye.
  • the ocular administration of the pharmaceutical composition or the nucleic add is performed after tamponade, gas or silicone extraction.
  • the invention provides a kit or kit of parts comprising at least one nucleic acid of the first aspect or at least one pharmaceutical composition of the second aspect, e.g., for use in a method described herein.
  • the kit may further comprise additional components as described in the context of the pharmaceutical composition of the second aspect.
  • kits may contain information about administration and dosage and patient groups.
  • kits preferably kits of parts, may be applied e.g. for any of the applications or uses mentioned herein, preferably for the use of the nucleic add of the first aspect or the pharmaceutical composition of the second aspect for the treatment or prophylaxis of diseases, disorder, or condition.
  • the kit or kit of parts as defined herein comprises at least one syringe or application device.
  • a syringe or application device for ocular delivery e.g. intravitreal delivery.
  • the present invention relates to the medical use of the nucleic acid as defined herein, the pharmaceutical composition as defined herein, or the kit or kit of parts as defined herein.
  • embodiments relating to any of the previous aspects may likewise be read on and be understood as suitable embodiments of medical uses of the invention.
  • embodiments relating to medical uses as described herein of course also relate to methods of treatments (fifth aspect).
  • the invention provides a nucleic acid encoding a RUNX3 transcription factor of the first aspect, a pharmaceutical composition comprising a nucleic acid encoding a RUNX3 transcription factor of the second aspect, or a kit or kit of the third aspect, for use as a medicament in treating or preventing a disease, disorder, or condition in a subject.
  • the RUNX3 transcription factor comprises or consists of an amino acid sequence being identical or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 129-163, or a fragment or variant thereof, preferably an amino acid sequence being identical or at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 129.
  • the nucleic add preferably the mRNA encoding the RUNX3 transcription factor comprises or consists of a nucleic acid sequence which is identical or at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence SEQ ID NOs: 269-458, 462-462, or a fragment or variant of that sequence, preferably an mRNA sequence that is identical or at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the nucleic acid sequences SEQ ID NOs: 456 or 462.
  • the nucleic of the pharmaceutical composition is an mRNA encapsulated in a lipid-based carrier as defined herein, preferably an LNP as defined herein.
  • the use may be for human medical purposes and also for veterinary medical purposes, preferably for human medical purposes. In other embodiments, the use may be for human medical purposes, in particular for young infants, newborns, immunocompromised recipients, pregnant and breast-feeding women, and elderly people.
  • the nucleic acid, the pharmaceutical composition, or the kit or kit of parts is administered by intramuscular, intranasal, transdermal, intradermal, intralesional, intracranial, subcutaneous, intracardial, intratumoral, intravenous, intrapulmonal, intraarticular, sublingual, pulmonary, intrathecal, or ocular administration.
  • the present invention provides a nucleic add of the first aspect, or a pharmaceutical composition of the second aspect, or a kit or kit of parts of the third aspect, for use as a medicament in treating or preventing an ocular disease, disorder, or condition in a subject.
  • the disease, disorder, or condition is associated with or caused by an overexpressed and/or an overactive RUNX1 transcription factor.
  • administration of a nucleic acid encoding RUNX3 leads to a reduction of the cellular expression of EMT-assodated genes including TGFbeta2, SMAD3, and/or COL1A1 .
  • administration of a nucleic acid encoding RUNX3 leads to a reduction of the EMT markers and pathological cell proliferation.
  • treatment with nucleic add encoding the RUNX3 transcription factor also promote wound healing. This was illustrated by a reduction of fibroblast, which are increased to tissue damage and play a critical role in wound healing, following administration of nucleic acid encoding the RUNX3 transcription factor.
  • the disease, disorder, or condition is associated with or caused by a downregulated and/or inhibited RUNX3, e.g. human autoimmune diseases, cancer, chronic inflammatory diseases (e.g. colitis) or inflammation.
  • a downregulated and/or inhibited RUNX3 e.g. human autoimmune diseases, cancer, chronic inflammatory diseases (e.g. colitis) or inflammation.
  • the present invention provides an nucleic acid of the first aspect, or a pharmaceutical composition of the second aspect, or a kit or kit of parts of the third aspect, for use as a medicament in treating or preventing a disease, disorder, or condition that is associated with or caused by pathological EMT, pathological cell proliferation, aberrant angiogenesis, aberrant neovascularization, degeneration, fibrosis and/or solid tumors and/or aberrant proliferation and migration of RPE cells in a subject.
  • EMT-assodated diseases include pathologic ocular fibrosis and proliferation, for example PVR, conjunctival fibrosis (e.g. ocular dcatricial pemphigoid), corneal scarring, corneal epithelial down growth, and/or aberrant fibrosis, diseases in the anterior segment of the eye (e.g., comeal opadfication and glaucoma), corneal dystrophies, herpetic keratitis, inflammation (e.g., pterygium), macula edema, retinal and vitreous hemorrhage, fibrovascular scarring, neovascular glaucoma, age-related macular degeneration (ARMD), geographic atrophy, diabetic retinopathy (DR), retinopathy of prematurity (ROP), subretinal fibrosis, epiretinal fibrosis, and gliosis.
  • pathologic ocular fibrosis and proliferation for example PVR,
  • EMT epithelial graft-versus-host disease
  • corneal scarring corneal epithelial downgrowth
  • conjunctival scarring eye tumors such as melanoma and metastatic tumors, or fibrosis.
  • the present invention provides an nucleic acid of the first aspect, or a pharmaceutical composition of the second aspect, or a kit or kit of parts of the third aspect, for use as a medicament in treating or preventing a disease, disorder, or condition that is associated with or caused by aberrant angiogenesis.
  • angiogenesis is observed in numerous diseases, such as proliferative diabetic retinopathy, ROP, DR, AMD, retinal vein occlusions, ocular ischemic syndrome, neovascular glaucoma, retinal hemangiomas, and cancer (especially in solid tumors) and cerebral small vessel disease. It is also observed in genetic diseases such as Coats’ disease,
  • Aberrant angiogenesis includes any angiogenesis that is not a normal (nonpathological) part of an organism’s development, growth, or healing.
  • Ocular neovascularization includes retinal neovascularization as well as neovascularization in the anterior segment of the eye.
  • aberrant angiogenesis may manifest itself as anterior ocular neovascularization, e.g., aberrant angiogenesis that occurs as a part of corneal graft rejection. Corneal angiogenesis is involved in corneal graft rejection.
  • Any disease, disorder, or condition associated with or caused by pathological EMT, pathological cell proliferation, aberrant angiogenesis, aberrant neovascularization, and/or fibrosis may be inhibited, treated, or prevented using methods and compositions disclosed herein, e.g. using the nucleic acid or composition encoding a RUNX3 transcription factor.
  • RPE cells may be misplaced from their anatomical location and induced to undergo EMT under the stimuli of growth factors, inflammatory cytokines, and exposure to vitreous, a collagenous gel that fills the space between the lens and the retina.
  • EMT of RPE cells plays a critical role in the pathobiology of PVR.
  • the ocular disease, disorder or condition may be selected from, neovascularization, retinal degenerative disease, diabetic eye disease, retinal detachment, optic nerve disease, endocrine disorders, cancer disease, infectious disease, parasitic disease, in particular, pigmentary uveitis (PU), branch retinal vein occlusion (BRVO), central retinal vein occlusion (CRVO), macular edema, cystoid macular edema (CME), uveitic macular edema (UME), cytomegalovirus retinitis, endophthalmitis, scleritis, choriotetinitis, dry eye syndrome, Norris disease, Coat's disease, persistent hyperplastic primary vitreous, familial exudative vitreoretinopathy, Leber congenital amaurosis, X- linked retinoschisis, Leber's hereditary optic neurophathy, uveitis, refraction and accommodation disorders, kera
  • the ocular disease, disorder, or condition is selected from proliferative diabetic retinopathy (PDR), macular edema, non-proliferative diabetic retinopathy, age-related macular degeneration, geographic atrophy, ocular neovascularization, retinopathy of prematurity (ROP), a retinal vein occlusion, ocular ischemic syndrome, neovascular glaucoma, a retinal hemangioma, Coats' disease, FEVR, or Norrie disease, Von Hippel-Lindau disease or persistent hyperplastic primary vitreous (PHPV), or epiretinal membrane (ERM), small vessel disease, thyroid eye disease, induction of epithelial cell differentiation, osteoarthritis, ocular fibrosis, retinal degeneration, osteoporosis, cancer or metastasis, or PVR.
  • PDR proliferative diabetic retinopathy
  • macular edema macular edema
  • the ocular disease, disorder, or condition is selected from age-related macular degeneration (AMD).
  • AMD is an eye disease that is a leading cause of vision loss in older people in developed countries. The vision loss usually becomes noticeable in a person's sixties or seventies and tends to worsen overtime. AMD mainly affects central vision. The vision loss in this condition results from a gradual deterioration of light-sensing cells in the tissue at the back of the eye that detects light and colour (the retina). Specifically, AMD affects a small area near the center of the retina, called the macula, which is responsible for central vision. Side (peripheral) vision and night vision are generally not affected.
  • the dry form is much more common, accounting for 85 to 90 percent of all cases of AMD. It is characterized by a build-up of yellowish deposits called drusen beneath the retina and slowly progressive vision loss. The condition typically affects vision in both eyes, although vision loss often occurs in one eye before the other.
  • the wet form of age-related macular degeneration is associated with severe vision loss that can worsen rapidly. This form of the condition is characterized by the growth of abnormal, fragile blood vessels underneath the macula. These vessels leak blood and fluid, which damages the macula and makes central vision appear blurry and distorted. Any symptom, type, or stage of AMD may be inhibited, treated, or prevented using methods and compositions disclosed herein, e.g. using the nucleic acid or composition encoding a RUNX3 transcription factor.
  • the ocular disease, disorder, or condition is selected from diabetic retinopathy.
  • Diabetic retinopathy is a condition that occurs in people who have diabetes. It causes progressive damage to the retina, which is the light-sensitive lining at the back of the eye. Over time, diabetes damages the blood vessels in the retina. Diabetic retinopathy occurs when these tiny blood vessels leak blood and other fluids. This causes the retinal tissue to swell, resulting in cloudy or blurred vision. The condition usually affects both eyes. The longer a person has diabetes, without being properly treated, the more likely they will develop diabetic retinopathy. If left untreated, diabetic retinopathy can cause blindness.
  • Symptoms of diabetic retinopathy include (i) seeing spots or floaters; (ii) blurred vision; (iii) having a dark or empty spot in the center of vision; and (iv) difficulty seeing well at night. Often the early stages of diabetic retinopathy have no visual symptoms. Early detection and treatment can limit the potential for significant vision loss from diabetic retinopathy. PDR is a more advanced form of the disease. At this stage, new fragile blood vessels can begin to grow in the retina and into the vitreous. The new blood vessels may leak blood into the vitreous, clouding vision. Without wishing to be bound by any scientific theory, diabetic retinopathy results from the damage diabetes causes to the small blood vessels located in the retina. These damaged blood vessels can cause vision loss.
  • Diabetic retinopathy is classified into two types: (1) Non-proliferative diabetic retinopathy (PDR) is the early stage of the disease in which symptoms will be mild or nonexistent. In NPDR, the blood vessels in the retina are weakened. Tiny bulges in the blood vessels, called microaneurysms, may leak fluid into the retina.
  • PDR Non-proliferative diabetic retinopathy
  • PDR is the more advanced form of the disease. At this stage, circulation problems deprive the retina of oxygen. As a result, new, fragile blood vessels can begin to grow in the retina and into the vitreous, the gel-like fluid that fills the eye. The new blood vessels may leak blood into the vitreous, clouding vision. Both NPDR and PDR may also result in macular edema. Other complications of PDR include detachment of the retina due to scar tissue formation and the development of neovascular glaucoma. Glaucoma is an eye disease in which there is progressive damage to the optic nerve. In PDR, new blood vessels grow into the area of the eye that drains fluid from the eye.
  • retinopathy of prematurity is a potentially blinding eye disorder that primarily affects premature infants. The smaller a baby is at birth, the more likely that baby is to develop ROP.
  • Retinal detachment is the main cause of visual impairment and blindness in ROP. Without wishing to be bound by any scientific theory, several complex factors may be responsible for the development of ROP.
  • the eye starts to develop at about 16 weeks of pregnancy, when the blood vessels of the retina begin to form at the optic nerve in the back of the eye. The blood vessels grow gradually toward the edges of the developing retina, supplying oxygen and nutrients. During the last 12 weeks of a pregnancy, the eye develops rapidly. When a baby is born full-term, the retinal blood vessel growth is mostly complete (the retina usually finishes growing a few weeks to a month after birth). If a baby is born prematurely, before these blood vessels have reached the edges of the retina, normal vessel growth may stop.
  • aspects of the present invention relate to inhibiting, preventing, or treating the onset of or the progression of a ROP in a premature infant using a nucleic acid or composition of the invention. Any symptom or stage of ROP may be inhibited, treated, or prevented using methods and compositions disclosed herein, e.g. using the nucleic acid or composition encoding a RUNX3 transcription factor.
  • Risk factors for RVO include: (i) atherosclerosis; (ii) diabetes; (iii) high blood pressure (hypertension, e.g., a systolic pressure of at least 140 mmHg or a diastolic pressure of at least 90 mmHg); and (iv) other eye conditions, such as glaucoma, macular edema, or vitreous hemorrhage.
  • the risk of these disorders increases with age, therefore RVO most often affects older people.
  • Blockage of retinal veins may cause other eye problems, including: (i) glaucoma (high pressure in the eye), caused by new, abnormal blood vessels growing in the front part of the eye; (ii) neovascularization.
  • RVO can cause the retina to develop new, abnormal blood vessels, a condition called neovascularization. These new vessels may leak blood or fluid into the vitreous, the jelly-like substance that fills the inside of the eye. Small spots or clouds, called floaters, may appear in the field of vision. With severe neovascularization, the retina may detach from the back of the eye.); (iii) macular edema, caused by the leakage of fluid in the retina; and (iv) neovascular glaucoma (New blood vessels in certain parts of the eye can cause pain and a dangerous increase in pressure inside the eye.). Any symptom, type, or stage of RVO may be inhibited, treated, or prevented using methods and compositions disclosed herein, e.g.
  • the ocular disease, disorder, or condition is selected from ocular ischemic syndrome (OIS).
  • OIS encompasses the ocular signs and symptoms that result from chronic vascular insufficiency.
  • Common anterior segment findings include advanced cataract, anterior segment inflammation, and iris neovascularization.
  • Posterior segment signs include narrowed retinal arteries, dilated but no tortuous retinal veins, midperipheral dot-and-blot retinal haemorrhages, cotton-wool spots, and optic nerve/retinal neovascularization.
  • the presenting symptoms include ocular pain and abrupt or gradual visual loss.
  • OIS ocular ischemic syndrome
  • a symptom, type, or stage of ocular ischemic syndrome may be inhibited, treated, or prevented using methods and compositions disclosed herein, e.g. using the nucleic acid or composition encoding a RUNX3 transcription factor.
  • the ocular disease, disorder, or condition is selected from neovascular glaucoma (NVG).
  • NVG neovascular glaucoma
  • This angiogenesis factor causes new blood vessel growth from pre-existing vascular structure.
  • it can cause glaucoma either through secondary open-angle or secondary closed-angle mechanisms. This is accomplished through the growth of a fibrovascular membrane over the trabecular meshwork in the anterior chamber angle, resulting in obstruction of the meshwork and/or associated peripheral anterior synechiae.
  • NVG is a potentially devastating glaucoma, where delayed diagnosis or poor management can result in complete loss of vision or, quite possibly, loss of the globe itself.
  • IOP intraocular pressure
  • Retinal ischemia is the most common and important mechanism in most, if not all, cases that result in the anterior segment changes causing NVG.
  • Various predisposing conditions cause retinal hypoxia and, consequently, production of an angiogenesis factor. Once released, the angiogenic factors) diffuses into the aqueous and the anterior segment and interacts with vascular structures in areas where the greatest aqueous-tissue contact occurs.
  • fibrovascular membranes which may be invisible on gonioscopy, accompany NVA and progressively obstruct the trabecular meshwork. This causes secondary open-angle glaucoma.
  • the fibrovascular membranes along the NVA tend to mature and contract, thereby tenting the iris toward the trabecular meshwork and resulting in peripheral anterior synechiae and progressive synechial angle closure. Elevated IOP is a direct result of this secondary angle-closure glaucoma. Any symptom, type, or stage of neovascular glaucoma may be inhibited, treated, or prevented using methods and compositions disclosed herein, e.g. using the nucleic acid or composition encoding a RUNX3 transcription factor.
  • the ocular disease, disorder, or condition is selected from retinal hemangiomas.
  • Retinal hemangiomas also known as retinal capillary hemangiomas (RCHs) and retinal hemangioblastomas, occur most frequently in conjunction with von Hippel-Lindau (VHL) syndrome. These lesions are characterized by plump, but otherwise normal, retinal capillary endothelial cells with normal pericytes and basement membrane. Astrocytes with lipid vacuoles are found in the tumor interstitia. Isolated RCH outside of VHL do occur, although they are more likely to be single, unilateral, and present later.
  • RCHs retinal capillary hemangiomas
  • VHL von Hippel-Lindau
  • Von Hippel-Lindau syndrome has an autosomal dominant inheritance pattern, with an incidence of 1 in 36,000 live births. These lesions can occur either singly, or more often, multiply and bilaterally, with a greater than 80% predilection for peripheral location. Vision loss can occur from exudation, strabismus, hemorrhage, and retinal detachment, as well secondary causes such as macular edema, lipid maculopathy, and epiretinal membrane. Early lesions often present as indistinct areas of redness in the retina, which appear to be retinal hemorrhages.
  • a subject at risk of developing a retinal hemangioma such as a subject with VHL, is treated to delay or prevent the onset of a retinal hemangioma, e.g. using the nucleic acid or composition encoding a RUNX3 transcription factor.
  • the ocular disease, disorder, or condition is selected from Coats’ disease.
  • Coats’ disease also known as exudative retinitis or retinal telangiectasis, sometimes spelled Coates' disease
  • Coats’ disease is a rare congenital, nonhereditary eye disorder, causing full or partial blindness, characterized by abnormal development of blood vessels behind the retina.
  • Coats’ disease results in a gradual loss of vision. Blood leaks from the abnormal vessels into the back of the eye, leaving behind cholesterol deposits and damaging the retina.
  • Coats’ disease normally progresses slowly. At advanced stages, retinal detachment is likely to occur. Glaucoma, atrophy, and cataracts can also develop secondary to Coats’ disease.
  • Coats disease Initially, these may be mistaken for psychological hallucinations, but are actually the result of both retinal detachment and foreign fluids mechanically interacting with the photoreceptors located on the retina.
  • One early warning sign of Coats’ disease is yellow-eye in flash photography. An eye affected by Coats’ will glow yellow in photographs as light reflects off cholesterol deposits. Coats’ disease is thought to result from breakdown of the blood-retinal barrier in the endothelial cell, resulting in leakage of blood products containing cholesterol crystals and lipid-laden macrophages into the retina and subretinal space. Over time, the accumulation of this proteinaceous exudate thickens the retina, leading to massive, exudative retinal detachment.
  • a subject at risk of developing Coats' disease is treated to delay or prevent the onset of Coats' disease, e.g. using the nucleic acid or composition encoding a RUNX3 transcription factor.
  • the ocular disease, disorder, or condition is selected from Norrie disease.
  • Norrie disease is an inherited eye disorder that leads to blindness in male infants at birth or soon after birth. It causes abnormal development of the retina, the layer of sensory cells that detect light and colour, with masses of immature retinal cells accumulating at the back of the eye. As a result, the pupils appear white when light is shone on them, a sign called leukocoria.
  • the irises (coloured portions of the eyes) or the entire eyeballs may shrink and deteriorate during the first months of life, and cataracts (cloudiness in the lens of the eye) may eventually develop. About one third of individuals with Norrie disease develop progressive hearing loss, and more than half experience developmental delays in motor skills such as sitting up and walking.
  • NDP norrin cystine knot growth factor
  • the NDP gene provides instructions for making a protein called norrin. Mutations in the Norrie gene are often unique to a family and have been described throughout the extent of the Norrie gene. Although Norrie disease itself does not seem to shorten lifespan, individuals with blindness, deafness and/or mental disability may have a reduced lifespan as a result of these conditions. Any symptom, type, or stage of Norrie disease may be inhibited, treated, or prevented using methods and compositions disclosed herein, e.g. using the nucleic acid or composition encoding a RUNX3 transcription.
  • the ocular disease, disorder, or condition is selected from familial exudative vitreoretinopathy (FEVR).
  • FEVR is a rare hereditary ocular disorder characterized by a failure of peripheral retinal vascularization which may be abnormal or incomplete.
  • FEVR is a condition with fundus changes similar to those in retinopathy of prematurity but appearing in children who had been born full-term with normal birthweight. With respect to genetics, about 50% of cases can be linked to 4 causative genes (DP, LRP5, FZD4, and TSPAN12), all of which form part of the Wnt signalling pathway, which is vital for normal retinal vascular development.
  • any symptom, type, or stage of FEVR may be inhibited, treated, or prevented using methods and compositions disclosed herein.
  • a subject at risk of developing FEVR is treated to delay or prevent the onset or progression of FEVR.
  • a subject at risk of developing FEVR is treated to delay or prevent the onset or progression of aberrant angiogenesis due to FEVR, e.g. using the nucleic add or composition encoding a RUNX3 transcription factor.
  • PVR occurs in about 8-10% of patients undergoing primary retinal detachment surgery and prevents the successful surgical repair of rhegmatogenous retinal detachment. PVR can be treated with surgery to reattach the retina, however, the visual outcome of the surgery is very poor. If PVR is progressive, then despite complex surgery, low vision in the eye results. PVR is characterized by proliferation or migration of cells derived from RPE, glia, or inflammatory recruitment on the retinal surface and within the vitreous. These cells transdifferentiate and take on contractile properties. The process of PVR can start when there is an interruption to the surface lining (e.g., through posterior vitreous detachment and local preretinal membrane formation or retinal tears in the periphery).
  • the PVR process is selfpropagating and is often considered an inappropriate excess wound-healing response.
  • the cellular proliferation can increase the influx of inflammatory cytokines and inflammatory cells.
  • proliferation or migration of RPE cells describes their transdifferentiation to assume contractile properties through internal cellular contractile proteins and by laying down extracellular collagen.
  • the cells can multiply and grow along any available scaffolding (e.g., the retinal surfaces or elements of the residual vitreous).
  • the mass contraction can lead to retinal wrinkles, folds, tears, and traction retinal detachment.
  • fluid from the vitreous humor enters a retinal hole.
  • the accumulation of fluid in the subretinal space and the fractional force of the vitreous on the retina result in rhegmatogenous retinal detachment.
  • the retinal cell layers come in contact with vitreous cytokines.
  • These cytokines trigger the ability of the retinal pigmented epithelium (RPE) to proliferate and migrate.
  • the process involved resembles fibrotic wound healing by the RPE cells.
  • the RPE cells undergo EMT and develop the ability to migrate out into the vitreous.
  • RPE cell layer-neural retinal adhesion and RPE-ECM (extracellular matrix) adhesions are lost.
  • the RPE cells lay down fibrotic membranes while they migrate and these membranes contract and pull at the retina.
  • Risk factors and clinical signs As described above, the most common development of PVR is after a retinal detachment surgery and/or repair, although patients can develop PVR spontaneously with retinal detachment prior to surgery or with longstanding primary detachments. Multiple factors have been associated with the formation of PVR. In general, processes that increase vascular permeability are more likely to increase the probability of PVR formation. Specific risk factors that have been identified include: uveitis; large, giant, or multiple tears; vitreous hemorrhage, preoperative or postoperative choroidal detachments; aphakia; multiple previous surgeries; and large detachments involving greater than 2 quadrants of the eye.
  • PVR early signs of PVR are often subtle and can include cellular dispersion in the vitreous and on the retinal surface, which can appear as a white opacification of the retinal surface and small wrinkles or folds. More developed PVR is characteristic with fixed folds and retinal detachment. Diagnosis is typically done by indirect ophthalmoscopy and slit-lamp biomicroscopy. Additionally, an ultrasound can help visualize immobile retinal folds of detachment and prominent vitreous membranes. Also, wide-field fundus photography can be used to visualize retinal detachments. However, the clinical history and exam is often enough to make the diagnosis of a retinal detachment.
  • Ocular wound healing typically occurs in 3 stages: (1) an inflammatory stage, (2) a proliferative stage, and (3) a modulatory stage.
  • PVR can be viewed in a similar fashion, with the wound being the retinal detachment. This healing response often takes place over many weeks.
  • preretinal PVR adopts an immature appearance and consistency.
  • the retina may still remain compliant, and the PVR membrane may be difficult to remove due to its amorphous form.
  • the PVR membrane becomes more mature, taking on a white, fibrotic appearance.
  • the PVR is more easily identifiable, causes rigidity of the retina, and can be more identifiably removed.
  • Grade A The extent of PVR in patients is often classified (or graded) depending on the severity. The most commonly used classification system was published by the Retina Society Terminology Committee. It classifies the appearance of PVR based on clinical signs and its geographic location (Grade A, B, C, or D). Grade A is characterized by the appearance of vitreous haze and RPE cells in the vitreous, or by pigment clumping. Grade B is characterized by wrinkling of the edges of the retinal tear or the inner retinal surface. Grade C is characterized by posterior or anterior full thickness retinal folds with the presence of epi/subretinal membranes/bands. Grade D is characterized by fixed retinal folds in all four quadrants.
  • PVR proliferative diabetic retinopathy
  • PDR proliferative diabetic retinopathy
  • PVR is a condition distinct from a small blood vessel disease.
  • the fundamental process involved in PDR is aberrant angiogenesis, and therefore impacting vascular endothelial cells.
  • the fundamental processes is the aberrant EMT of retinal pigment epithelial derived cells, and other cells within the eye.
  • any symptom, type, or stage of PVR may be inhibited, treated, or prevented using methods and compositions disclosed herein, e.g. using the nucleic acid or composition encoding a RUNX3 transcription factor.
  • the ocular disease, disorder, or condition is epiretinal membrane, a very common disease that occurs after retinal detachment surgery and can be considered a very mild form of PVR, that can cause visual change, due to a very thin membrane forming on top of the retina.
  • the ocular disease, disorder, or condition is PVR, preferably the prevention of PVR.
  • the nucleic add or the pharmaceutical composition or the kit or kit of parts for use as a medicament reduces the cell proliferation and/or cell growth in eyes with PVR.
  • C-PVR cells human primary cell cultures obtained from surgically removed PVR membranes
  • HMRECs primary human retinal microvascular endothelial cells
  • transfection using formulated RUNX3 mRNA can reduce proliferation markers expression which could be detected as well as a modulation of EMT and fibrotic markers in C-PVR ( Figure 3).
  • intravitreal injection of RUNX3 mRNA to a PVR model in rabbits effectively reduces the pathology severity in vivo and decreased the accumulation of fibrotic membranes on top of the retina ( Figure 4).
  • RUNX3 mRNA reduces lesion size in a laser-CNV mouse model 7 days after treatment (Figure 5).
  • cell proliferation and/or cell growth is reduced in eyes with PVR.
  • the ocular disease, disorder, or condition is selected from Epiretinal Membrane (ERM).
  • ERM Epiretinal Membrane
  • Idiopathic ERMs affect the architecture of the macula. There can be blunting of the foveal contour or wrinkling on the retinal surface from membrane contracture. Most commonly it involves the foveal and parafoveal area. They most commonly cause minimal symptoms and can be simply observed, but in some cases they can result in painless loss of vision and metamorphopsia (visual distortion).
  • ERMs are most symptomatic when affecting the macula, which is the central portion of the retina that helps us to distinguish fine detail used for reading and recognizing faces. There are no eye drops, medications or nutritional supplements to treat ERMs. A surgical procedure called vitrectomy is the only option in eyes that require treatment.
  • the invention provides an nucleic acid, or a pharmaceutical composition, or a kit or kit of parts, for use as a medicament in treating or preventing a disease, disorder, or condition in a subject, wherein the subject has suffered a trauma to the eye, comprises a retinal hole, a retinal tear, a retinal detachment disorder, or has undergone an ocular surgery.
  • Retinal detachment disorder is a disorder of the eye in which the neurosensory retina separates from the retinal pigment epithelial layer underneath.
  • the mechanism most commonly involves a break in the retina that then allows the fluid in the eye to get behind the retina.
  • a break in the retina can occur from a posterior vitreous detachment, injury to the eye, or inflammation of the eye.
  • Other risk factors include being short sighted and previous cataract surgery.
  • diagnosis is accomplished by either looking at the back of the eye with an ophthalmoscope or by ultrasound. Symptoms include an increase in the number of floaters, flashes of light, and worsening of the outer part of the visual field, which may be described as a curtain over part of the field of vision. In about 7% of cases both eyes are affected.
  • Retinal detachments affect between 0.6 and 1 .8 people per 10,000 per year. About 0.3% of people are affected at some point in their life. It is most common in people who are in their 60s or 70s, and males are more often affected than females. The long-term outcomes depend on the duration of the detachment and whether the macula was detached. If treated before the macula detaches outcomes are generally good.
  • the subject has not been diagnosed or characterized with some other ocular disorder comprising age-related macular degeneration or an ocular angiogenesis disease or disorder.
  • the vitreous moves away from the retina without causing problems. But sometimes the vitreous pulls hard enough to tear the retina in one or more places, and thus causing a retinal tear. Fluid may pass through a retinal tear, lifting the retina off the back of the eye.
  • vitreous separation retinal tear, and retinal detachment
  • the patient may notice the floaters and flashing lights (photopsia) more commonly associated with isolated vitreous separation.
  • An ophthalmologist, optometrist, or primary care physician may be suspicious about a more serious problem if symptoms are of very recent or sudden onset and are accompanied by a shower of spots or “cobwebs”.
  • peripheral vision which may present as a shadow moving toward the center of one’s field of vision.
  • retinal detachment a retinal hole may develop. Because the vitreous is attached to the retina with tiny strands of collagen, it can pull on the retina as it shrinks.
  • this shrinkage can tear off a small piece of the retina in the periphery, causing a hole or tear of the periphery retina. If this missing piece of retina is in the macula, it is called a macular hole.
  • another direct cause of macular holes due to vitreous shrinkage is when the collagen strands stay attached to the retina forming an epi retinal membrane. These membranes can contract around the macula, causing the macula to develop a hole from the traction.
  • Retinal detachments commonly occur secondary to peripheral retinal tears/holes, and rarely form macular holes.
  • a minority of retinal detachments result from trauma, including blunt blows to the orbit, penetrating trauma, and concussions to the head.
  • rhegmatogenous retinal detachment There are three types of retinal detachment: (1) rhegmatogenous retinal detachment - a rhegmatogenous retinal detachment occurs due to a break in the retina (e.g., a retinal tear) that allows fluid to pass from the vitreous space into the subretinal space between the sensory retina and the retinal pigment epithelium.
  • Retinal breaks are divided into three types - holes, tears and dialyses. Holes form due to retinal atrophy especially within an area of lattice degeneration. Tears are due to vitreoretinal traction. Dialyses are very peripheral and circumferential and may be either fractional or atrophic.
  • Exudative, serous, or secondary retinal detachment an exudative retinal detachment occurs due to inflammation, injury or vascular abnormalities that results in fluid accumulating underneath the retina without the presence of a hole, tear, or break.
  • exudative detachment can be caused by the growth of a tumor on the layers of tissue beneath the retina, namely the choroid. This cancer is called a choroidal melanoma.
  • T ractional retinal detachment - a fractional retinal detachment occurs when fibrous (from PVR membrane) or fibrovascular (from neovascular disorders such as proliferative diabetic retinopathy) tissue, caused by an injury, inflammation or neovascularization, pulls the sensory retina from the retinal pigment epithelium.
  • the retinal detachment disorder is selected from rhegmatogenous retinal detachment, exudative retinal detachment, or fractional retinal detachment.
  • the nucleic add, the pharmaceutical composition, or the kit or kit of parts is administered by local administration, preferably by ocular administration as defined herein.
  • the nucleic acid or composition of the invention may be administered using an ocular delivery device.
  • the ocular delivery device may be designed for the controlled release of the nucleic acid or the pharmaceutical composition with multiple defined release rates and sustained dose kinetics and permeability.
  • the ocular administration is selected from intravitreal administration, by administration prior to an ocular surgery, during an ocular surgery, or after an ocular surgery. In particularly preferred embodiments, the ocular administration is selected from intravitreal administration.
  • At least one ocular administration is prior to an ocular surgery, during an ocular surgery, and/or after an ocular surgery.
  • Some disease, disorders or conditions in the eye occur after an ocular surgery or operation as described herein (e.g. PVR).
  • administration of the pharmaceutical composition or nucleic add e.g. an RNA encoding RUNX3 via intraoperative administration is preferred.
  • a first dose of the nucleic acid or composition is administered during an ocular surgery, and second and further doses are administered via intravitreal administration.
  • a nucleic acid encoding a RUNX3 transcription factor of the invention may be administered at the time of diagnosis of a retinal detachment, or during an ocular surgery (e.g. to prevent the development of a disease, e.g. PVR) and a second and optional further doses are administered via intravitreal administration (e.g. to prevent the development of a disease, e.g. PVR).
  • the disease, disorder, or condition is associated with or caused by overexpressed and/or overactive RUNX transcription factor.
  • RUNX1 a ocular disease.
  • RUNX1 has non-detectable basal expression in the healthy retina, whereas in pathologies such as proliferative diabetic retinopathy and choroidal neovascularization, aberrant RUNX1 signalling occurs and is believed to drive the angiogenic process.
  • pathologies such as proliferative diabetic retinopathy and choroidal neovascularization
  • aberrant RUNX1 signalling occurs and is believed to drive the angiogenic process.
  • RUNX1 expression which is normally silent, is strongly induced to drive control pathological processes associated with aberrant angiogenesis, EMT, and fibrosis in the eye and elsewhere. These processes are fundamental to prevalent conditions including cancer, proliferative diabetic retinopathy, exudative age-related macular degeneration, proliferative vitreoretinopathy, lung fibrosis, and virus-caused lung fibrosis e.g. COVID-19.
  • ocular diseases characterized by aberrant angiogenesis or pathologic EMT are associated with RUNX1 overexpression.
  • diseases that involve aberrant angiogenesis that are associated with RUNX1 overexpression include non-proliferative diabetic retinopathy, diabetic macular edema, exudative age-related macular degeneration, retinal neovascularization, iris neovascularization, neovascular glaucoma, central retinal vein occlusion, branch retinal vein occlusion, Coats’ disease, familial exudative vitreoretinopathy (FEVR), Von Hippel-Lindau disease, retinal hemangioma, Leber's military aneurysms, macula telangiectasia, polypoidal choroidal vasculopathy, myopic choroidal neovascularization, idiopathic choroidal neovascularization, corneal neovascularization, thyroid eye disease, small vessel disease.
  • FEVR
  • diseases that involve aberrant EMT that are associated with RUNX1 overexpression include PVR, open angle glaucoma, exudative age-related macular degeneration (fibrosis of CNV lesions), uveal metastatic cancers, geographic atrophy. Further, examples of diseases that may be associated with RUNX1 overexpression include primary ocular tumors including uveal melanoma, retinoblastoma, astrocytomas.
  • nucleic add or composition of the invention may be administered only once or multiple times.
  • a nucleic acid encoded RUNX3 transcription factor may be administered using a method disclosed herein at least about once, twice, three times, four times, five times, six times, or seven times per day, week, month, or year.
  • a nucleic acid encoded RUNX3 transcription factor is administered once per month. In certain embodiments, a nucleic add encoded RUNX3 transcription factor is administered once per week, once every two weeks, once a month via intravitreal injection.
  • the administration of the nucleic add, the pharmaceutical composition, or the kit or kit of parts is performed more than once, for example two times, three times, or four times, for example periodically.
  • a composition is self-administered.
  • the present invention relates to a method of treating or preventing a disease, disorder or condition.
  • embodiments relating to the previous aspects may likewise be read on and be understood as suitable embodiments of method of treatments of the invention.
  • specific features and embodiments relating to method of treatments as provided herein may also apply for medical uses of the invention and vice versa.
  • Preventing (inhibiting) or treating a disease relates to inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as an infection.
  • Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.
  • the term “ameliorating”, with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment.
  • Inhibiting a disease can include preventing or reducing the risk of the disease.
  • the beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters that are specific to the particular disease.
  • a “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.
  • the present invention relates to a method of treating or preventing a disease, disorder or condition, wherein the method comprises applying or administering to a subject in need thereof an effective amount of the nucleic acid of the first aspect, the pharmaceutical composition of the second aspect, the kit or kit of parts of the third aspect.
  • effective when referring to an amount of a therapeutic compound refers to the quantity of the compound that is sufficient to yield a desired therapeutic response without undue adverse side effects (e.g. toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used according to the invention.
  • the present invention relates to a method of reducing the activity of RUNX1 in a cell or a subject.
  • Embodiments relating to the previous aspects may likewise be read on and be understood as suitable embodiments of the method of reducing the activity of RUNX1 in a cell or a subject of the present aspect.
  • the method of reducing the activity of a RUNX1 in a cell or a subject comprises a) applying or administering a nucleic acid comprising at least one cds encoding at least one RUNX3 transcription factor or a fragment or variant thereof as defined in the first aspect; or b) applying or administering a pharmaceutical composition comprising the nucleic acid comprising at least one cds encoding at least one RUNX3 transcription factor or a fragment or variant thereof as defined in the second aspect; to a cell, tissue, or subject, wherein the RUNX3 transcription factor is produced in the cell, tissue, or subject after administration or application to said cell, tissue, or subject.
  • the applying or administering is selected from intramuscular, intranasal, transdermal, intradermal, intralesional, intracranial, subcutaneous, intracardial, intratumoral, intravenous, intrapulmonal, intraarticular, sublingual, pulmonary, intrathecal, local administration, or ocular administration.
  • the ocular administration is selected from topical, intravitreal, intracameral, subconjunctival, subretinal, subtenon, retrobulbar, topical, orbital, suprachoroidal, posterior juxtascleral, or intraoperative administration, preferably intravitreal or intraoperative administration.
  • an ocular administration leads to a production of the RUNX3 transcription factor cornea, lens, ciliary body, vitreous, sclera, choroid, retina, optic nerve, macula, scleral cells, choroid cells, retinal cells, inflammatory cells, retinal pigment epithelium (RPE), Muller cells, microglia, photoreceptors, amacrine cells, choroidal melanocytes retinal ganglion cells, horizontal cells, bipolar cells, astrocytes, vitreous, trabecular mesh, conjunctiva, corneal endothelium, Bruch’s membrane, conjunctiva, and retinal or choroidal blood vessels or hyaloid vessels, or in cells of the brain comprising choroid plexus epithelial cells.
  • RPE retinal pigment epithelium
  • Muller cells microglia, photoreceptors
  • amacrine cells choroidal melanocytes retinal ganglion cells
  • horizontal cells bipolar cells
  • an ocular administration leads to a production of the RUNX3 in cells and/or tissues of the eye, preferably in RPE cells or cells derived from RPE cells.
  • an ocular administration leads to a production of the RUNX3 transcription factor in retinal cells, preferably selected from photoreceptor cells, bipolar cells, ganglion cells, horizontal cells, Muller cells, mural cells, vascular endothelial cells, microglia, and amacrine cells. Particularly preferred are Muller cells, and microglia.
  • the reduction of the activity of RUNX1 is a transient reduction. This is particularly important in the context of the whole invention, as a permanent reduction of the activity of RUNX1 would potentially be associated with side effects.
  • a transient molecule such as RNA is particularly suitable in that context as RNA, in particular mRNA, is typically degraded, and the encoded RUNX3 transcription factor protein has also a limited half-life (depending on the tissue, the protein sequence etc.).
  • the RUNX3 transcription factor is produced in the cell, tissue, or subject after administration or application of the nucleic acid or the composition to said cell, tissue, or subject, wherein
  • RUNX3 is a dominant negative inhibitor of RUNX1 ;
  • the produced RUNX3 reduces or prevents nuclear translocation of RUNX1 ;
  • RUNX3 reduces the cellular expression of RUNX1 ;
  • RUNX3 reduces the cellular expression of proteins that are controlled or regulated by RUNX1 ;
  • the produced RUNX3 reduces the cellular expression of TGFbeta2, SMAD3, and/or COL1 A1 ; and/or - the produced RUNX3 increase the transcription rate of MARVELD2; and/or
  • the produced RUNX3 reduces or prevents pathological EMT, pathological cell proliferation, aberrant angiogenesis, aberrant neovascularization, degeneration, and/or fibrosis; and/or
  • Item 1 An artificial nucleic acid comprising at least one cds encoding a RUNX3 transcription factor or a fragment or variant thereof, wherein the nucleic acid comprises at least one heterologous untranslated region (UTR).
  • UTR heterologous untranslated region
  • Item 2 The artificial nucleic acid factor of item 1 , wherein the RUNX3 transcription factor is selected from a full- length RUNX3 protein, or an N-terminally and/or a C-terminally truncated RUNX3 protein fragment.
  • Item 3 The artificial nucleic acid of items 1 or 2, wherein the RUNX3 transcription factor, or a fragment or variant thereof, comprises a Runt domain (RD).
  • RD Runt domain
  • Item 4 The artificial nucleic acid of item 3, wherein the Runt domain (RD) mediates binding of RUNX3 to DNA as well as an interaction of RUNX3 with the core-binding factor subunit beta (CBFbeta).
  • RD Runt domain
  • CBFbeta core-binding factor subunit beta
  • Item 5 The artificial nucleic acid of item 1 or 2, wherein the RUNX3 transcription factor, or a fragment or variant thereof, comprises a transactivation domain (AD) and/or an inhibition domain (ID).
  • AD transactivation domain
  • ID inhibition domain
  • Item 6 The artificial nucleic acid of item 5, wherein the RUNX3 transcription factor, or a fragment or variant thereof, activates or represses transcription regulation of genes involved in pathological epithelial to mesenchymal transition (EMT), induction of epithelial cell differentiation, pathological cell proliferation, aberrant angiogenesis, aberrant neovascularization, degeneration, osteoarthritis, cancer or metastasis and/or fibrosis.
  • EMT epithelial to mesenchymal transition
  • Item 7 The artificial nucleic acid of any one of the preceding items, wherein the RUNX3 transcription factor, or a fragment or variant thereof, comprises an amino acid sequence which comprises at least one, two, or more amino acid substitutions, deletions or insertions selected from K162R, K200R, K206R, K162Q, K200Q, K206Q, P323R, P323del, P324del, P325del, Y326del or 430insKKK, or any functionally equivalent amino acid substitution at position K162, K200, K206, K162, K200, K206, P323, P324, P325, Y326 or 430.
  • Item 8 The nucleic acid of any one of the preceding items, wherein the RUNX3 transcription factor, or a fragment or variant thereof, comprises or consists of an amino acid sequence selected from or derived from the GenBank® accession number NM_004350.3, NM_001031680.2, NM_001320672.1 , XM_005246024.5, XM_011542351 .2, XM_047433131.1 , XM_054339349.1 or XM_054339350.1 .
  • Item 9 The artificial nucleic acid of any one of the preceding items, wherein the RUNX3 transcription factor comprises or consists of an amino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 129-163, or fragments or variants of any of these, preferably SEQ ID NO: 129, or fragments or variant.
  • Item 10 The artificial nucleic acid of any one of the preceding items, wherein the at least one cds is a codon modified cds, preferably wherein codon modified cds is selected from a C maximized cds, a CAI maximized cds, human codon usage adapted cds, a G/C content modified cds, and a G/C optimized cds, or any combination thereof, preferably wherein the at least one codon modified cds is a G/C optimized cds.
  • codon modified cds is selected from a C maximized cds, a CAI maximized cds, human codon usage adapted cds, a G/C content modified cds, and a G/C optimized cds, or any combination thereof, preferably wherein the at least one codon modified cds is a G/C optimized cds.
  • Item 11 The artificial nucleic acid of any one of the preceding items, wherein the at least one cds comprises or consists of a nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 164-268, or a fragment or a variant of any of these, preferably SEQ ID NO: 199, or a fragment or a variant
  • Item 12 The artificial nucleic acid of any one of the preceding items, wherein the at least one heterologous untranslated region (UTR) is selected from at least one heterologous 5’-UTR and/or at least one heterologous 3-UTR.
  • UTR heterologous untranslated region
  • Item 13 The artificial nucleic acid of item 12, wherein the at least one heterologous 3-UTR comprises or consists of a nucleic acid sequence derived from a 3-UTR of a gene selected from PSMB3, ALB7, alpha-globin, betaglobin, ANXA4, CASP1 , COX6B1 , FIG4, GNAS, NDUFA1 , RPS9, SLC7A3, TUBB4B, or from a homolog, a fragment or a variant of any one of these genes, preferably wherein the at least one heterologous 3’-UTR comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 66-95, 112-123, or a fragment or a variant of any of these.
  • Item 14 The artificial nucleic acid of items 12 or 13, wherein the at least one heterologous 3-UTR comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 67, or a fragment or a variant thereof.
  • Item 15 The artificial nucleic acid of item 12, wherein the at least one heterologous 5-UTR comprises or consists of a nucleic acid sequence derived from a 5-UTR of a gene selected from HSD17B4, RPL32, AIG1 , alphaglobin, ASAH1 , ATP5A1 , COX6C, DPYSL2, MDR, MP68, NDUFA4, NOSIP, RPL31 , RPL35A, SLC7A3, TUBB4B, UBQLN2, or from a homolog, a fragment or variant of any one of these genes, preferably wherein the at least one heterologous 5-UTR comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 12-45, 64, 65, or a fragment or a variant
  • Item 17 The artificial nucleic acid of items 12 to 16, wherein the at least one heterologous 5-UTR is selected from HSD17B4 and the at least one heterologous 3’ UTR is selected from PSMB3.
  • Item 19 The artificial nucleic acid of item 18, wherein the at least one poly(A) sequence comprises about 100 adenosine nucleotides.
  • Item 20 The artificial nucleic acid of item 18 or 19, wherein the at least one poly(A) sequence is located at the 3’ terminus, optionally, wherein the 3’ terminal nucleotide is an adenosine.
  • Item 21 The artificial nucleic acid of any one of the preceding items, wherein the nucleic acid comprises at least one poly(C) sequence and/or at least one miRNA binding site and/or at least one histone stem-loop sequence.
  • Item 22 The artificial nucleic acid of any one of the preceding items, wherein the nucleic acid comprises at least one histone stem-loop sequence, wherein said histone stem-loop sequence comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 3,4, or a fragment or variant of any of these.
  • Item 23 The artificial nucleic acid of any one of the preceding items, wherein the nucleic acid is selected from a DNA vector, preferably an AAV vector, or an RNA.
  • Item 24 The artificial nucleic acid of any one of the preceding items, wherein the nucleic acid is an RNA selected from mRNA, circular RNA, replicon RNA, or viral RNA.
  • Item 25 The artificial nucleic acid of any one of the preceding items, wherein the nucleic acid is an mRNA.
  • Item 26 The artificial nucleic acid of items 23 or 24, wherein the RNA comprises at least one modified nucleotide, preferably selected from N1 -methylpseudouridine (ml i ) or pseudouridine (i ), more preferably selected from N1 -methylpseudouridine (m1ip).
  • modified nucleotide preferably selected from N1 -methylpseudouridine (ml i ) or pseudouridine (i ), more preferably selected from N1 -methylpseudouridine (m1ip).
  • Item 27 The artificial nucleic acid of any one of the preceding items, wherein the nucleic acid is a modified RNA wherein each uracil is substituted by N1 -methylpseudouridine (m1ip).
  • Item 28 The artificial nucleic acid of any one of the preceding items, wherein the nucleic acid is an RNA that comprises a 5’-cap structure.
  • Item 29 The artificial nucleic acid of item 28, wherein the 5’-cap structure is selected from a cap1 structure or a modified cap1 structure.
  • Item 30 The artificial nucleic acid of any one of the preceding items, wherein the nucleic acid is an in vitro transcribed RNA, preferably wherein RNA in vitro transcription has been performed in the presence of a sequence optimized nucleotide mixture.
  • Item 31 The artificial nucleic acid of any one of the preceding items, wherein the nucleic acid is a purified RNA, preferably wherein the RNA has been purified by RP-HPLC, AEX, SEC, hydroxyapatite chromatography, TFF, filtration, precipitation, core-bead flowthrough chromatography, oligo(dT) purification, cellulose-based purification, or any combination thereof.
  • Item 32 The artificial nucleic acid of item 31 , wherein the at least one step of purification is selected from RP-HPLC and/or TFF.
  • Item 33 The artificial nucleic acid of any one of the preceding items, wherein the nucleic acid, preferably the RNA, has an integrity of at least about 50%, preferably of at least about 60%, more preferably of at least about 70%, most preferably of at least about 80%.
  • Item 34 The artificial nucleic acid of any one of the preceding items, wherein the nucleic acid, preferably the RNA, is suitable for use in treatment or prevention of a disease, disorder or condition, preferably an ocular disease, disorder or condition.
  • Item 35 The artificial nucleic acid of any one of the preceding items, wherein the nucleic acid, preferably the RNA, comprises the following sequence elements preferably in 5’- to 3’-direction:
  • a 5-UTR preferably selected or derived from a 5’-UTR of a HSD17B4 gene
  • a 3-UTR preferably selected or derived from a 3 -UTR of a PSMB3 gene
  • Item 36 The artificial nucleic acid of any one of the preceding items, wherein the nucleic acid, preferably the RNA, comprises or consists of a nucleic acid sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 269-458, 461-462, or a fragment or variant of any of these sequences.
  • Item 37 The artificial nucleic acid of any one of the preceding items, wherein the nucleic acid, preferably the RNA, comprises or consists of a nucleic acid sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 269-273, 300-304, 331-335, 362-366, 393-397, 424428, 455-458, 461 62, or a fragment or variant of any of these sequences.
  • nucleic acid of any one of the preceding items, wherein the nucleic acid, preferably the RNA, comprises or consists of a nucleic acid sequence which is identical or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 274-299, 305-330, 336-361 , 367-392, 398423, 429-454 or a fragment or variant of that sequence.
  • nucleic acid of item 37 wherein the nucleic acid, preferably the RNA, comprises or consists of a nucleic acid sequence which is identical to the nucleic acid sequence SEQ ID NOs: 269, 455, 300, 456, 461- 462, preferably SEQ ID NOs: 456 or 462, or a fragment or variant of any of these sequences.
  • Item 40 A pharmaceutical composition comprising at least one artificial nucleic acid comprising at least one cds encoding a RUNX3 transcription factor or a fragment or variant thereof as defined in any one of the items 1 to 39.
  • Item 41 The pharmaceutical composition of item 40, comprising at least one pharmaceutically acceptable carrier or excipient.
  • Item 42 The pharmaceutical composition of item 40 or 41 , wherein the at least one artificial nucleic acid, preferably the RNA, is formulated in at least one cationic or polycationic compound.
  • Item 43 The pharmaceutical composition of item 42, wherein the at least one cationic or polycationic compound is selected from a cationic or polycationic polymer, a cationic or polycationic polysaccharide, a cationic or polycationic lipid, a cationic or polycationic protein, a cationic or polycationic peptide, or any combinations thereof.
  • Item 44 The pharmaceutical composition of item 43, wherein the one or more cationic or polycationic peptides are selected from SEQ ID NOs: 124-128, or any combinations thereof.
  • Item 45 The pharmaceutical composition of item 43, wherein the cationic or polycationic polymer is selected from a polyethylene glycol/peptide polymer, preferably comprising HQ-PEG5000-S-(S- CHHHHHHRRRRHHHHHHC-S-)7-S-PEG5000-QH (SEQ ID NO: 127 as peptide monomer), HO- PEG5000-S-(S-CHHHHHHRRRRHHHHHHC-S-)4-S-PEG5000-OH (SEQ ID NO: 127 as peptide monomer), HO-PEG5000-S-(S-CGHHHHHRRRRHHHHHGC-S-)7-S-PEG5000-OH (SEQ ID NO: 128 as peptide monomer), or HO-PEG5000-S-(S-CGHHHHHRRRRHHHHHGC-S-)4-S-PEG5000-OH (SEQ ID NO: 128 as peptide monomer).
  • Item 46 The pharmaceutical composition of item 43 or 45, wherein the cationic or polycationic polymer additionally comprises at least one lipidoid component.
  • Item 47 The pharmaceutical composition of item 46, wherein the at least one lipidoid component is selected from 3- C12-OH, 3-C12-OH-cat, or 3-C12-C3-OH.
  • Item 48 The pharmaceutical composition of items 40 to 43, wherein the at least one artificial nucleic acid, preferably the RNA, is formulated in lipid-based carriers.
  • Item 49 The pharmaceutical composition of item 48, wherein the lipid-based carriers are selected from liposomes, lipid nanoparticles, lipoplexes, solid lipid nanoparticles, lipo-polylexes, and/or nanoliposomes.
  • Item 50 The pharmaceutical composition of item 48 or 49, wherein the lipid-based carriers are lipid nanoparticles, preferably wherein the lipid nanoparticles encapsulate the artificial nucleic acid.
  • Item 51 The pharmaceutical composition of items 48 to 50, wherein the lipid-based carriers comprise at least one aggregation-reducing lipid, at least one cationic lipid or ionizable lipid, at least one neutral lipid or phospholipid, and at least one steroid or steroid analog.
  • Item 52 The pharmaceutical composition of item 51 , wherein the aggregation reducing lipid is selected from a polymer conjugated lipid.
  • Item 53 The pharmaceutical composition of item 52, wherein the polymer conjugated lipid is selected from a PEG- conjugated lipid or a PEG-free lipid.
  • Item 54 The pharmaceutical composition of item 48 to 53, wherein the lipid-based carriers comprise a polymer conjugated lipid selected or derived from DMG-PEG 2000, C10-PEG2K, Cer8-PEG2K, POZ-lipid.
  • Item 55 The pharmaceutical composition of items 51 to 54, wherein the cationic lipid or ionizable lipid is selected from an amino lipid, preferably wherein the amino lipid comprises a tertiary amine group.
  • Item 57 The pharmaceutical composition of items 48 to 56, wherein the lipid-based carriers comprise a cationic lipid selected or derived from SM-102, SS-33/4PE-15, HEXA-C5DE-PipSS, or compound C26.
  • Item 58 The pharmaceutical composition of items 48 to 57, wherein the lipid-based carriers comprise a neutral lipid selected or derived from DSPC, DHPC, or DphyPE.
  • Item 59 The pharmaceutical composition of items 48 to 58, wherein the lipid-based carriers comprise a steroid or steroid analog selected or derived from cholesterol, cholesteryl hemisuccinate (CHEMS), preferably cholesterol.
  • CHEMS cholesteryl hemisuccinate
  • Item 60 The pharmaceutical composition of items 48 to 59, wherein the lipid-based carriers comprise
  • Item 61 The pharmaceutical composition of items 48 to 60, wherein the lipid-based carriers comprise about 20-60% cationic lipid, about 5-25% neutral lipid, about 25-55% steroid or steroid analogue, and about 0.5-15% aggregation reducing lipid.
  • Item 62 The pharmaceutical composition of items 48 to 61 , wherein the wt/wt ratio of lipid to nucleic acid in the lipid- based carrier is from about 10:1 to about 60:1 .
  • Item 63 The pharmaceutical composition of items 48 to 62, wherein the N/P ratio of the lipid-based carriers encapsulating the nucleic acid, preferably the RNA, is in a range from about 1 to about 20.
  • Item 64 The pharmaceutical composition of items 48 to 63, wherein the lipid-based carriers have a Z-average size in a range of about 50nm to about 200nm, preferably 50nm to 120nm.
  • Item 65 The pharmaceutical composition of items 40 to 64, wherein the composition comprises at least one antagonist of at least one RNA sensing pattern recognition receptor selected from a Toll-like receptor, preferably a TLR7 antagonist and/or a TLR8 antagonist.
  • Item 66 The pharmaceutical composition of items 40 to 65, wherein the composition comprises an anti- inflammatory agent, preferably wherein the anti-inflammatory agent comprises a steroid or a nonsteroidal antiinflammatory drug (NSAID).
  • the anti-inflammatory agent comprises a steroid or a nonsteroidal antiinflammatory drug (NSAID).
  • NSAID nonsteroidal antiinflammatory drug
  • Item 67 The pharmaceutical composition of items 45 to 66, wherein the composition comprises at least one RUNX1 inhibitor.
  • Item 68 The pharmaceutical composition of item 68, wherein the RUNX1 inhibitor is selected from a small molecule inhibitor of RUNX1 , an inhibitory nucleic acid (siRNA) of RUNX1 , or a nucleic acid encoding a RUNX1 inhibitor (e.g. a RUNX-Trap).
  • the RUNX1 inhibitor is selected from a small molecule inhibitor of RUNX1 , an inhibitory nucleic acid (siRNA) of RUNX1 , or a nucleic acid encoding a RUNX1 inhibitor (e.g. a RUNX-Trap).
  • Item 69 The pharmaceutical composition of item 68, wherein the nucleic acid encoding a RUNX1 inhibitor encodes a CBFbeta-SMMHC fusion protein.
  • Item 70 The pharmaceutical composition of items 40 to 69, wherein the composition is a liquid composition or a dried composition.
  • Item 71 The pharmaceutical composition or the artificial nucleic acid of any one of the preceding items, wherein upon intramuscular, intranasal, transdermal, intradermal, intralesional, intracranial, subcutaneous, intracardial, intratumoral, intravenous, intrapulmonal, intraarticular, sublingual, pulmonary, intrathecal, retronasal or ocular administration of the composition or nucleic acid to a cell, tissue, or subject, the RUNX3 transcription factor is produced.
  • Item 72 The pharmaceutical composition or the artificial nucleic acid of item 71 , wherein the administration is an ocular administration.
  • Item 73 The pharmaceutical composition or the artificial nucleic acid of any one of the preceding items, wherein ocular administration of the composition or the nucleic acid leads to a production of the RUNX3 transcription factor or a fragment or variant thereof in cells and/or tissues of the eye, preferably in cells and/or tissues selected from cornea, lens, ciliary body, vitreous, sclera, choroid, retina, optic nerve, macula, scleral cells, retinal cells, inflammatory cells, retinal pigment epithelium (RPE), Bruch’s membrane, and retinal or choroidal blood vessels, or cells in the brain comprising choroid plexus epithelial cells.
  • RPE retinal pigment epithelium
  • Item 74 The pharmaceutical composition or the artificial nucleic acid of any one of the preceding items, wherein ocular administration of the composition or the nucleic acid leads to a production of the RUNX3 transcription factor or a fragment or variant thereof in retinal pigment epithelial (RPE) cells or cells derived from them.
  • RPE retinal pigment epithelial
  • Item 75 The pharmaceutical composition or the artificial nucleic acid of any one of the preceding items, wherein ocular administration of the composition or the nucleic acid leads to a production of the RUNX3 transcription factor in retinal cells selected from photoreceptor cells, bipolar cells, ganglion cells, horizontal cells, Muller cells, mural cells, vascular endothelial cells, microglia, and amacrine cells.
  • Item 76 The pharmaceutical composition or the artificial nucleic acid of any one of the preceding items, wherein ocular administration of the composition or the nucleic acid reduces or inhibits the cellular expression of RUNX1 , TGFbeta2, SMAD3, and/or COL1 A1 .
  • Item 77 The pharmaceutical composition or the artificial nucleic acid of any one of the preceding items, wherein ocular administration of the composition or the nucleic acid reduces or prevents pathological epithelial to mesenchymal transition (EMT), pathological cell proliferation, aberrant angiogenesis, aberrant neovascularization, degeneration, and/or fibrosis.
  • Item 78 The pharmaceutical composition or the artificial nucleic acid of item 72 to 77, wherein the ocular administration is selected from topical, intravitreal, intracameral, subconjunctival, subretinal, subtenon, retrobulbar, into the ciliary body, orbital, suprachoroidal, posterior juxtascleral, or intraoperative administration.
  • Item 79 The pharmaceutical composition or the artificial nucleic acid of item 72 to 78, wherein the ocular administration is an intravitreal or intraoperative administration.
  • Item 80 The pharmaceutical composition or the artificial nucleic acid of items 72 to 79, wherein the ocular administration is performed into a tamponade agent-filled human eye.
  • Item 81 The pharmaceutical composition or the artificial nucleic acid of item 80, wherein the tamponade agent is a gas agent or a silicone agent.
  • Item 82 A Kit or kit of parts comprising at least one artificial nucleic acid of any one of items 1 to 39, and/or at least one pharmaceutical composition of any one of items 40 to 81 , optionally comprising a liquid vehicle for solubilising, and optionally comprising technical instructions providing information on administration and dosage of the components.
  • Item 83 An artificial nucleic acid of any one of items 1 to 39, or a pharmaceutical composition of any one of items 40 to 81 , or a kit or kit of parts of item 82, for use as a medicament in treating or preventing a disease, disorder, or condition in a subject.
  • Item 84 The artificial nucleic acid of any one of items 1 to 39, or the pharmaceutical composition of any one of items 40 to 81 , or the kit or kit of parts of item 82, for use as a medicament of item 83, wherein the disease, disorder, or condition is associated with or caused by an overexpressed and/or an overactive RUNX1 .
  • Item 85 The artificial nucleic acid of any one of items 1 to 39, or the pharmaceutical composition of any one of items 40 to 81 , or the kit or kit of parts of item 82, for use as a medicament of item 83, wherein the disease, disorder, or condition is associated with or caused by a downregulated and/or inhibited RUNX3.
  • Item 86 The artificial nucleic acid of any one of items 1 to 39, or the pharmaceutical composition of any one of items 40 to 81 , or the kit or kit of parts of item 82, for use as a medicament of item 83 to 85, wherein the disease, disorder, or condition is associated with or caused by pathological EMT, pathological cell proliferation, aberrant angiogenesis, aberrant neovascularization, degeneration, fibrosis, solid tumors, and/or aberrant proliferation and migration of RPE cells in a subject.
  • Item 87 The artificial nucleic acid of any one of items 1 to 39, or the pharmaceutical composition of any one of items 40 to 81 , or the kit or kit of parts of item 82, for use as a medicament of item 83 to 86, wherein the disease, disorder, or condition is an ocular disease, disorder, or condition selected from PDR, macular edema, nonproliferative diabetic retinopathy, age-related macular degeneration, geographic atrophy, ocular neovascularization, ROP, a retinal vein occlusion, ocular ischemic syndrome, neovascular glaucoma, a retinal hemangioma, Coats' disease, FEVR, or Norrie disease, persistent hyperplastic primary vitreous (PHPV), thyroid eye disease, epiretinal membrane, small vessel disease, induction of epithelial cell differentiation, osteoarthritis, cancer or metastasis, inflammation, ERM or PVR.
  • PDR ocular disease, disorder, or condition selected
  • Item 88 The artificial nucleic acid of any one of items 1 to 39, or the pharmaceutical composition of any one of items 40 to 81 , or the kit or kit of parts of item 82, for use as a medicament of item 87, wherein the ocular disease, disorder, or condition is ERM and/or PVR, preferably the prevention of ERM and/or PVR.
  • Item 89 The artificial nucleic acid of any one of items 1 to 39, or the pharmaceutical composition of any one of items 40 to 81 , or the kit or kit of parts of item 82, for use as a medicament of item 87 or 88, wherein cell proliferation and/or cell growth is reduced in eyes with ERM or PVR.
  • Item 90 The artificial nucleic acid of any one of items 1 to 39, or the pharmaceutical composition of any one of items 40 to 81 , or the kit or kit of parts of item 82, for use as a medicament of item 83 to 89, wherein the subject has suffered a trauma to the eye, comprises a retinal hole, a retinal tear, a retinal detachment disorder, or has undergone an ocular surgery.
  • Item 91 The artificial nucleic acid of any one of items 1 to 39, or the pharmaceutical composition of any one of items 40 to 81 , or the kit or kit of parts of item 82, for use as a medicament of item 90, wherein the retinal detachment disorder is selected from rhegmatogenous retinal detachment, exudative retinal detachment, or fractional retinal detachment.
  • Item 92 The artificial nucleic acid of any one of items 1 to 39, or the pharmaceutical composition of any one of items 40 to 81 , or the kit or kit of parts of item 82, for use as a medicament of item 83 to 91 , wherein the use comprises administration of the artificial nucleic acid, the pharmaceutical composition, or the kit or kit of parts by local administration, preferably by ocular administration.
  • Item 93 The artificial nucleic acid of any one of items 1 to 39, or the pharmaceutical composition of any one of items 40 to 81 , or the kit or kit of parts of item 82, for use as a medicament of item 92, wherein the ocular administration is selected from intravitreal administration, administration prior to an ocular surgery, administration during an ocular surgery, or administration after an ocular surgery.
  • Item 94 The artificial nucleic acid of any one of items 1 to 39, or the pharmaceutical composition of any one of items 40 to 81 , or the kit or kit of parts ofitem 82 for use as a medicament of item 83 to 93, wherein the administration of the artificial nucleic acid, the pharmaceutical composition, or the kit or kit of parts is performed more than once, for example two times, three times, or four times, for example periodically.
  • Item 95 A method of treating or preventing a disease, disorder or condition, wherein the method comprises applying or administering to a subject in need thereof an effective amount of the artificial nucleic acid of any one of items 1 to 39, or the pharmaceutical composition of any one of items 40 to 81 , or the kit or kit of parts of item 82.
  • Item 96 The method of treating or preventing a disease, disorder or condition of item 95, wherein the method is further characterized by any of the features of items 83 to 94.
  • the present example provides methods of obtaining the RNA of the invention as well as methods of generating composition of the invention comprising nucleic acid, in particular RNA formulated in polyethylene glycol/peptide polymers or RNA formulated in lipid-based carriers such as LNPs.
  • Linearized DNA templates were used for DNA dependent RNA in vitro transcription (IVT) using T7 RNA polymerase in the presence of a sequence optimized nucleotide mixture (ATP/GTP/CTP/UTP) and cap analog (for cap1 : m7G(5’)ppp(5’)(2’OMeA)pG; TriLink) under suitable buffer conditions.
  • IVT was performed in the presence of a nucleotide mixture where UTP is replaced by N1 -Methylpseudouridine (m1MJ) or pseudouridine (MJ).
  • m1MJ N1 -Methylpseudouridine
  • MJ pseudouridine
  • RNA formulations The obtained polyethylene glycol/peptide polymers (HO-PEG 5000-S-(S-CGH5R4H5GC-S-)7-S-PEG 5000-GH), dissolved in water, were used to prepare RNA formulations. RNA was complexed with the polymer at the 1 :2 ratio (W/W). 3-C12-OH lipidoid was used. Formulated RNA was lyophilized and stored at -80°C. Particle size was measured using a zeta-sizer.
  • LNPs used in the working examples were prepared using a microfluidic system (NanoAssemblr, Precision NanoSystems) according to standard protocols which enables controlled, bottom-up, molecular self-assembly of nanoparticles via custom-engineered microfluidic mixing chips.
  • Example 2 Methods used in the experiments
  • the present example provides methods used fortesting the nucleic acid based RUNX3 transcription factor formulations (obtained according to Example 1).
  • Patient-derived PVR membranes were immediately processed after the surgery for single-cell isolation according to Delgado-Tirado et al., 2020. Briefly, C-PVR were seeded with 3 x 104A/vell density in 48 well-plates at 37°C and 5% CO2. The cells were treated with the formulation or PVR basal control (control) in triplicates for each condition and incubated for 4h. Next, the cells were washed with PBS, cultured in PVR media with the growth factors for 24h, and then collected for further analysis.
  • Primary human microvascular endothelial cells were purchased from Cell Systems (ACBR1 181). Cells were seeded with 1 x 104 and 3 x 106/well density in a 96 or 12-well plate and incubated at 37°C and 5% CO2. Cells were treated with the formulations in the same fashion than C-PVR.
  • C-PVR cells were cultured and maintained in black, clear-bottom 96-well plates. Cells were incubated with the formulations for 4h in serum free media was switched to complete media and cells incubated for 24h. 24h post treatment, the proliferation rate was measured using CyQUANT Direct Cell Proliferation Assay (ThermoFisher Scientific) according to the manufacturer's recommendation. C-PVR cells were cultured in a 24-well plate to form a confluent monolayer. Following the proliferation assay a lactate dehydrogenase (LDH) assay (Roche) was used to assess toxicity of the formulations according to the manufacturer's guidelines. For migration, a scratch wound assay was performed.
  • LDH lactate dehydrogenase
  • C- PVR or HMRECs were seeded in 12 well plates and grown to confluency. Cells were treated following the same regime as before. A scratch was made in the center of the well with a 200pl tip. After washes with PBS, fresh media was added to the wells and the wound was imaged in a EVOS M7000 imaging system (ThermoFisher Scientific).
  • Protein concentration was measured using the BCA assay. 10pg of protein were prepared in 10pl RIPA buffer, 4pl of 1M DTT and 10pl Laemmli buffer to reach a final volume of40pl. Samples were denatured for 5 min at 90 °C and were fractionated for 1 h at 70 V using 4-20% pre-cast Mini-PROTEAN TGX (Bio-Rad) and Criterion TGX (Bio-Rad) gradient gels with a SDS-Tris-Glycine buffer. Proteins were transferred to Poly (vinylidene fluoride) membranes (BIO-RAD) using a Trans-Blot® TurboTM semi-dry transfer system (BIORAD) for 7min at 25V.
  • Poly vinylidene fluoride
  • the counts matrix is filtered to only include the top 5,000 variable features. Differential expression genes analysis was used to identify the cell types and compared each cluster to all others using the Wilcoxon method in Seurat with each retained marker expressed at a minimum log fold change threshold of 0.25 for cluster-specific marker genes identification.
  • PVR induction in rabbits was carried out described before (Delgado-Tirado et al., 2020). Male New Zealand White rabbits (2.3kg of weight, 6-8-week-old) were purchased from Charles River. Briefly, gas displacement of the vitreous was induced by intravitreal injection of 0.2mL of perfluoropropane (C3F8) (Alcon) 2.5mm behind the limbus. 3d after gas injection, 0.1 mL of BSS containing approximately 1 *10 A 6 C-PVR cells were administrated via intravitreal injection along with 50pL of mRNA formulation. A second dose of 50pL of mRNA was performed 7d later.
  • C3F8 perfluoropropane
  • the eyes were enucleated, after euthanasia, and incubated in Davidson’s fixative at room temperature (Millipore Sigma). Then, a small 1 *1 mm scleral window was made to facilitate fixative penetration within the eye and were left in fixative for another 24h at room temperature. Following, the eyes were transferred to 70% ethanol. Eyes were paraffin embedded and 5pm serial sections were stained with Hematoxylin and eosin 9and assessed for retina morphology.
  • CNV laser-induced choroidal neovascularization
  • Example 3 Formulated RUNX3 mRNA induced the expression of RUNX3 and inhibited the proliferation and migration of C-PVR and HMREC
  • RNA formulations used in the experiment were prepared according to Example 1 , and methods used herein are further described in Example 2.
  • C-PVR were seeded with 3 x 10 A 4/well density in 48 well-plates and treated with 5pg/ml CVCM-formulated RUNX3 mRNA and incubated for 4 hours.
  • the cells were washed with PBS, cultured in media with the growth factors for 24h, and then collected for further analysis via western blot, proliferation, cell viability (lactate dehydrogenase (LDH) assay) and migration assays.
  • LDH lactate dehydrogenase
  • Bioanalyzer High Sensitivity DNA Assay (Agilent Technologies) was used to assess size distribution and molarity of resulting cDNA libraries which were then sequenced on an Illumina NextSeq 500 instrument according to Illumina and 10x Genomics guidelines with 1 .4-1 .8pM input and 1 % PhiX control library spike-in (Illumina).
  • HMREC HMREC were seeded with 1x10 A 4 and 3x10 A 6/well density in a 96 or 12-well plate, treated with 5pg/ml formulated RUNX3 or Luciferase mRNA and incubated for 4h.
  • the cells were washed with PBS, cultured in media with the growth factors for 24h, and collected for further analysis via western blot, proliferation, cell viability (LDH) and migration assays.
  • Feature maps showed the expression of RUNX3 across the cells and treatments.
  • Example 4 Single-cell RNA sequencing analysis of C-PVR cells treated with formulated RUNX3 mRNA Proliferative vitreoretinopathy (PVR) is an excessive wound repair response characterized by high migration capability and proliferation of cells that are vital for the EMT epithelial-to-mesenchymal transition process.
  • the aim of this experiment was to analyze the effect of RUNX3 overexpression in the C-PVR model via single cell RNA sequencing.
  • RNA formulations used in the experiment were prepared according to Example 1 , and methods used herein are further described in Example 2.
  • Figure 3A and 3B show the distribution of the cells of the two groups, control and formulated RUNX3 mRNA (RUNX3_A) treated C-PVR cells.
  • Treatment with RUNX3_A completely abolished the cluster that identifies as fibroblast (black arrow). Fibroblasts are the most common cell type represented in connective tissue and play a critical role in wound healing as tissue damage stimulates fibrocytes and induces the production of fibroblasts.
  • the treatment with formulated RUNX3 mRNA (RUNX3_A) was confirmed by the RUNX3 expression across the clusters in the RUNX3_A group compared with the control group ( Figure 3C - 3E).
  • a reduction of RUNX1 expression in formulated RUNX3 mRNA (RUNX3_A) treated cells could also be shown ( Figure 3C).
  • Fibrosis is a key component of PVR pathology. Intraretinal fibrosis leads to stiffness of the retina and can prevent the retina from flattening after surgical membrane removal.
  • Figure 3F showed a major reduction in the expression of proliferation biomarkers such as marker of proliferation KI-67 (MKI67) and proliferating cell nuclear antigen (PCNA), both actively expressed during the phase G2 and DNA synthesis of replicating cells.
  • proliferation biomarkers such as marker of proliferation KI-67 (MKI67) and proliferating cell nuclear antigen (PCNA), both actively expressed during the phase G2 and DNA synthesis of replicating cells.
  • epithelial cell markers expression like KRT7 and KRT8 were increased (Figure 3G) after treatment with formulated RUNX3_A mRNA, while mesenchymal biomarkers such as SNAI2 and CDH2 were downregulated (Figure 3H).
  • COL1 A1 a relevant fibrotic marker, was downregulated.
  • RUNX3_A mRNA modulates the transforming growth factor beta 2 (TGFB2) signaling by reducing the expression of TGFbeta2 and SMAD3 ( Figure 3H).
  • TGFB2 transforming growth factor beta 2
  • RUNX3_B mRNA CCND2, CDK4, ASPN, COL14A1 , data not shown.
  • Single-cell RNA sequencing of C-PVR cells treated with Luciferase, RUNX3 demonstrated the change in the distribution across the cell cycles (data not shown).
  • MKI67 expression a proliferation marker, localized with cells in phase G2M and S, indicating that those cells are actively proliferating.
  • RUNX1 expression is present in cells undergoing phase S and G2M, denoting the role of RUNX1 in the proliferation of C-PVR cells and the progression of the pathology (data not shown).
  • Single-cell RNA sequencing of C-PVR treated with RUNX3 demonstrated the reduction of RUNX1 expression compared with the luciferase and NC control groups.
  • Proliferation markers such as Cyclin D1 and D2 (CCND1 , CCND2), and PCNA and CDK4 were also reduced, indicating that the RUNX3 treatment inhibits proliferation of C-PVR.
  • fibrotic markers from the collagen family were downregulated with the RUNX3 therapy (Figure 3I).
  • Example 5 In vivo assessment of CVCM-formulated RUNX3 mRNA in a PVR rabbit model
  • RNA formulations used in the experiment were prepared according to Example 1 , and methods used herein are further described in Example 2.
  • Example 6 Effect of formulated RUNX3 mRNA in a laser-induced choroidal neovascularization mice model
  • the aim of this experiment was to analyze the therapeutic effect of RUNX3 on angiogenesis via the mouse laser-induced choroidal neovascularization (CNV) model.
  • CNV mouse laser-induced choroidal neovascularization
  • the mice laser-induced CNV model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research.
  • AMD neovascular age-related macular degeneration
  • RPE retinal pigment epithelium
  • Bruch’s membrane By administering targeted laser injury to the retinal pigment epithelium (RPE) and Bruch’s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD.
  • RNA formulations used in the experiment were prepared according to Example 1 , and methods used herein are further described in Example 2.
  • CVCM-formulated RUNX3_B mRNA significantly reduced the leakage associated with abnormal angiogenesis in a laser L-CNV model ( Figure 5).
  • Example 7 Expression and functionality of RUNX3 protein after mRNA-LNP transfection in vitro
  • RNA formulations used in the experiment are prepared according to Example 1 , methods used herein are described in Example 2.
  • RUNX3 protein For a dose response of RUNX3 protein expression, 25ng, 50ng, 100ng, 200ng LNP-formulated unmodified mRNA (SEQ ID: 300) and m li -modified mRNA (SEQ ID: 462) was added to HUVECs or ARPE-19 cells per well in a 96-well format in 3 copies per condition (HUVECs 8,000 cells per well, ARPE-19 15,000 cells per well). The formulations were added to seeded cells (total volume 150pL) and left on the cells for the experimental duration until cell lysis 24h after transfection. Medium-treated only cells were used as controls. RUNX3 protein was detected via capillary Western Blot using an anti-RUNX3 antibody (mouse anti-RUNX3, abeam, Cat.No. ab135248, dilution 1 :500), normalization to
  • GAPDH detected via anti-GAPDH antibody Cell signaling, Cat.No. 2118, dilution 1 :100
  • secondary antibodies Protein Simple: anti-mouse, Cat.No. 042-205 and anti-rabbit, Cat.No. 042-206, both undiluted Cell signaling, Cat.No. 2118, dilution 1 :100
  • the respective cells 4000 HUVECs were seeded in 10OpI culture medium (2% FCS and full VEGF 10ng/ml) in 96-well format. Transfection was performed with 50pl LNPs (dose 50ng and 10Ong per 96-well). As a positive control, 10% FBS was used to induce proliferation. For inhibiting proliferation 2pg/ml in DMSO Mitomycin was added. DMSO was also used as vehicle control (for Mitomycin). The proliferation assay was performed in Incucyte and imaging was done every 3 hours up to 72 hours.
  • C-PVR patient-derived PVR cells
  • HMRECs primary human retinal microvascular endothelial cells
  • RUNX3 positive cells localized with cells in G1 phase (Example 3, Figure 1 and 2).
  • transfection using formulated RUNX3 mRNA reduces proliferation markers expression as well as a modulation of EMT and fibrotic markers in C-PVR could be detected.
  • EMT leads to the differentiation of fibroblast/myofibroblast contributing to the accumulation of fibrous connective tissue in damaged tissue, which generates permanent scarring or organ malfunction.
  • the downregulation of EMT contributes to resolving the pathology in ocular diseases such as PVR.
  • Intravitreal injection of formulated RUNX3 mRNA to a PVR model in rabbits effectively reduced the pathology severity in vivo and decreased the accumulation of fibrotic membranes on top of the retina (Example 5, Figure 4).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne, entre autres, des constructions d'acide nucléique artificielles, de préférence de l'ARN, comprenant au moins une séquence codante codant pour un facteur de transcription RUNX3 ou un fragment ou un variant de celui-ci, l'acide nucléique comprenant de préférence au moins une région non traduite (UTR) hétérologue. L'invention concerne en outre des compositions pharmaceutiques comprenant l'acide nucléique, de préférence formulées en des polymères de polyéthylène glycol/peptide, des supports polymères ou des supports à base de lipides. L'invention concerne également des méthodes de traitement ou de prévention de troubles, de maladies ou d'affections, et des utilisations médicales, en particulier des troubles, maladies ou affections oculaires tels que la vitréorétinopathie proliférative (PVR) ou des membranes épirétiniennes (EPR), à l'aide des acides nucléiques codant pour RUNX3 décrits ici.
PCT/EP2024/071649 2023-07-31 2024-07-31 Facteur de transcription runx3 codé par un acide nucléique Pending WO2025027060A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363516617P 2023-07-31 2023-07-31
US63/516,617 2023-07-31

Publications (1)

Publication Number Publication Date
WO2025027060A1 true WO2025027060A1 (fr) 2025-02-06

Family

ID=92214397

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/071649 Pending WO2025027060A1 (fr) 2023-07-31 2024-07-31 Facteur de transcription runx3 codé par un acide nucléique

Country Status (1)

Country Link
WO (1) WO2025027060A1 (fr)

Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002098443A2 (fr) 2001-06-05 2002-12-12 Curevac Gmbh Composition pharmaceutique contenant un arnm stabilise et optimise pour la traduction dans ses regions codantes
WO2008077592A1 (fr) 2006-12-22 2008-07-03 Curevac Gmbh Procédé de purification d'arn à l'échelle préparative par hplc
WO2009030481A1 (fr) 2007-09-04 2009-03-12 Curevac Gmbh Complexes d'arn et de peptides cationiques pour transfection et immunostimulation
WO2011026641A1 (fr) 2009-09-03 2011-03-10 Curevac Gmbh Conjugués de polyéthylèneglycol/peptide à liaison disulfure pour la transfection d'acides nucléiques
WO2012019780A1 (fr) 2010-08-13 2012-02-16 Curevac Gmbh Acide nucléique comprenant ou codant pour une tige-boucle d'histone et une séquence poly(a) ou un signal de polyadénylation pour augmenter l'expression d'une protéine codée
WO2015101416A1 (fr) 2013-12-30 2015-07-09 Curevac Gmbh Procédés d'analyse d'arn
WO2016180430A1 (fr) 2015-05-08 2016-11-17 Curevac Ag Procédé de production d'arn
WO2016193206A1 (fr) 2015-05-29 2016-12-08 Curevac Ag Procédé de production et de purification d'arn, comprenant au moins une étape de filtration à flux tangentiel
WO2017053297A1 (fr) 2015-09-21 2017-03-30 Trilink Biotechnologies, Inc. Compositions et procédés de synthèse d'arn coiffés en 5'
WO2017191274A2 (fr) * 2016-05-04 2017-11-09 Curevac Ag Arn codant pour une protéine thérapeutique
WO2017212009A1 (fr) 2016-06-09 2017-12-14 Curevac Ag Supports hybrides pour cargo d'acides nucléiques
WO2018078053A1 (fr) 2016-10-26 2018-05-03 Curevac Ag Vaccins à arnm à nanoparticules lipidiques
WO2018093797A1 (fr) 2016-11-15 2018-05-24 The Schepens Eye Research Institute, Inc. Compositions et méthodes pour le traitement de l'angiogenèse aberrante
WO2019077001A1 (fr) * 2017-10-19 2019-04-25 Curevac Ag Nouvelles molécules d'acide nucléique artificielles
WO2019099560A1 (fr) 2017-11-14 2019-05-23 The Schepens Eye Research Institute, Inc. Inhibition de runx1 pour le traitement de la vitréorétinopathie proliférative et d'états associés à une transition épithéliale à mésenchymateuse
WO2019099595A1 (fr) 2017-11-14 2019-05-23 The Schepens Eye Research Institute, Inc. NANO-ÉMULSION D'INHIBITEUR DE CBFβ-RUNX1 POUR L'ADMINISTRATION DE MÉDICAMENTS POUR LES YEUX
WO2020145465A1 (fr) * 2019-01-08 2020-07-16 주식회사바이오러넥스 Composition pharmaceutique comprenant un gène ou une protéine runx3 en tant que principe actif pour la prévention ou le traitement d'un cancer du poumon avec mutation du gène k-ras
WO2020161342A1 (fr) * 2019-02-08 2020-08-13 Curevac Ag Arn codant administré dans l'espace suprachoroïdien pour le traitement de maladies ophtalmiques
WO2021123332A1 (fr) 2019-12-20 2021-06-24 Curevac Ag Nanoparticules lipidiques pour l'administration d'acides nucléiques
WO2021156267A1 (fr) 2020-02-04 2021-08-12 Curevac Ag Vaccin contre un coronavirus
WO2021216378A1 (fr) 2020-04-23 2021-10-28 The Schepens Eye Research Institute, Inc. Procédés et matériaux pour le traitement de la fibrose
WO2021239880A1 (fr) 2020-05-29 2021-12-02 Curevac Ag Vaccins combinés à base d'acide nucléique
WO2023007019A1 (fr) 2021-07-30 2023-02-02 CureVac SE Analogues de coiffe ayant un lieur acyclique à la nucléobase dérivée de guanine
WO2023031394A1 (fr) 2021-09-03 2023-03-09 CureVac SE Nouvelles nanoparticules lipidiques pour l'administration d'acides nucléiques
WO2023073228A1 (fr) 2021-10-29 2023-05-04 CureVac SE Arn circulaire amélioré pour exprimer des protéines thérapeutiques
WO2023144330A1 (fr) 2022-01-28 2023-08-03 CureVac SE Inhibiteurs de facteurs de transcription codés par un acide nucleique

Patent Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002098443A2 (fr) 2001-06-05 2002-12-12 Curevac Gmbh Composition pharmaceutique contenant un arnm stabilise et optimise pour la traduction dans ses regions codantes
WO2008077592A1 (fr) 2006-12-22 2008-07-03 Curevac Gmbh Procédé de purification d'arn à l'échelle préparative par hplc
WO2009030481A1 (fr) 2007-09-04 2009-03-12 Curevac Gmbh Complexes d'arn et de peptides cationiques pour transfection et immunostimulation
WO2011026641A1 (fr) 2009-09-03 2011-03-10 Curevac Gmbh Conjugués de polyéthylèneglycol/peptide à liaison disulfure pour la transfection d'acides nucléiques
WO2012019780A1 (fr) 2010-08-13 2012-02-16 Curevac Gmbh Acide nucléique comprenant ou codant pour une tige-boucle d'histone et une séquence poly(a) ou un signal de polyadénylation pour augmenter l'expression d'une protéine codée
WO2015101416A1 (fr) 2013-12-30 2015-07-09 Curevac Gmbh Procédés d'analyse d'arn
WO2016180430A1 (fr) 2015-05-08 2016-11-17 Curevac Ag Procédé de production d'arn
WO2016193206A1 (fr) 2015-05-29 2016-12-08 Curevac Ag Procédé de production et de purification d'arn, comprenant au moins une étape de filtration à flux tangentiel
WO2017053297A1 (fr) 2015-09-21 2017-03-30 Trilink Biotechnologies, Inc. Compositions et procédés de synthèse d'arn coiffés en 5'
WO2017191274A2 (fr) * 2016-05-04 2017-11-09 Curevac Ag Arn codant pour une protéine thérapeutique
WO2017212009A1 (fr) 2016-06-09 2017-12-14 Curevac Ag Supports hybrides pour cargo d'acides nucléiques
WO2018078053A1 (fr) 2016-10-26 2018-05-03 Curevac Ag Vaccins à arnm à nanoparticules lipidiques
WO2018093797A1 (fr) 2016-11-15 2018-05-24 The Schepens Eye Research Institute, Inc. Compositions et méthodes pour le traitement de l'angiogenèse aberrante
WO2019077001A1 (fr) * 2017-10-19 2019-04-25 Curevac Ag Nouvelles molécules d'acide nucléique artificielles
WO2019099560A1 (fr) 2017-11-14 2019-05-23 The Schepens Eye Research Institute, Inc. Inhibition de runx1 pour le traitement de la vitréorétinopathie proliférative et d'états associés à une transition épithéliale à mésenchymateuse
WO2019099595A1 (fr) 2017-11-14 2019-05-23 The Schepens Eye Research Institute, Inc. NANO-ÉMULSION D'INHIBITEUR DE CBFβ-RUNX1 POUR L'ADMINISTRATION DE MÉDICAMENTS POUR LES YEUX
WO2020145465A1 (fr) * 2019-01-08 2020-07-16 주식회사바이오러넥스 Composition pharmaceutique comprenant un gène ou une protéine runx3 en tant que principe actif pour la prévention ou le traitement d'un cancer du poumon avec mutation du gène k-ras
WO2020161342A1 (fr) * 2019-02-08 2020-08-13 Curevac Ag Arn codant administré dans l'espace suprachoroïdien pour le traitement de maladies ophtalmiques
WO2021123332A1 (fr) 2019-12-20 2021-06-24 Curevac Ag Nanoparticules lipidiques pour l'administration d'acides nucléiques
WO2021156267A1 (fr) 2020-02-04 2021-08-12 Curevac Ag Vaccin contre un coronavirus
WO2021216378A1 (fr) 2020-04-23 2021-10-28 The Schepens Eye Research Institute, Inc. Procédés et matériaux pour le traitement de la fibrose
WO2021239880A1 (fr) 2020-05-29 2021-12-02 Curevac Ag Vaccins combinés à base d'acide nucléique
WO2023007019A1 (fr) 2021-07-30 2023-02-02 CureVac SE Analogues de coiffe ayant un lieur acyclique à la nucléobase dérivée de guanine
WO2023031394A1 (fr) 2021-09-03 2023-03-09 CureVac SE Nouvelles nanoparticules lipidiques pour l'administration d'acides nucléiques
WO2023073228A1 (fr) 2021-10-29 2023-05-04 CureVac SE Arn circulaire amélioré pour exprimer des protéines thérapeutiques
WO2023144330A1 (fr) 2022-01-28 2023-08-03 CureVac SE Inhibiteurs de facteurs de transcription codés par un acide nucleique

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. NM 001 031680.2
"UniProt", Database accession no. Q01196-11
CECILIA WEI LIN LEE ET AL: "RUNX3 functions as an oncogene in ovarian cancer", GYNECOLOGIC ONCOLOGY, ACADEMIC PRESS, LONDON, GB, vol. 122, no. 2, 28 April 2011 (2011-04-28), pages 410 - 417, XP028380605, ISSN: 0090-8258, [retrieved on 20110503], DOI: 10.1016/J.YGYNO.2011.04.044 *
IKUNO YASUSHI ET AL: "An in vivo gene therapy approach for experimental proliferative vitreoretinopathy using the truncated pletelet-derived growth factor [alpha] receptor", INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, vol. 43, no. 7, 1 July 2002 (2002-07-01), US, pages 2406 - 2411, XP093218392, ISSN: 0146-0404 *
LU XI ET AL: "RUNX3 expression in the retina and its effect on retinal neovascularization | IOVS | ARVO Journals", 1 June 2022 (2022-06-01), XP093217295, Retrieved from the Internet <URL:https://iovs.arvojournals.org/article.aspx?articleid=2780129> *
no. 2089251-47-6
YOKOMIZO-NAKANO TAKAKO ET AL: "Overexpression of RUNX3 Represses RUNX1 to Drive Transformation of Myelodysplastic Syndrome", CANCER RESEARCH, vol. 80, no. 12, 15 June 2020 (2020-06-15), San Diego, CA . Philadelphia (PA, pages 2523 - 2536, XP093218385, ISSN: 0008-5472, Retrieved from the Internet <URL:https://aacrjournals.org/cancerres/article-pdf/80/12/2523/2871720/2523.pdf> DOI: 10.1158/0008-5472.CAN-19-3167 *

Similar Documents

Publication Publication Date Title
JP7234283B2 (ja) 翼状片を治療するための組成物及び方法
AU2008289552B2 (en) Therapeutic compositions for treatment of inflammation of ocular and adnexal tissues
JP7416700B2 (ja) 増殖性硝子体網膜症および上皮間葉転換と関連付けられる状態の治療のためのｒｕｎｘ１阻害の方法
CN107109410B (zh) 通道调节剂
JP2011516400A5 (fr)
KR101951787B1 (ko) mTOR 저해제를 함유하는 황반변성 치료용 의약조성물
CN114845718B (zh) 用于治疗与过度血管形成相关的眼部疾病的化合物
TW201641105A (zh) 醫藥組成物及其用途
Shahlaee et al. Translational and clinical advancements in management of proliferative vitreoretinopathy
Gu et al. Geniposide alleviates choroidal neovascularization by downregulating HB-EGF release from RPE cells by downregulating the miR-145-5p/NF-κB axis
Daftarian et al. Intravitreal connective tissue growth factor neutralizing antibody or bevacizumab alone or in combination for prevention of proliferative vitreoretinopathy in an experimental model
Ding et al. Evaluation of nintedanib efficacy: Attenuating the lens fibrosis in vitro and vivo
WO2025027060A1 (fr) Facteur de transcription runx3 codé par un acide nucléique
US20150148383A1 (en) Methods for Inhibiting or Reversing Epiretinal Membrane Formation
CA2640986C (fr) Compositions therapeutiques pour le traitement de l&#39;inflammation des tissus oculaires et annexiels
AU2014227534B2 (en) Therapeutic compositions for treatment of inflammation of ocular and adnexal tissues
US20220280473A1 (en) Compositions and methods for modulating epithelial-mesenchymal transition
Mautino et al. Side effects of drugs used in ocular treatment
이석재 Studies on Treatment of Disease Phenotypes of Vision-Threatening Ocular Disease Mouse Models via Intraocular Drugs Delivery
EA045318B1 (ru) Соединения для лечения глазных болезней, связанных с избыточной васкуляризацией
CN113768929A (zh) Fdi化合物在眼科疾病中的用途
BR112017025122B1 (pt) Uso de nintedanib para tratar pterígio

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24751662

Country of ref document: EP

Kind code of ref document: A1