WO2025027060A1 - Nucleic acid encoded runx3 transcription factor - Google Patents

Abstract

The present invention is inter alia directed to artificial nucleic acid constructs, 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). Further provided are pharmaceutical compositions comprising the nucleic acid, preferably formulated in polyethylene glycol/peptide polymers, polymeric carriers, or lipid-based carriers. Also provided are methods of treating or preventing disorders, diseases, or conditions, and medical uses, in particular ocular disorders, diseases, or conditions such as proliferative vitreoretinopathy (PVR) or epiretinal membranes (EPR), using the RUNX3 encoding nucleic acids described herein.

Description

Nucleic acid encoded RUNX3 transcription factor

Introduction

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. However, the use of 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.

Accordingly, transcription factors may represent powerful therapeutic targets for treating or preventing numerous diseases, and nucleic add sequences, for example RNA, may represent a promising class of molecules to provide the information for expressing intracellular proteins such as transcription factors.

Thus, 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. The objects mentioned above are solved by the underlying description and the accompanying claims.

Short description of the invention

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). Further provided are pharmaceutical compositions comprising the nucleic add, preferably formulated in polyethylene glycol/peptide polymers, polymeric carriers, or lipid-based carriers. Also provided are methods of treating or preventing disorders, diseases, or conditions, and medical uses, in particular ocular disorders, diseases, or conditions such as proliferative vitreoretinopathy (PVR) or epiretinal membranes (EPR), using the RUNX3 nucleic acids described herein.

An epiretinal membrane (ERM) is 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. 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.

As shown in the Example section, 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. In a screening approach, several transcription factors have been identified capable of reducing RUNX1 expression and/or activity. Surprisingly, 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. The inventors generated an effective nucleic acid that suitable for transiently expressing RUNX3. Without wishing to be bound to theory, 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. Surprisingly, after administration of the nucleic acid, the produced 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). In addition, RUNX3 reduced cell clusters identified as fibroblasts, as well as lead to a reduction of proliferation marker expression (Figure 3). As further shown herein, intraocular administration of the RNA-encoded RUNX3 strongly reduced proliferation and ocular pathology triggered by injection of human PVR cells in a rabbit eye (Figure 4). Additionally, formulated RUNX3 mRNA reduced lesion size in a laser-CNV mouse model, 7 days after treatment (Figure 5). Moreover, LNP formulated mRNA encoding RUNX3 was effectively expressed in various and reduced proliferation in HUVEC cells (Figures 6-7).

Accordingly, the present invention therefore demonstrates that nuclei acid, in particular 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. In particular, nuclei acid such as RNA providing RUNX3 may be used in treating or preventing ocular diseases, e.g. PVR and/or epiretinal membranes (ERM).

In a first aspect, 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. Preferably, the nucleic add is an RNA, more preferably an mRNA. Preferably, the RUNX3 transcription factor is human.

In a second aspect, 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.

Preferably, the nucleic add is formulated in polyethylene glycol/peptide polymers, polymeric carriers, or lipid-based carriers (e.g. LNPs). Suitably, the formulation is selected from LNPs.

In a third aspect, 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.

In further aspects provide methods of treating or preventing disease, disorder or condition and first and further medical uses of the nucleic acid, the pharmaceutical composition, or the kit or kit of parts. Preferably, 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. Brief description of the figures

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.

Figure 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. RUNX3 overexpression induced notable changes in the profile of hallmark EMT genes as shown by featured dot plots of proliferation markers (3F), epithelial markers (3G), and mesenchymal markers (3H). Single-cell RNA sequencing of C-PVR treated with RUNX3 CVCM1-B 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. Additionally, several 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.

Figure 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.

Figure 6: Dose-dependent RUNX3 protein expression in HUVEC and ARPE-19 cells for LNP-fiormulated ml i - modified mRNA (6A and 6C) and unmodified mRNA (6B and 6D). Further information is provided in the Example section, Example 7.

Figure 7: Proliferation in HUVECs after transfection with LNP-formulated RUNX3 mRNA. Further information is provided in the Example section, Example 7. Definitions

For the sake of clarity and readability the following definitions are provided. Any technical feature mentioned for these definitions may be read on each and every embodiment of the invention. Additional definitions and explanations may be specifically provided in the context of these embodiments.

Percentages in the context of numbers should be understood as relative to the total number of the respective items.

About: The skilled person knows that e.g. certain parameters or determinants can slightly vary based on the method by which the parameter is determined. As used herein “about” refers to this small variance in a determinant or value based on the method used to measure the determinant or value.

Angiogenesis: The term 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. Moreover, 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.

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. Suitably in the context of the invention, the cds encodes at least one RUNX3 transcription factor, or a fragment or variant thereof.

Core binding factors: The term “Core binding factors” or “CBFs” 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.

Derived from: The term “derived from” as used herein in the context of a nucleic acid, i.e. for a nucleic acid “derived from” (another) nucleic acid, means that the 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. In the context of amino acid sequences, 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 (EMT): 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. 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. Thus, 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. Many transcription factors (TFs) that can repress E-cadherin directly or indirectly are considered as 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) and krueppel-like factor 8 (KLF8) can bind to the E-cadherin promoter and repress its transcription, whereas factors such as Twist (also referred to as class A basic helix-loop-helix protein 38; bHLHa38), Goosecoid, transcription factor 4 (TCF4), homeobox protein Sineoculis homeobox homolog 1 (SIX1) and fork-head box protein C2 (FOXC2) repress E-cadherin indirectly. Several signaling pathways (transforming growth factor beta (TGFbeta), fibroblast growth factor (FGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), Wnt/beta-catenin and Notch) and hypoxia may induce EMT. In particular, Ras-MAPK (mitogen- activated protein kinases) activates Snail and Slug. 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. 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. However, on the other hand, tumor protein 53 (p53), a well-known tumor suppressor, 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. After the initial stage of embryogenesis, 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. Later in embryogenesis, during gastrulation, 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. During wound healing, 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. Similarly, in each menstrual cycle, 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. 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. Cells that undergo EMT gain stem cell-like properties, thus giving rise to cancer stem cells (CSCs).

Fibrosis: The term "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. Repeated injuries, 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. Physiologically, 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. The most well characterized 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. However, the entire process, although necessary, can lead to a progressive irreversible fibrotic response if tissue injury is severe or repetitive, or if the wound healing response itself becomes deregulated. 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: The term “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.

Identity (of a sequence): The term “identity” as used herein in the context of a nucleic acid sequence, or an amino acid sequence, 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: The term “neovascularization” has to be understood as the (natural) process of formation of new blood vessels. Typically, 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. Typically, there are three different pathways that comprise neovascularization: (1) vasculogenesis, (2) angiogenesis, and (3) arteriogenesis. Several 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: The term “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. 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: The term “RNA in vitro transcription” (IVT) relates to a process wherein RNA is synthesized in a cell-free system in vitro. In IVT, 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).

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. E.g., 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.

The term “variant' as used herein in the context of proteins or peptides 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). Preferably, 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.

Detailed description of the invention

The present application is filed together with a sequence listing in electronic format, which is part of the description (WIPO standard ST.26). Where reference is made to “SEQ ID NOs” of other patent applications or patents, said sequences, e.g. amino acid sequences or nucleic acid sequences, are explicitly incorporated herein by reference.

1. Nucleic acid encoding at least one RUNX3 transcription factor

In a first aspect, 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. It has to be noted that 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.

The 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. In mammals, three RUNX genes have been identified, including RUNX1 , RUNX2 and RUNX3. 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. 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.

In particularly preferred embodiments, 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.

The term “artificial nucleic acid” as used herein refers to a nucleic acid that does not occur naturally. In other words, 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. Typically, an artificial nucleic acid may be designed and/or generated by genetic engineering to correspond to a desired artificial sequence of nucleotides. In this context, an artificial nucleic acid is a sequence that may not occur naturally, i.e. a sequence that differs from the wild type sequence or reference sequence by at least one nucleotide. The term “artificial nucleic acid” is not restricted to mean “one single molecule” but is understood to comprise an ensemble of essentially identical nucleic acid molecules. The term “artificial nucleic acid” as used herein may relate to artificial DNA or, preferably, to artificial RNA.

In embodiments, 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).

In preferred embodiments, the full length RUNX3 protein is RUNX3 protein variant or protein isoform 1 .

In preferred embodiments, 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.

Preferably, N-terminally an/or a C-terminally truncated RUNX3 protein fragments as defined herein comprise at least the Runt domain (RD).

In preferred embodiments, the Runt domain (RD) recognizes and binds the DNA motif or DNA core consensus binding sequence 5’- TGTGGT-3’ or 5-TGCGGT-3’.

In other preferred embodiments, the Runt domain (RD) 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. Despite that 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.

In other preferred embodiments, 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.

In preferred embodiments, 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.

Preferably, 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.

For example, although 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. In some embodiments, 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 . The following 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

In preferred embodiments, 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. In preferred embodiments, 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. In particularly preferred embodiments, 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.

In preferred embodiments, 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.

Accordingly, the administration of the nucleic add (e.g. RNA) to a cell, tissue, or subject leads to a translation of the at least one cds into at least one transcription factor protein. Whenever reference is made to a “produced transcription factor”, 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. Accordingly, functional and structural features and embodiments that are described herein relating to the “RUNX3 transcription factor'’ or relating to the “(produced) RUNX3 transcription factor'’ should be understood to refer to RUNX3 transcription factor proteins as defined herein that are produced/translated in the cytosol upon administration of the nucleic acid of the invention to a cell, tissue, or subject. In embodiments, 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.

Preferred in that context is 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).

In other embodiments, 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).

In embodiments, the RUNX3 transcription factor, or a fragment or variant thereof (that is provided by the nucleic acid) 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.

In preferred embodiments, 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). For example, for example the RUNX3 that comprises residues 68 to 196 may comprise an amino acid sequence according to SEQ ID NO: 134. For example, the RUNX3 that comprises residues 76 to 197 may comprise an amino acid sequence according to SEQ ID NO: 136. For example, the RUNX3 that comprises residues 68 to 201 may comprise an amino acid sequence according to SEQ ID NO: 138. For example, for example 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 .

In preferred embodiments the amino add sequence of RUNX3 comprises the full-length isoform 1 amino add sequence according to SEQ ID NO: 129. For example, the preferred RUNX3 comprises the Runt domain (RD) sequence according to SEQ ID NO: 134. For example, the preferred RUNX3 comprises the Runt domain (RD) sequence according to SEQ ID NO: 136. For example, the preferred RUNX3 comprises the Runt domain (RD) sequence according to SEQ ID NO: 138. For example, 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.

In other embodiments, the RUNX3 transcription factor, or a fragment or variant thereof (that is provided by the nucleic add) 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. In preferred embodiments, the RUNX3 transcription factor, or a fragment or variant thereof (that is provided by the nucleic add) 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.

In preferred embodiments, the RUNX3 transcription factor (that is provided by the nucleic acid) 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. 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, epireti nal fibrosis, and gliosis. Other conditions associated with EMT including cancer, e.g., mesothelioma, ocular chronic graft-versus-host disease, corneal scarring, corneal epithelial downgrowth, conjunctival scarring, eye tumors such as melanoma and metastatic tumors, or fibrosis.

In preferred embodiments, 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.

In other preferred embodiments, the RUNX3 (provided by the nucleic acid) reduces proliferation and/or migration of retinal pigment epithelial (RPE) cells in a subject. 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. In other preferred embodiments, the RUNX3 (provided by the nucleic acid) reduces EMT in a subject.

In preferred embodiments, the RUNX3 (provided by the nucleic acid) displays an anti-angiogenic effect in a subject. Preferably, 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.

In preferred embodiments, 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.

The term “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. In preferred embodiments, the target transcription factor is selected from a transcription factor that has an aberrant transcription factor activity or pathologic transcription factor activity. Suitably, the aberrant or pathologic target transcription factor activity is an overexpression and/or an overactivation.

In preferred embodiments, 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. In preferred embodiments, the target transcription factor is selected from a transcription factor that is overexpressed and/or overactive in a disease, disorder, or condition. In preferred embodiments, the target transcription factor is selected from a transcription factor that is overexpressed or overactive in an ocular disease, disorder, or condition. In preferred embodiments, 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.

In particularly preferred embodiments, the target transcription factor is RUNX1 .

Accordingly, in particularly preferred embodiments, 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.

Accordingly, in embodiments of the invention, the nucleic acid encoding the RUNX3 transcription factor as defined herein is a RUNX1 antagonist or a RUNX1 inhibitor.

Runt-related transcription factor 1 (RUNX1), 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. As described above, 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 .

In preferred embodiments, upon administration of the nucleic add to a cell or subject, the produced RUNX3 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.

Accordingly, in preferred embodiments, upon administration of the nucleic acid to a cell or subject, the produced RUNX3 reduces the cellular expression of RUNX1 controlled genes or gene products.

In particularly preferred embodiments, upon administration of the nucleic acid to a cell or subject, the produced RUNX3 reduces the cellular expression of TGFbeta2 (TGF|32), SMAD3, and/or COL1 A1. In preferred embodiments, upon administration of the nucleic acid to a cell or subject, the produced RUNX3 increase the transcription rate of MARVELD2. MARVELD2 is a tight junction associated epithelial marker, as a predictor of the future state of the cell.

In preferred embodiments, upon administration of the nucleic acid to a cell or subject, the produced RUNX3, reduces or prevents pathological EMT, pathological cell proliferation, aberrant angiogenesis, aberrant (ocular) neovascularization, degeneration, and/or fibrosis. In embodiments, the administered nucleic acid encoding the RUNX3 transcription factor reduces or prevents pathological EMT, pathological cell proliferation, aberrant angiogenesis, aberrant (ocular) neovascularization, degeneration, and/or fibrosis in a more effective way as a small molecule inhibitor of RUNX (e.g. Ro5-335) or at least comparably effective as a small molecule inhibitor of RUNX (e.g. Ro5-335).

In preferred embodiments, upon administration of the nucleic acid to a cell or subject, the produced RUNX3 transcription factor reduces the cellular expression of the RUNX1 target transcription factor. In cells, 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). As 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.

Suitable coding sequences

According to preferred embodiments, the nucleic acid of the invention comprises at least one cds encoding at least one RUNX3 transcription factor or a fragment or variant thereof. In that context, any cds encoding at least one RUNX3 transcription factor as defined herein, or fragments and variants thereof, may be understood as suitable cds and may therefore be comprised in the nucleic add of the invention.

In embodiments, 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

In embodiments, 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.

In embodiments, 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. Accordingly, in preferred embodiments in this context, 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. In preferred embodiments, the nucleic acid is a modified and/or stabilized nucleic acid. According to preferred embodiments, 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.

Preferably, the nucleic of the invention may be provided as a “stabilized nucleic acid”, preferably a “stabilized RNA”.

In the following, suitable modifications/adaptations are described that are capable of “stabilizing” the nucleic acid.

In particularly preferred embodiments, the nucleic add comprises at least one codon modified cds. Suitably, 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.

The term “codon modified cds” as used herein 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. Suitably, 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.

In particularly preferred embodiments, 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.

In particularly preferred embodiments, the nucleic acid may be modified, wherein the G/C content of the at least one cds may be optimized compared to the G/C content of the corresponding wild type or reference cds (herein referred to as “G/C optimized cds”). “Optimized” in that context refers to a cds wherein the G/C content is preferably increased to the essentially highest possible G/C content. The generation of a G/C content optimized nucleic acid sequence may be carried out using a method according to W02002098443. In this context, the disclosure of W02002098443 is included in its full scope in the present invention. G/C optimized cds are indicated by the abbreviations “opt1”.

In preferred embodiments, the at least one cds has a G/C content of at least about 50%, 55%, or 60%. In particular embodiments, the at least one cds has a G/C content of at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%.

In preferred embodiments, 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.

In particularly preferred embodiments, 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.

UTR

Preferably, 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.

The term “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.

In preferred embodiments, 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.

The term “untranslated region” or “UTR” or “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.

In preferred embodiments, the nucleic acid comprises a cds, and a 5-UTR and/or 3-UTR. Notably, UTRs may harbour regulatory sequence elements that determine RNA turnover, stability, and localization. Moreover, UTRs may harbour sequence elements that enhance translation. In medical applications, 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.

Suitably, 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. In preferred embodiments, 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.

In preferred embodiments, 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.

Preferably, the nucleic acid comprises at least one 3 -UTR, which may be derivable from a gene that relates to an RNA with enhanced half-life (i.e. that provides a stable RNA).

In some embodiments, 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.

In some embodiments, the at least one 3-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: 66-123, or a fragment or a variant of any of these. In preferred embodiments, 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. In preferred embodiments, 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. In particularly preferred embodiments, the nucleic acid comprises a 3’-UTR derived or selected from a PSMB3 gene. Preferably, the at least one heterologous 3’-UTR derived or selected from PSMB3, 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 ofSEQ ID NOs: 66, 67, 112-123, or a fragment or a variant thereof, preferably SEQ ID NO: 67, or a fragment or a variant thereof.

In preferred embodiments, 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. Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites.

Preferably, 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).

In some embodiments, 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).

In some embodiments, 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.

In preferred embodiments, 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. In preferred embodiments, 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. In particularly preferred embodiments, the nucleic acid comprises a 5 -UTR derived or selected from a HSD17B4 gene. Preferably, 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.

In embodiments, 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. In particularly preferred embodiments, 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)).

Accordingly, in particularly preferred embodiments, 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)). It has been shown by the inventors that this embodiment is particularly beneficial for expressing the RUNX3 transcription factor in human cells of the eye e.g. retinal pigment epithelium (RPE) cells.

In various embodiments, the nucleic acid is monocistronic, bidstronic, or multicistronic, preferably monocistronic.

In preferred embodiments, 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. In preferred embodiments, the “Kozak sequence” comprises or consists of RNA sequence ACC.

Poly(N)sequences, histone stem loops:

In preferred embodiments, 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.

In preferred embodiments, the nucleic add, e.g. the RNA, comprises at least one poly(A) sequence. In some embodiments, the nucleic acid comprises least two, three, or more poly(A) sequences.

The term “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.

In embodiments, 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.

In particularly preferred embodiments, the at least one poly(A) sequence comprises about 100 adenosine nucleotides (A100), preferably about 100 consecutive adenosine nucleotides.

In alternative embodiments, the nucleic acid comprises at least one interrupted poly(A) sequence wherein the poly(A) sequence is interrupted by at least one non-adenosine nucleotide (N), preferably by about 10 non-adenosine (N10) nucleotides. In that context, a poly(A) sequence A30-N10-A70 is preferred.

In preferred embodiments, the at least one poly(A) sequence as defined herein is located directly at the 3’ terminus of the nucleic acid, preferably the RNA. Accordingly, 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. In other words, 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. Advantageously, 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.

In particularly preferred embodiments, the at least one poly(A) sequence comprises about 100 adenosine nucleotides (A100), preferably about 100 consecutive adenosine nucleotides. In preferred embodiments, the at least one poly(A) sequence is obtained from a DNA template during RNA in vitro transcription. In other embodiments, the at least one poly(A) sequence is obtained in vitro by common methods of chemical synthesis. Alternatively, the at least one poly(A) sequence is generated by enzymatic polyadenylation of the RNA (after RNA in vitro transcription). In some embodiments, the at least one poly(A) sequence is obtained by enzymatic polyadenylation, wherein the majority of RNA molecules preferably comprise about 100 (+/- 20) to about 500 (+/- 100) adenosine nucleotides, preferably about 100 (+/- 20) to about 200 (+/- 40) adenosine nucleotides.

In preferred embodiments, the nucleic acid, preferably the RNA, comprises at least one histone stem-loop (hSL). A 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. According, the nucleic acid may comprise at least one hSL sequence derived from the specific formulae (la) or (Ila) of W02012019780. In preferred embodiments, 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.

In preferred embodiments, 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. In embodiments, 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.

In preferred embodiments, the nucleic acid is an isolated nucleic acid. The term “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. The “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.

In particularly preferred embodiments, 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.

In embodiments, the nucleic acid of the invention is selected from a DNA or an RNA.

In some embodiments, 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. In preferred embodiments, the DNA a viral DNA, preferably an adeno-assodated virus DNA.

In preferred embodiments, 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. In embodiments, the RNA is selected from mRNA, circular RNA, replicon RNA or self-replicating RNA, or viral RNA.

In embodiments, the RNA is a circular RNA. As “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. In other embodiments, 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.

In particularly preferred embodiments, the nucleic acid of the invention is an mRNA.

In the context of the invention, 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. Preferably, the mRNA of the invention is non-replicative.

In embodiments, 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.

Modified nucleotides

According to various embodiments, the nucleic add, preferably the RNA, is modified, wherein the modification refers to chemical modifications comprising backbone modifications as well as sugar modifications or base modifications.

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.

Accordingly, in preferred embodiments, the nucleic acid is an RNA that comprises at least one modified nucleotide.

In embodiments, 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. In embodiments, 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.

Particularly preferred in that context are pseudouridine (ip) and N1 -methylpseudouridine (m1ip). Accordingly, in preferred embodiments, the nucleic acid is an RNA that comprises at least one modified nucleotide, preferably a modified nucleotide selected from pseudouridine (ip) or N1 -methylpseudouridine (m1ip). In some embodiments, essentially all, e.g. essentially 100% of the uracil in the cds (or the full nucleic acid sequence) have a chemical modification, preferably a chemical modification in the 5-position of the uracil. In preferred embodiments, 100% of the uracil in the frill nucleic acid sequence, preferably the RNA sequence, are substituted with N1 -methylpseudouridine (m1ip). Alternatively, 100% of the uracil in the frill nucleic add sequence, preferably the RNA sequence are substituted with pseudouridine (ip).

In alternative embodiments, 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. In such embodiments, the nucleic acid is an RNA comprising a sequence consisting of G, C, A and U, and optionally, comprises a 5’-cap structure.

Cap structure

In preferred embodiments, the nucleic acid, preferably the RNA, comprises a 5’-cap structure.

Accordingly, in preferred embodiments, the nucleic acid, preferably the RNA, comprises a 5’-cap structure, preferably m7G, capO, cap1 , cap2, a modified capO or a modified cap1 structure. In preferred embodiments, 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.

Suitably, the 5’-cap (capO or cap1) structure may be formed in RNA in vitro transcription using a cap analog.

The term “cap analogue” refers to a non-polymerizable di-nucleotide or tri-nucleotide that has cap functionality in that it facilitates translation or localization, and/or prevents degradation of an RNA molecule when incorporated at the 5’-end of the molecule. Non-polymerizable means that the cap analog will be incorporated only at the 5’-terminus because it does not have a 5’ triphosphate and therefore cannot be extended in the 3’-direction e.g. by an RNA polymerase.

In embodiments, a cap1 or modified cap1 structure is generated using 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 .

In preferred embodiments, 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. In other preferred embodiments, 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 .

In other embodiments, 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.

In preferred embodiments, about 70%, 75%, 80%, 85%, 90%, 95% of the 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).

In preferred embodiments, 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. Such a 5’-terminal sequence element may comprise e.g. a binding site for T7 RNA polymerase. Further, 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.

Further RNA features

In preferred embodiments, the nucleic add is an in vitro transcribed RNA, preferably an in vitro transcribed mRNA.

In preferred embodiments, the nucleic add is a purified RNA, preferably a purified mRNA.

The term “purified RNA” refers to RNA which has a higher purity after certain purification steps than the starting material. Typical impurities comprise peptides, proteins, spermidine, RNA fragments, dsRNA, free nucleotides, DNA, etc. It is desirable for the “degree of RNA purity” to be as close as possible to 100%. Preferably, a “purified RNA” has a degree of purity of more than 75%, 80%, 85%, 90%, or 95%. The degree of purity may be determined by an analytical HPLC.

In embodiments, 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. Preferably, 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.

In embodiments, the RNA has an integrity of at least 60%, 70%, 80%, 90%. The term “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.

In preferred embodiments, the nucleic add, preferably the RNA, is suitable for use in treatment or prevention of a disease, disorder or condition. In particularly preferred embodiments, the nucleic acid, preferably the RNA, is suitable for use in treatment or prevention of an ocular disease, disorder or condition.

Preferred nucleic acid constructs

In various embodiments, the nucleic acid, preferably the RNA, comprises at least the following elements:

A) a 5’-cap structure, preferably as spedfied herein;

B) a 5’-UTR and/or a 3-UTR, preferably as spedfied herein;

C) at least one cds encoding at least one RUNX3 transcription factor as specified herein;

D) a 3’-UTR, preferably as specified herein;

E) optionally, a histone stem-loop as spedfied herein; and

F) at least one poly(A) sequence, preferably as spedfied herein.

In preferred embodiments, 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;

B) a 5’-UTR, preferably selected or derived from a 5’-UTR of a HSD17B4 gene, or a fragment thereof;

C) a cds encoding a RUNX3 transcription factor as spedfied herein;

D) a 3’-UTR, preferably selected or derived from a 3’-UTR of a PSMB3 gene, or a fragment thereof;

E) optionally, a histone stem-loop as specified herein;

F) a poly(A) sequence as specified herein, preferably comprising about 100 A nucleotides; and

G) optionally, chemically modified nucleotides, suitably selected from ip or ml ip, preferably ml ip.

Particularly preferred RNA sequences are provided in 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. Columns D and G relates to RNA sequences comprising a wild type or reference cds, columns E and H relates to RNA sequences comprising a G/C optimized (opt1) cds, columns F and I relates to RNA sequences comprising a human codon usage adapted (opt3) cds. Further information is provided under '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. Preferred are RNA sequences of column E.

Table 2: RNA sequences encoding the preferred RUNX3 transcription factor

In preferred embodiments, 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.

Preferably, 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.

In preferred embodiments, 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. In particularly preferred embodiments, 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. 2. Composition comprising at least one nucleic acid encoding a RUNX3 transcription factor

In a second aspect, 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.

Notably, features and embodiments described in the context of the first aspect (the nucleic acid encoding RUNX3) have to be read on and understood as suitable embodiments of the composition of the second aspect and vice versa.

In the context of the invention, a “composition” 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. Alternatively, 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.

Preferably, the at least one nucleic add of the pharmaceutical composition is selected from an RNA as further defined in the first aspect. In particularly preferred embodiments, the at least one nucleic acid of the pharmaceutical composition is selected from an mRNA as further defined in first aspect.

In embodiments, 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.

In various embodiments, 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.

Formulation/Complexation

In preferred embodiments, the at least one nucleic acid, preferably the RNA, is complexed or assodated with at least one compound to obtain a formulated composition. A formulation in that context may have the function of a transfection agent and/or may protect the nucleic add from degradation. Suitably a compound for formulation is selected from peptides, proteins, lipids, polysaccharides, and/or polymers.

In preferred embodiments, the at least one nucleic acid, preferably the RNA, is formulated with at least one cationic (cationic or preferably ionizable) or polycationic compound (cationic or preferably ionizable).

In preferred embodiments, 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.

The term “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. The terms “cationic”, “cationisable”, and “permanently cationic” must be understood as defined in WO2023/031394 [p.12, line 32 to p.13, line 16]. The term “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].

In preferred embodiments, 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. Further 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.

In embodiments, 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.

In embodiments, the at least one cationic or polycationic proteins or peptides preferably selected from SEQ ID NOs: 124-128, or any combinations thereof.

In some embodiments, the pharmaceutical composition comprises at least one nucleic acid and a polymeric carrier.

The term “polymeric carried’ as used herein 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).

In embodiments, the at least one nucleic acid, preferably the RNA, 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).

In preferred embodiments, 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. Typically, the lipidoid is a compound that comprises two or more cationic nitrogen atoms and at least two lipophilic tails. In contrast to cationic lipids, 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. In embodiments, 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. In embodiments, 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. Preferably, said formulations are particularly suitable for ocular administration.

Formulation in lipid-based carriers

In preferred embodiments, the at least one nucleic acid, preferably the RNA, is formulated in lipid-based carriers.

The term “lipid-based carriers” as used herein 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".

The term “encapsulation” as used herein 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.

In preferred embodiments, 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. In particularly preferred embodiments, 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. In an LNP, 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.

In embodiments, 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. In preferred embodiments, 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.

Cationic lipids

In embodiments, 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. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.

In preferred embodiments, the at least one cationic or ionizable lipid may carry a net positive charge at physiological pH. Preferably the cationic or ionizable lipid comprises a tertiary nitrogen group or quaternary nitrogen group, most preferably a tertiary nitrogen group. Accordingly, the at least one cationic or ionizable lipid may be selected from an amino lipid.

Preferably, 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.

In preferred embodiments, the at least one cationic or ionizable lipid is a lipid selected or derived from formula (111-1)

preferably, wherein one of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -S-S-, -C(=O)S-, SC(=O)-, - NRaC(=O)-, -C(=O)NRa- -NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- and the other of L1 or L2 is -O(C=O)-, - (C=O)O- -C(=O)-, -O-, -S(O)x- -S S-, -C(=O)S- SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- or a direct bond; G1 and G2 are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene; G3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; Ra is H or C1- C12 alkyl; R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl; R3 is H, OR5, CN, C(=O)OR4, OC(=O)R4 or -NR5C(=O)R4; R4 is C1 -C12 alkyl; R5 is H or C1 -C6 alkyl; and x is 0, 1 or 2.

In embodiments, the at least one cationic or ionizable lipid is selected from lipids disclosed in WO2018078053 (i.e. lipids derived from formula I, II, and III, or lipids as specified in claims 1-12), the disclosure of WO2018078053 hereby incorporated by reference. In that context, lipids disclosed in Table 7 of WO2018078053 (e.g. lipids derived from formula 1-1 to 1-41) and lipids disclosed in Table 8 of WO2018078053 (e.g. lipids derived from formula 11-1 to II-36) may be suitably used in the context of the invention. Accordingly, formula 1-1 to formula 1-41 and formula 11-1 to formula II-36 of WO2018078053, and the specific disclosure relating thereto, are herewith incorporated by reference.

In embodiments, the at least one cationic or ionizable lipid is selected or derived from structures 111-1 to HI-36 of Table 9 of WO2018078053. Accordingly, formula 111-1 to HI-36 of WO2018078053, and the specific disclosure relating thereto, are herewith incorporated by reference.

In embodiments, the at least one cationic or ionizable lipid selected or derived from formula HI-3 of WO2018078053. A preferred lipid of said formula HI-3 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.

Further 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). In other embodiments, 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.

In other embodiments, the at least one cationic or ionizable lipid is selected or derived from 9-Heptadecanyl 8-{(2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate, also referred to as SM-102, i.e. CAS Number 2089251 -47-6.

Accordingly, in preferred embodiments, the at least one cationic or ionizable lipid is selected or derived from ALC-0315, SM-102, SS-33/4PE-15, HEXA-C5DE-PipSS, or C26.

Neutral lipids

In preferred embodiments, the lipid-based carrier comprises at least one neutral lipid or phospholipid.

The term “neutral lipid” refers to any lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Neutral lipids may be selected from DHPC, DHPC, DOPC, DPPC, DOPG, DPPG, DOPE, POPC, POPE, DOPE-mal, DPPE, DMPE, DSPE, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, SOPE, transDOPE, 1 ,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE), DPhyPS (1 ,2-diphytanoyl-sn-glycero-3- phospho-L-serine), or mixtures thereof. In preferred embodiments, the at least one neutral lipid is selected or derived from DSPC, DHPC, DPhyPE, or DPhyPS.

Steroids, steroid analogs or sterols

In preferred embodiments, the lipid-based carrier comprises a steroid, steroid analog or sterol.

Preferably, the steroid or steroid analog is selected or derived from cholesterol, cholesteryl hemisucdnate (CHEMS), or any derivate of these. In preferred embodiments, the steroid, steroid analog or sterol is cholesterol.

Aggregation reducing lipids / polymer conjugated lipids

In preferred embodiments, the lipid-based carriers comprise at least one aggregation reducing lipid or moiety.

The term “aggregation reducing moiety” refers to a molecule comprising a moiety suitable of reducing or preventing aggregation of the lipid-based carrier. The term “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. Under storage conditions or during formulation, 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. In the context of the invention, 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).

In preferred embodiments, the aggregation reducing lipid is a polymer conjugated lipid.

The term “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.

In embodiments, the polymer conjugated lipid is selected from a PEG-conjugated lipid or a PEG-free lipid.

In preferred embodiments, 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. In embodiments, 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.

In other embodiments, the polymer conjugated lipid is selected or derived from formula (IV) of WO2018078053, preferably from formula (IVa) of WO2018078053. In that context, a preferred polymer-conjugated lipid is selected from ALC-0159 (2[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide).

In embodiments, the aggregation reducing lipid is selected from a PEG-free lipid, e.g. a PEG-free polymer conjugated lipid. Preferably, 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”. In embodiments, the “POZ lipid” or respectively preferred polymer conjugated lipids are described in W02023031394, the full disclosure herewith incorporated by reference. In particular, the disclosure relating to polymer conjugated lipids as defined in any one of claims 1 to 8 of WO2023031394 is herewith incorporated by reference.

In some embodiments, the polymer conjugated lipid is a PEG-free lipid selected from a POZ-lipid. In preferred embodiment in that context, the aggregation-reducing lipid is selected or derived from PMOZ 1 , PMOZ 2, PMOZ 3, PMOZ 4, or PMOZ 5 of W02023031394. In a particularly preferred embodiment, the polymer conjugated lipid is selected or derived from PMOZ 4 according to formula “PMOZ4” of W02023031394, herewith incorporated by reference.

Accordingly, in preferred embodiments, the at least one aggregation-reducing lipid is selected or derived from DMG- PEG 2000, C10-PEG2K, Cer8-PEG2K, a POZ-lipid such as PMOZ4, or ALC-0159.

Lipid-based carrier compositions

In preferred embodiments, 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.

In embodiments, the lipid-based carrier, preferably the LNP, comprising the nucleic acid, preferably the RNA, comprise

(i) at least one cationic lipid or ionizable lipid, preferably as defined herein;

(ii) at least one neutral lipid or phospholipid, preferably as defined herein;

(iii) at least one steroid or steroid analogue, preferably as defined herein; and

(iv) at least one aggregation reducing lipid, preferably as defined herein.

In embodiments, the lipid-based carrier, preferably the LNP, comprising the nucleic acid, preferably the RNA, comprise

(i) at least one cationic lipid selected or derived from ALC-0315, SM-102, SS-33/4PE-15, HEXA-C5DE-PipSS or C26;

(ii) at least one neutral lipid selected or derived from DSPC, DHPC, or DPhyPE;

(iii) at least one steroid or steroid analog selected or derived from cholesterol; and

(iv) at least one aggregation reducing lipid selected or derived from ALC-0159, DMG-PEG 2000, C10-PEG2K, Cer8- PEG2K, or “PMOZ 4”; and wherein the lipid-based carriers encapsulate the nucleic acid (e.g. the RNA).

In embodiments, 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.

In preferred embodiments, 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.

In embodiments, the lipid-based carrier, preferably the LNP, comprising the nucleic add, preferably the RNA, comprise

(i) a cationic lipid ALC-0315; (ii) a neutral lipid DSPC; (iii) a steroid or steroid analog cholesterol; and (iv) an aggregation redudng lipid ALC-0159; preferably wherein (i) to (iv) are in a molar ratio of about 47.4% cationic lipid, about 10% neutral lipid, about 40.9% steroid or steroid analog, and about 1 .7% aggregation reducing lipid, preferably wherein the 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.

In embodiments, 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.

In various embodiments, 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. The term “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. The term “dynamic light scattering” or “DLS” refers to a method for analyzing particles in a liquid, wherein the liquid is typically illuminated with a monochromatic light source and wherein the light scattered by particles in the liquid is detected. Suitable DLS protocols and instruments are known in the art.

In preferred embodiments, 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.

In preferred embodiments, 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.

In embodiments, 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.

In embodiments, 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.

In preferred embodiments, the surface of the lipid-based carrier, preferably the LNP, is uncharged at pH 7.

In embodiments, 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.

Any of the 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.

In embodiments, the composition additionally comprises a RUNX1 transcription factor inhibitor that is an RNA encoding a CBFbeta-SMMHC fusion protein according to WO2023144330. Accordingly, 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.

In other embodiments, the composition comprises an anti- inflammatory agent that suitably comprises a steroid or a nonsteroidal anti-inflammatory drug (NSAID). Administration of the pharmaceutical composition orthe nucleic acid

Suitably, upon administration of the pharmaceutical composition or nucleic add to a cell, tissue, or subject, the encoded RUNX3 transcription factor as defined herein is produced in said cell, tissue, or subject.

In preferred embodiments, upon intramuscular, intranasal, transdermal, intradermal, intralesional, intracranial, subcutaneous, intracardial, intratumoral, intravenous, intrapulmonal, intraarticular, retronasa, sublingual, pulmonary, intrathecal, or ocular administration of the pharmaceutical composition or nucleic acid to a 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.

In preferred embodiments, upon local administration of the pharmaceutical composition or nucleic acid to a 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.

In preferred embodiments of the invention, 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).

In preferred embodiments in that context, ocular administration is selected from intravitreal or intraoperative administration.

In another embodiment, the ocular administration may be performed via a device, for example a device for intravitreal delivery or suprachoroidal delivery. Suitably, the device is configured to be a depot for the pharmaceutical composition. Such a device allows controlled administration to the eye (e.g. in regular intervals, e.g. one a day) e.g. via a port.

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. In the context of intravitreal administration, 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.

In preferred embodiments, the ocular administration is intraoperative administration. Some disease, disorders or conditions in the eye occur after an ocular surgery or operation (e.g. PVR). Accordingly, administration of the pharmaceutical composition or nucleic add (e.g. an RNA encoding a RUNX3 transcription factor) via intraoperative administration is preferred in medical applications where a disease, disorders or condition occurs after an ocular surgery or operation (e.g. PVR).

In preferred embodiments, 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. In particularly preferred embodiments, ocular administration of the pharmaceutical composition or the nucleic acid (e.g. an RNA encoding a RUNX3 transcription factor) leads to a production of the RUNX3 transcription factor in retinal pigment epithelium (RPE) cells or cells derived from RPE cells.

The retinal pigment epithelium (RPE) 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. When wound repair is complete, 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. In some instances, 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.

Accordingly for some disease, disorders or conditions (e.g. PVR) it is preferred that the RUNX3 transcription factor is produced in RPE cells upon ocular administration to e.g. inhibit an overactive and/or overexpressed RUNX1 .

In particularly preferred embodiments, ocular administration of the pharmaceutical composition or the nucleic acid (e.g. an RNA encoding a RUNX3 transcription factor) 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. Particularly preferred are Muller cells, and microglia.

In particularly preferred embodiments, ocular administration of the pharmaceutical composition or the nucleic acid (e.g. an RNA encoding a RUNX3 transcription factor) reduces or inhibits the cellular expression of RUNX1 , TGFbeta2, SMAD3, and/or COL1 A1 . Accordingly, the administration leads to a production of the RUNX3 transcription factor which (directly or indirectly) reduces or inhibits the cellular expression of RUNX1 , TGFbeta2, SMAD3, and/or COL1 A1 .

In particularly preferred embodiments, ocular administration of the pharmaceutical composition or the nucleic acid (e.g. an RNA encoding a RUNX3 transcription factor) 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.

In some embodiments, the ocular administration of the pharmaceutical composition or the nucleic acid (e.g. an RNA encoding a RUNX3 transcription factor) is performed into a tamponade agent-filled human eye, a silicone-fi lied human eye, or a gas-filled human eye. In some embodiments, the ocular administration of the pharmaceutical composition or the nucleic add (e.g. an RNA encoding a RUNX3 transcription factor) is performed after tamponade, gas or silicone extraction.

3. A kit or kit of parts

In a third aspect, 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.

Embodiments relating to the nucleic acid of the first aspect and embodiments relating to the pharmaceutical composition of the second aspect may likewise be read on and be understood as suitable embodiments of the kit or kit of parts. In preferred embodiments, the kit or kit of parts comprises at least one nucleic acid of the first aspect, preferably at least one RNA, and/or at least one pharmaceutical composition of the second aspect.

In addition, the kit or kit of parts may comprise a liquid vehicle for solubilising, and/or technical instructions providing information on administration and dosage of the components.

The kit may further comprise additional components as described in the context of the pharmaceutical composition of the second aspect.

The technical instructions of said kit may contain information about administration and dosage and patient groups. Such 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.

In preferred embodiments, the kit or kit of parts as defined herein comprises at least one syringe or application device. Suitably, a syringe or application device for ocular delivery (e.g. intravitreal delivery).

4. Medical uses

In a further aspect, 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.

Notably, 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. In addition, embodiments relating to medical uses as described herein of course also relate to methods of treatments (fifth aspect).

Accordingly, 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.

Herein “preventing” of a disease or condition is defined by reducing the risk of and/or delaying development of a disease.

Preferably, 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. More preferably, the at least one cds encoding the RUNX3 transcription factor comprises or consists of a nucleic acid 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 add sequences SEQ ID NOs: 164-268, or a fragment or a variant of any of these, preferably a nucleic acid 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 add sequences SEQ ID NOs: 199. Even more preferably, 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. Suitably, the nucleic of the pharmaceutical composition is an mRNA encapsulated in a lipid-based carrier as defined herein, preferably an LNP as defined herein. In embodiments, 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.

In various embodiments, 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.

In a further aspect, 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.

In preferred embodiments, the disease, disorder, or condition is associated with or caused by an overexpressed and/or an overactive RUNX1 transcription factor.

As shown in the example section, 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 . In addition, administration of a nucleic acid encoding RUNX3 leads to a reduction of the EMT markers and pathological cell proliferation. Additionally, 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.

In other embodiments, 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.

Accordingly, in preferred embodiments, 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.

Examples of 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. Other conditions assodated with EMT including cancer, e.g., mesothelioma, ocular chronic graft-versus-host disease, corneal scarring, corneal epithelial downgrowth, conjunctival scarring, eye tumors such as melanoma and metastatic tumors, or fibrosis.

In other preferred embodiments, 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.

Aberrant 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,

Nome’s Disease, FEVR and Von Hippel-Lindau. 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.

In some instances, 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 (e.g. lung fibrosis, and fibrosis in virus infections, e.g. COVID-19) 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.

Metabolic conditions that trigger RUNX1 hyperactivation such as diabetes (e.g. high blood sugar) or genetic conditions leading to RUNX1 overexpression such as Down 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. In preferred embodiments, the ocular disease, disorder, or condition is associated with or caused by aberrant proliferation or migration of RPE cells in a subject.

For example, upon retinal detachment or trauma, 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. For example, EMT of RPE cells plays a critical role in the pathobiology of PVR.

In preferred embodiment, 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, keratoconus, amblyopia, conjunctivitis, comeal ulcers, dacryocystitis, Duane retraction syndrome, optic neuritis, ocular inflammation, glaucoma, macular degeneration, and uveitis, or any disease, disorder or condition related or associated thereto.

In particularly preferred embodiments, 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.

In preferred embodiments, 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. There are two major types of age-related macular degeneration, known as the dry form and the wet form. 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.

In preferred embodiments, 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. For example, fluid can leak into the macula, the area of the retina responsible for clear central vision. Although small, the macula is the part of the retina that allows us to see colours and fine detail. The fluid causes the macula to swell, resulting in blurred vision. In an attempt to improve blood circulation in the retina, new blood vessels may form on its surface. These fragile, abnormal blood vessels can leak blood into the back of the eye and block vision. 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. This leakage may lead to swelling of the macula. (2) 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. This greatly raises the eye pressure, which damages the optic nerve. If left untreated, PDR can cause severe vision loss and even blindness. Any symptom, type, or stage of diabetic retinopathy may be inhibited, treated, or prevented using methods and compositions disclosed herein, e.g. using the nucleic add or composition encoding a RUNX3 transcription factor. In preferred embodiments, the ocular disease, disorder, or condition is selected from retinopathy of prematurity. Retinopathy of prematurity (ROP) 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. This disorder, which usually develops in both eyes, is one of the most common causes of visual loss in childhood and can lead to lifelong vision impairment and blindness. These infants are at a much higher risk for ROP. About 90 percent of all infants with ROP are in the milder category and do not need treatment. However, infants with more severe disease can develop impaired vision or even blindness. About 1 , 100-1 ,500 infants annually develop ROP that is severe enough to require medical treatment. About 400-600 infants each year in the US become legally blind from ROP. ROP occurs when abnormal blood vessels grow and spread throughout the retina, the tissue that lines the back of the eye. These abnormal blood vessels are fragile and can leak, scarring the retina and pulling it out of position. This causes a retinal detachment. 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. The edges of the retina (the periphery) may not get enough oxygen and nutrients. The periphery of the retina may then send out signals to other areas of the retina for nourishment. As a result, new abnormal vessels begin to grow. These new blood vessels are fragile and weak and can bleed, leading to retinal scarring. When these scars shrink, they pull on the retina, causing it to detach from the back of the eye. 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.

In preferred embodiments, the ocular disease, disorder, or condition is selected from retinal vein occlusion (RVO). RVO is a blockage of the small veins that carry blood away from the retina. RVO is most often caused by hardening of the arteries (atherosclerosis) and the formation of a blood clot. Blockage of smaller veins (branch veins or BRVO) in the retina often occurs in places where retinal arteries that have been thickened or hardened by atherosclerosis cross over and place pressure on a retinal vein. 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. using the nucleic add or composition encoding a RUNX3 transcription factor. In preferred embodiments, 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. Without wishing to be bound by any scientific theory, the most common etiology of OIS is severe unilateral or bilateral atherosclerotic disease of the internal carotid artery or marked stenosis at the bifurcation of the common carotid artery. OIS may also be caused by giant cell arteritis. The decreased vascular perfusion results in tissue hypoxia and increased ocular ischemia, leading to neovascularization. Any 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.

In preferred embodiments, the ocular disease, disorder, or condition is selected from neovascular glaucoma (NVG). NVG is classified as a secondary glaucoma. Numerous secondary ocular and systemic diseases that share one common element, retinal ischemia/hypoxia and subsequent release of an angiogenesis factor, cause NVG. This angiogenesis factor causes new blood vessel growth from pre-existing vascular structure. Depending on the progression of NVG, 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. In managing NVG, it is essential to treat both the elevated intraocular pressure (IOP) and the underlying cause of the disease. 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. The resultant growth of new vessels at the pupillary border and iris surface [neovascularization of the iris (NVI)] and over the iris angle [neovascularization of the angle (NVA)] ultimately leads to formation of fibrovascular membranes. The fibrovascular membranes, which may be invisible on gonioscopy, accompany NVA and progressively obstruct the trabecular meshwork. This causes secondary open-angle glaucoma. As the disease process continues, 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.

In preferred embodiments, 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. 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. Patients may be relatively asymptomatic until the lesions achieve larger size, and it is imperative to perform life-long surveillance of even asymptomatic individuals with VHL because smaller lesions are more easily eradicated than larger lesions. Any symptom, type, or stage of retinal hemangioma may be inhibited, treated, or prevented using methods and compositions disclosed herein. In some embodiments, 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.

In preferred embodiments, 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), 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. In some cases, removal of the eye may be necessary (enucleation). The most common sign at presentation is leukocoria (abnormal white reflection of the retina). Symptoms typically begin as blurred vision, usually pronounced when one eye is closed (due to the unilateral nature of the disease). Often the unaffected eye will compensate for the loss of vision in the other eye; however, this results in some loss of depth perception and parallax. Deterioration of sight may begin in either the central or peripheral vision. Deterioration is likely to begin in the upper part of the vision field as this corresponds with the bottom of the eye where blood usually pools. Flashes of light, known as photopsia, and floaters are common symptoms. Persistent color patterns may also be perceived in the affected eye. 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. On funduscopic eye examination, the retinal vessels in early Coats’ disease appear tortuous and dilated, mainly confined to the peripheral and temporal portions of retina. In moderate to severe Coats’ disease, massive retinal detachment and hemorrhage from the abnormal vessels may be seen. Any symptom, type, or stage of Coats' disease may be inhibited, treated, or prevented using methods and compositions disclosed herein. In some embodiments, 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.

In preferred embodiments, 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. Other problems may include mild to moderate intellectual disability, often with psychosis, and abnormalities that can affect circulation, breathing, digestion, excretion, or reproduction. Mutations in the norrin cystine knot growth factor (NDP) gene cause Norrie disease. 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.

In preferred embodiments, 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. In some embodiments, a subject at risk of developing FEVR is treated to delay or prevent the onset or progression of FEVR. In some embodiments, 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.

In particularly preferred embodiments, the ocular disease, disorder, or condition is selected from PVR. PVR is a clinical syndrome that develops as a complication of rhegmatogenous retinal detachment and is also commonly associated with eye trauma. PVR is the most common cause of failure in retinal detachment surgery, however, it can also occur with untreated eyes with retinal detachment. In particular, PVR can occur with vitreous hemorrhage, after cryotherapy, after laser retinopexy, pneumatic retinopexy, scleral buckling, or vitrectomy, and after a variety of surgical complications. PVR is also common after eye traumas (e.g., penetrating injuries) and other conditions associated with prolonged inflammation. 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. In embodiments, as described herein 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. During rhegmatogenous 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. During this process 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. During this process the 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. Thus, this leads to secondary retinal detachment after primary retinal detachment surgery. During RPE disruption, inflammation may play an important role in the development of PVR. Cytokines IL-6, IL-I, TNFalpha have been identified in high concentrations in the vitreous in the early, proliferative stages of PVR, but they decrease to normal levels in the scarring phase. Other molecules involved in PVR include TGF and IL- 6. The transcription factor RUNX1 is involved in the development and progression of PVR.

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. 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.

Development Stages: 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. Early on, preretinal PVR adopts an immature appearance and consistency. During this phase, the retina may still remain compliant, and the PVR membrane may be difficult to remove due to its amorphous form. By 6 to 8 weeks, however, the PVR membrane becomes more mature, taking on a white, fibrotic appearance. In this stage, the PVR is more easily identifiable, causes rigidity of the retina, and can be more identifiably removed.

Classification: 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. Diagnosis via clinical examination and imaging for PVR is known in the art, e.g., as described in the classification of retinal detachment with proliferative vitreoretinopathy. PVR is a condition distinct from proliferative diabetic retinopathy (PDR). 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. To the contrary, in PVR, 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. In preferred embodiments, 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.

Accordingly, in particularly preferred embodiments, the ocular disease, disorder, or condition is PVR, preferably the prevention of PVR. Preferably, 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.

As shown in the example section, treatment of C-PVR cells (human primary cell cultures obtained from surgically removed PVR membranes) and primary human retinal microvascular endothelial cells (HMRECs) with nucleic acid encoding a RUNX3 transcription factor showed anti-proliferative and anti-migration effects in vitro (Figure 1 and 2, respectively). Additionally, 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). Furthermore, 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). Additionally, it has also been shown, that RUNX3 mRNA reduces lesion size in a laser-CNV mouse model 7 days after treatment (Figure 5).

In preferred embodiments in that context, cell proliferation and/or cell growth is reduced in eyes with PVR.

In embodiments, the ocular disease, disorder, or condition is selected from Epiretinal Membrane (ERM). ERM is 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. 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). Generally, 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.

In preferred embodiments, 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. Typically, 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. Without treatment permanent loss of vision may occur. 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. Optionally, 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. When the retina is pulled away from the back of the eye, it is a retinal detachment. Typically, 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.

The symptoms of vitreous separation, retinal tear, and retinal detachment are similar and sometimes can overlap. On occasion, 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”. Of even greater concern is the loss of peripheral vision, which may present as a shadow moving toward the center of one’s field of vision. Additionally, in 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. Sometimes, 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. Additionally, 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. 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. The atrophic form most often occurs as idiopathic dialysis of the young. (2) 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. In evaluation of retinal detachment, it is critical to exclude exudative detachment as surgery will make the situation worse, not better. Although rare, 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. (3) 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.

Accordingly, in preferred embodiments, the retinal detachment disorder is selected from rhegmatogenous retinal detachment, exudative retinal detachment, or fractional retinal detachment.

In preferred embodiments, 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.

In some embodiments, 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.

In preferred embodiments, 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.

In particularly preferred embodiments in that context, 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). Accordingly, administration of the pharmaceutical composition or nucleic add (e.g. an RNA encoding RUNX3) via intraoperative administration is preferred.

In some embodiments, 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. For example, 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).

In particularly preferred embodiments, the disease, disorder, or condition is associated with or caused by overexpressed and/or overactive RUNX transcription factor. Preferred in that context is RUNX1 . Suitably, the disease, disorder, or condition is associated with or caused by overexpressed and/or overactive RUNX transcription factor is an 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. These data indicate that use of the nucleic acid encoding a RUNX inhibitor may be applicable in a very broad range of pathologies characterized by increased RUNX1 transcription factor activity. RUNX1 functions as a transcriptional switch allowing organisms to control for delicate cell fate decisions in multiple critical developmental processes including hematopoiesis. In addition, our group uncovered multiple instances where 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.

For example, essentially all ocular diseases characterized by aberrant angiogenesis or pathologic EMT are associated with RUNX1 overexpression. Examples of 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. Examples of 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. Additional diseases that may be associated with RUNX1 overexpression include comeal scarring after trauma, infection, chemical injury, cataracts, epiretinal membranes, corneal neovascularization, comeal scarring, which has implications for corneal transplants and corneal chemical injury / trauma, corneal viral infections, ocular cancers, uveal melanomas. In various embodiments of the invention, the nucleic add or composition of the invention may be administered only once or multiple times. For example, 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. In some embodiments, 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.

Accordingly, in preferred embodiments, 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.

In various embodiments, such as embodiments involving eye drops, a composition is self-administered.

5. Methods of treatment

In a further aspect, the present invention relates to a method of treating or preventing a disease, disorder or condition.

Notably, embodiments relating to the previous aspects may likewise be read on and be understood as suitable embodiments of method of treatments of the invention. In particular, 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.

In preferred embodiments, 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. As used herein, “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.

As mentioned above, further features and embodiments of the present aspect may be taken from the aspect “medical uses” as described in detail in the context of the fourth aspect.

6. A method of reducing the activity of RUNX1 in a cell ora subject

In a further aspect, 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 (e.g. nucleic add, pharmaceutical composition) 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.

I n preferred embodiments, 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.

In embodiments, 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.

In preferred embodiments, 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.

In embodiments, 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. In preferred embodiments, 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. In particularly preferred embodiments, 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.

In particularly preferred embodiments, 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. Using 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.).

In preferred embodiments of all aspects of the invention, 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

- the produced RUNX3 is a dominant negative inhibitor of RUNX1 ; and/or

- the produced RUNX3 binds to CBFbeta; and/or

- the produced RUNX3 reduces or prevents interaction of RUNX1 with its target DNA; and/or

- the produced RUNX3 reduces or prevents interaction of RUNX1 with CBFbeta; and/or

- the produced RUNX3 reduces cellular RUNX1 -CBFbeta complex formation and/or activity; and/or

- the produced RUNX3 reduces or prevents nuclear translocation of RUNX1 ; and/or

- the produced RUNX3 reduces the activity of RUNX1 ; and/or

- the produced RUNX3 reduces the cellular expression of RUNX1 ; and/or

- the produced RUNX3 reduces the cellular expression of proteins that are controlled or regulated by RUNX1 ; and/or

- 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

- the produced RUNX3 reduces or prevents cell proliferation and/or cell growth in eyes with PVR. Item List

Preferred embodiments of the present invention are provided in the following item list:

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).

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).

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).

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).

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.

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.

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.

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 of any of these.

Item 16: The artificial nucleic acid of items 12 or 15, 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 NO: 13, or a fragment or a variant thereof.

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 18: The artificial nucleic acid of any one of the preceding items, wherein the nucleic acid comprises at least one poly(A) sequence, preferably wherein the at least one poly (A) sequence comprises about 40 to about 500 adenosine nucleotides, preferably about 60 to about 250 adenosine nucleotides, more preferably about 60 to about 150 adenosine nucleotides.

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).

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) a 5’-cap structure;

B) a 5-UTR, preferably selected or derived from a 5’-UTR of a HSD17B4 gene;

C) a coding sequence encoding a RUNX3 transcription factor or a fragment or variant thereof;

D) a 3-UTR, preferably selected or derived from a 3 -UTR of a PSMB3 gene;

E) optionally, a histone stem-loop; and

F) a poly(A) sequence, preferably comprising about 100 A nucleotides.

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.

Item 38: 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%, 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.

Item 39: The artificial 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 56: The pharmaceutical composition of items 51 to 55, wherein the at least one cationic or ionizable lipid is a lipid selected or derived from formula (111-1), preferably, wherein one of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, - O-, -S(O)x-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa- or - NRaC(=O)O-, and the other of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -S S-, -C(=O)S- SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- or a direct bond; G1 and G2 are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene; G3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; Ra is H or C1-C12 alkyl; R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl; R3 is H, OR5, CN, C(=O)OR4, OC(=O)R4 or-NR5C(=O)R4; R4 is C1-C12 alkyl; R5 is H or C1-C6 alkyl; and x is 0, 1 or 2.

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.

Item 60: The pharmaceutical composition of items 48 to 59, wherein the lipid-based carriers comprise

(i) at least one cationic lipid, preferably as defined in items 55 to 57;

(ii) at least one neutral lipid, preferably as defined in item 58;

(iii) at least one steroid or steroid analogue, preferably as defined in item 49; and

(iv) at least one aggregation reducing lipid, preferably as defined in items 52 to 54.

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).

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).

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.

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.

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.

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.

Item 97: A method of reducing the activity of a RUNX1 transcription factor in a cell or a subject, wherein the method comprises a) applying or administering an artificial nucleic acid comprising at least one cds encoding at least one RUNX3 transcription factor or a fragment or variant thereof of items 1 to 39; or b) applying or administering a pharmaceutical composition comprising the artificial nucleic acid comprising at least one cds encoding at least one RUNX3 transcription factor or a fragment or variant thereof of items 40 to 81 ; to a cell, tissue, or subject, wherein the RUNX3 transcription factor or a fragment or variant thereof is produced in the cell, tissue, or subject after administration or application to said cell, tissue, or subject.

Examples

In the following, examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments presented herein and should rather be understood as being applicable to other compositions or uses as for example defined in the specification. Accordingly, the following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. Various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. Example 1 : Preparation of nucleic acid formulations encoding RUNX3 transcription factors

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.

1.1 Preparation of DNA templates for RNA in vitro transcription:

DNA sequences encoding a RUNX3 transcription factor were prepared and used for subsequent RNA in vitro transcription reactions. Some DNA sequences were prepared by modifying the wild type or reference encoding DNA sequences by introducing a G/C optimized cds for stabilization and expression optimization. Sequences were introduced into a DNA vector to comprise a stabilizing UTR sequence and a stretch of adenosines and optionally a histone stem-loop (hSL). The obtained plasmid DNA templates were transformed and propagated in bacteria using common protocols known in the art. Eventually, the plasmid DNA templates were extracted, purified, and linearized using a restriction enzyme and used for IVT. The herein used RNA constructs are provided in Table 3.

Table 3: RNA constructs used in the Examples

1.2. RNA in vitro transcription from plasmid DNA templates:

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. For producing modified RNA, IVT was performed in the presence of a nucleotide mixture where UTP is replaced by N1 -Methylpseudouridine (m1MJ) or pseudouridine (MJ). After RNA in vitro transcription, the obtained RNA IVT reaction was subjected to purification steps comprising RP-HPLC.

1.3. Preparation of polymer-lipidoid complexes carrying mRNA (CVCMs):

20mg of peptide (CGH5R4H5GC-NH2) was dissolved in N-methylpyrrolidone. After addition of 2ml of borate buffer (pH 8.5), the solution was stirred at RT for >18h. Subsequently, 12.6mg of PEG-SH 5000, dissolved in N-methylpyrrolidone, was added to the peptide solution and the vial filled up to 3ml with borate buffer (pH 8.5). After 18h, the reaction mixture was purified and further concentrated using Centricon filter units (MWCO 10kDa) and subsequently lyophilized. 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.

1 .4 Preparation of lipid-based carriers encapsulating the mRNA:

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. LNPs were composed of the following lipid components: SM-102 : cholesterol : DSPC : PEG2k-DMG = 50 : 38.5 O ,5mol%. 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).

2.1. C-PVR cell culture

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.

2.2 HMREC cell culture

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.

2.3 Proliferation, cell viability and migration assays

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. 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).

2.4. Western Blotting

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® Turbo™ semi-dry transfer system (BIORAD) for 7min at 25V. For imaging using the iBright 1500 (INVITROGEN), membranes were blocked with 5% dry milk in TBST-T 1 h at R.T probed overnight at 4°C with primary antibody and then 1 hour at room temperature with donkey anti-rabbit IgG-HRP conjugated secondary antibody. The antigen-antibody interaction was visualized with Clarity Western ECL Substrate (BIORAD).

2.5. Single ce// RNA sequencing of C-PVR cells in culture (scRNA-seq)

Single cell suspension of C-PVR primary culture treated with the formulations were prepared and scRNAseq was performed using 10x Genomics Chromium Single Cell 3’ Reagent Kits version 2 (PN-1000). Bioanalyzer High Sensitivity DNA Assay (Agilent Technologies) was used to assess size distribution and molarity of resulting cDNA libraries and 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). The data was processed using 10X Genomics’ Cell Ranger pipeline generating feature/barcode matrices from the raw counts data. Before clustering, 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.

2.6 PVR induction in rabbits and mRNA formulation administration

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^A6 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. A previously published PVR score grading system was used to assess severity of pathology (Delgado-Tirado et al., 2020). The score was determined by combination of the most severe phenotypes identified by indirect ophthalmoscopy, fundus imaging and optical coherence tomography 2 weeks after C-PVR cell injection as previously described (Delgado-Tirado et al., 2020).

2.7 Histology for rabbit eyes

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.

2.8 Laser-induced CNV mice model

For the laser-induced choroidal neovascularization (CNV) model, laser photocoagulation was performed using the Micron image-guide system and a 532nm laser; 4 laser spots were administered at the 3-, 6-, 9-, and 12-o’clock meridians at 2-to-3-disc diameters of distance from the optic nerve head. Briefly, CNV was induced in mice (Specific pathogen free (SPF) inbred male C57BL/6JRj mice (n=108) 8-10 weeks of age (Janvier Lab) on day 0 by perforating Bruch’s membrane using a diode laser, thereby triggering the growth of subretinal blood vessels recruited from the choroid. After CNV induction, a single unilateral intravitreal injection of 1 pl was administered. After 7d of treatment, fundus fluorescein angiography was recorded under general anesthesia. 0.1 ml of 2% sodium fluorescein (AKORN) was administered intraperitoneally, and serial photographs from the early (0 to 60s) and late phases (6 min) were captured using the Micron IV imaging system. Light source intensity and gain were standardized and maintained in all experiments. Leakage was quantified using Imaged V13, recording the difference between the early and late phases of the lesions. The lesion sizes were also quantified on choroidal flat-mounts after ILB4 (Isolectin-IB 4) vascular staining which was performed according to manufacturer’s description.

Example 3: Formulated RUNX3 mRNA induced the expression of RUNX3 and inhibited the proliferation and migration of C-PVR and HMREC

The aim of the experiment was to test whether a nucleic add encoding a RUNX3 transcription factor (RUNX3_A or RUNX3_B) induces the expression of RUNX3 and inhibits the proliferation and migration of C-PVR (patient derived proliferative vitreoretinopathy) and HMREC (primary human microvascular endothelial) cells. RNA formulations used in the experiment were prepared according to Example 1 , and methods used herein are further described in Example 2.

Table 4: Formulated mRNA constructs used in Example 3-5

C-PVR:

Briefly, C-PVR were seeded with 3 x 10^A4/well density in 48 well-plates and treated with 5pg/ml CVCM-formulated RUNX3 mRNA and incubated for 4 hours. Next, 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. To analyse the expression of RUNX3 in C-PVR Single cell suspension of C-PVR primary culture treated with the formulations were prepared, and scRNAseq was performed using 10x Genomics Chromium Single Cell 3' Reagent Kits version 2. 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^A4 and 3x10^A6/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.

Results:

A robust protein production of RUNX3 in C-PVR cells 24h after treated with formulation could be detected determined by Western blot (Figure 1 A). Immunocytochemistry staining showed that RUNX3 was primarily expressed in the cell nucleus in the C-PVR cells treated with RUNX3 formulations (data not shown). No basal expression of RUNX3 was detected in the C-PVR control group. No significant influence on toxicity in C-PVR determined by the lactate dehydrogenase (LDH) assay was observed between cells treated with mRNA-encoded RUNX3 and control 48h after treatment (Figure 1B).

Formulated RUNX3 mRNA display a significant decrease in proliferation and migration of C-PVR after 48h of treatment (Figure 1 C and 1 D). Single-cell RNA sequencing of C-PVR treated with RUNX3 CVCM1 -B demonstrated the expression of RUNX3 mRNA with 67% of positive cells expression compared with the luciferase and NT groups, where the expression was close to zero. Feature maps showed the expression of RUNX3 across the cells and treatments. RUNX3 positive cells localized with cells in G1 phase.

A distinct protein production on of RUNX3 on HMREC 24h after treated with formulation could be observed as shown by Western Blot (Fig 2A). Formulated RUNX3_B mRNA reduces proliferation and migration of HMREC cells (Fig 2B-2D).

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.

Briefly, single cell suspension of C-PVR primary culture treated with the formulations (5pg/ml formulated RNA, Table 4) were prepared and scRNAseq was performed. The Bioanalyzer High Sensitivity DNA Assay was used to assess size distribution and molarity of resulting cDNA libraries before sequencing. Louvain clustering was used to analyze the single cell sequencing data visualized by Uniform Manifold Approximation and Projection (UMAP). The cell percentage calculation was done at clustering resolution of 0.2. 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. Featured dot plots of proliferation markers, epithelial markers, mesenchymal markers and fibrotic markers were analyzed.

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). RUNX3 overexpression induced notable changes in the profile of hallmark EMT genes.

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. Likewise, 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). Also, COL1 A1 , a relevant fibrotic marker, was downregulated. Treatment with formulated RUNX3_A mRNA modulates the transforming growth factor beta 2 (TGFB2) signaling by reducing the expression of TGFbeta2 and SMAD3 (Figure 3H). in addition, further proliferation and fibrotic markers were also reduced after treatment using with formulated 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. Also, 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. Additionally, several 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

The aim of the experiment was to test whether the nucleic acid encoded RUNX3 (RUNX3_A) is also effective in a clinically relevant rabbit model for PVR. RNA formulations used in the experiment were prepared according to Example 1 , and methods used herein are further described in Example 2.

To induce PVR, gas displacement of the vitreous was induced by intravitreal injection of 0.2mL of perfluoropropane (C3F8) behind the limbus of male New Zealand White rabbits. Three days after gas injection, 0.1 mL of BSS containing approximately 1 *10^A6 C-PVR cells were administrated via intravitreal injection along with 36.5pg of RUNX3_A or Luciferase mRNA formulation (see Table 4). A second dose of 36.5pg of mRNA was performed 7 days later. The score was determined by combination of the most severe phenotypes identified by indirect ophthalmoscopy, fundus imaging and OCT (optical coherence tomography) 2 weeks after C-PVR cell injection. OCT, fundus, and were performed as previously described (Delgado-Tirado et al., 2020).

After two weeks, the PVR score was significantly lower in the group treated with the RUNX3_A mRNA, indicating a reduction in the pathology progression (Figure 4B). Interestingly, less PVR-related pathology has been observed in one of the RUNX3_A treated eyes in the OCT images (Figure 4A). The formation of aberrant membranes (pointed with black and white arrows) over the optic nerve could be detected in the luciferase group (Figure 4A). That was corroborated by the H&E staining of the rabbit retina section (Figure 4C). The staining showed a vast deposition of cells and extracellular matrix over the retina; meanwhile, the RUNX3_A treated eyes showed the presence of some floating cells with a remarkably less dense membrane (Figure 4C, right image).

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. The mice laser-induced CNV model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. 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.

Briefly, animals were treated via intravitreal administration with CVCM as control, or CVCM-formulated RUNX3_B mRNA (250ng/pl). Laser photocoagulation was used to break Bruch’s membrane and induced choroidal neovascularization. Then, 1 pl of CVCM formulated RUNX3_B and CVCM (0.9% saline solution) were intravitreally injected. In vivo fundus fluorescein angiography was recorded 7 days post-induction. Pixel density was measured in Image J for each lesion. Leakage was calculated as the difference between early (0-30 s post-injection of fluorescein) and late phase (6min). After 7 days of treatment, CVCM-formulated RUNX3_B mRNA significantly reduced the leakage associated with abnormal angiogenesis in a laser L-CNV model (Figure 5).

Table 5: Formulated mRNA constructs used in Example 6

After 7 days of treatment, 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

The aim of this experiment was to analyze the expression and functionality of LNP formulated RUNX3 mRNA in different cell lines (ARPE-19 and HUVECs). RNA formulations used in the experiment are prepared according to Example 1 , methods used herein are described in Example 2.

Dose-dependent expression ofRUNX3 in HUVEC and ARPE-19 cells

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).

Results:

The expression profile of ml ip-modified or unmodified RUNX3 in HUVECs and ARPE-19 cells is shown Figure 6. Both mRNAs, m li -modified (6A and 6C) or unmodified (6B and 6D), show an overall comparable dose dependent expression profile in the respective cell lines, with m1ip-modified mRNA having slightly higher expression levels.

Proliferation in HUVECs cells after transfection with LNP-formulated RUNX3 mRNA

For analysis of the proliferation of HUVECs after transfection LNP-formulated RUNX3 mRNA, 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.

Table 6: Formulated mRNA constructs used in Example 7

Results: Transfection using LNP-formulated RUNX3 mRNA show a distinct influence on cell proliferation in a concentration dependent manner in HUVEC cells.

Summary of the findings

Aberrant migration and proliferation are key characteristics of pathological cells. Both in vitro models, patient-derived PVR cells (C-PVR) and primary human retinal microvascular endothelial cells (HMRECs), as PVR and aberrant angiogenesis models demonstrated a robust expression of RUNX324 hours after treatment using RUNX3 mRNA; meanwhile, they showed anti-proliferative and anti-migration effects in vitro. RUNX3 positive cells localized with cells in G1 phase (Example 3, Figure 1 and 2). Additionally, 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. Additionally, several fibrotic markers from the collagen family were downregulated with the RUNX3 therapy (Example 4, Figure 3). 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). Furthermore, the in vitro observations correlate with the in vivo findings; minor density of the extracellular membrane is linked with decreased expression of fibrotic-related proteins and upstream pathways like EMT and cytokine signaling. After ? days of treatment, formulated RUNX3 mRNA reduces lesion size in a laser-CNV mouse model (Example 6, Figure 5). Moreover, LNP-formulated RUNX3 mRNA led to an RUNX3 expression in cells and reduced cell proliferation (Example 7, Figures 6 and 7).

Claims

1. An artificial nucleic acid comprising at least one coding sequence encoding a RUNX3 transcription factor or a fragment or variant thereof, wherein the nucleic acid comprises at least one heterologous untranslated region (UTR).

2. The artificial nucleic acid of claim 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.

3. The artificial nucleic acid of claim 1 or 2, wherein the RUNX3 transcription factor, or a fragment or variant thereof, comprises a Runt domain (RD), preferably wherein the RD mediates binding of RUNX3 to DNA as well as an interaction of RUNX3 with the core-binding factor subunit beta (CBFbeta).

4. The artificial nucleic acid of any one of claims 1 to 3, wherein the RUNX3 transcription factor, or a fragment or variant thereof, comprises a transactivation domain (AD) and/or an inhibition domain (ID).

5. The artificial nucleic acid of any one of claims 1 to 4, 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, inflammation, and/or fibrosis.

6. The artificial nucleic acid of any one of the preceding claims, 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 or430.

7. The artificial nucleic acid of any one of the preceding claims, wherein the RUNX3 transcription factor, or a fragment or variant thereof, comprises or consists of an amino acid sequence selected from or derived from a human RUNX3, preferably according to 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 .

8. The artificial nucleic acid of any one of the preceding claims, 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.

9. The artificial nucleic acid of any one of the preceding claims, wherein the at least one coding sequence is a codon modified coding sequence, preferably wherein codon modified coding sequence is selected from a C maximized coding sequence, a CAI maximized coding sequence, human codon usage adapted coding sequence, a G/C content modified coding sequence, and a G/C optimized coding sequence, or any combination thereof, preferably wherein the at least one codon modified coding sequence is a G/C optimized coding sequence.

10. The artificial nucleic acid of any one of the preceding claims, wherein the at least one coding sequence 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.

11. The artificial nucleic acid of any one of the preceding claims, 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.

12. The artificial nucleic acid of claim 11 , 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, beta-globin, 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 any one of SEQ ID NOs: 66-95, 112-123, or a fragment or a variant of any of these.

13. The artificial nucleic acid of claim 11 or 12, 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.

14. The artificial nucleic acid of claim 11 , 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 , 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, 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 any one of SEQ ID NOs: 12-45, 64, 65, or a fragment or a variant of any of these.

15. The artificial nucleic acid of claim 11 or 14, 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 NO: 13, or a fragment or a variant thereof.

16. The artificial nucleic acid of any one of the preceding claims, wherein the at least one at least one heterologous UTR is selected from a 5 -UTR from HSD17B4 and a 3’ UTR from PSMB3.

17. The artificial nucleic acid of any one of the preceding claims, wherein the nucleic acid comprises at least one poly(A) sequence, preferably wherein the at least one poly(A) sequence comprises about 40 to about 500 adenosine nucleotides, preferably about 60 to about 250 adenosine nucleotides, more preferably about 60 to about 150 adenosine nucleotides.

18. The artificial nucleic acid of claim 17, wherein the at least one poly(A) sequence comprises about 100 adenosine nucleotides.

19. The artificial nucleic acid of claim 17 or 18, wherein the at least one poly(A) sequence is located at the 3’ terminus, optionally, wherein the 3' terminal nucleotide is an adenosine.

20. The artificial nucleic acid of any one of the preceding claims, wherein the nucleic acid comprises at least one histone stem-loop sequence, preferably wherein the 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 SEQ ID NO: 3 or4, or a fragment or variant of any of these.

21. The artificial nucleic acid of any one of the preceding claims, wherein the nucleic acid is selected from a DNA vector, preferably an AAV vector, or an RNA.

22. The artificial nucleic acid of any one of the preceding claims, wherein the nucleic acid is an RNA selected from mRNA, circular RNA, replicon RNA, or viral RNA, preferably an mRNA.

23. The artificial nucleic acid of any one of the preceding claims, wherein the nucleic acid is an RNA that comprises at least one modified nucleotide, preferably selected from N1 -methylpseudouridine (m1i ) or pseudouridine (ip), more preferably selected from N1 -methylpseudouridine (m1i ).

24. The artificial nucleic acid of any one of the preceding claims, wherein the nucleic acid is an RNA wherein each uracil is substituted by N1 -methylpseudouridine (m1i ).

25. The artificial nucleic acid of any one of the preceding claims, wherein the nucleic acid is an RNA that comprises a 5’-cap structure.

26. The artificial nucleic acid of claim 25, wherein the 5’-cap structure is selected from a cap1 structure or a modified cap1 structure.

27. The artificial nucleic acid of any one of the preceding claims, 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.

28. The artificial nucleic acid of any one of the preceding claims, 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.

29. The artificial nucleic acid of any one of the preceding claims, 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%.

30. The artificial nucleic acid of any one of the preceding claims, 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.

31. The artificial nucleic acid of any one of the preceding claims, wherein the nucleic acid, preferably the RNA, comprises the following sequence elements preferably in 5’- to 3’-direction:

A) a 5’-cap structure;

B) a 5’-UTR, preferably selected or derived from a 5’-UTR of a HSD17B4 gene;

C) a coding sequence encoding a RUNX3 transcription factor or a fragment or variant thereof;

D) a 3’-UTR, preferably selected or derived from a 3’-UTR of a PSMB3 gene;

E) optionally, a histone stem-loop; and

F) a poly(A) sequence, preferably comprising about 100 A nucleotides.

32. The artificial nucleic acid of any one of the preceding claims, 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 any one of SEQ ID NOs: 269-458, or a fragment or variant of any of these sequences.

33. The artificial nucleic acid of any one of the preceding claims, 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: 300 or 456, or a fragment or variant of any of these sequences.

34. A pharmaceutical composition comprising at least one artificial nucleic acid comprising at least one coding sequence encoding a RUNX3 transcription factor or a fragment or variant thereof as defined in any one of the claims 1 to 33.

35. The pharmaceutical composition of claim 34, wherein the at least one artificial nucleic acid, preferably the RNA, is formulated in at least one cationic or polycationic compound, preferably 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.

36. The pharmaceutical composition of claim 34 or 35, wherein the at least one artificial nucleic acid, preferably the RNA, is formulated in lipid-based carriers.

37. The pharmaceutical composition of claim 36, wherein the lipid-based carriers are selected from liposomes, lipid nanoparticles, lipoplexes, solid lipid nanoparticles, lipo-polylexes, and/or nanoliposomes.

38. The pharmaceutical composition of claim 36 or 37, wherein the lipid-based carriers are lipid nanoparticles, preferably wherein the lipid nanoparticles encapsulate the artificial nucleic acid.

39. The pharmaceutical composition of any one of claims 36 to 39, 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.

40. The pharmaceutical composition of claim 39, wherein the aggregation reducing lipid is selected from a polymer conjugated lipid, preferably wherein the polymer conjugated lipid is selected from a PEG- conjugated lipid or a PEG-free lipid.

41. The pharmaceutical composition of any one of claims 39 to 40, wherein the cationic lipid or ionizable lipid is selected from an amino lipid, preferably wherein the amino lipid comprises a tertiary amine group.

42. The pharmaceutical composition of any one of claims 39 to 41 , wherein the steroid or steroid analog is selected from cholesterol, cholesteryl hemisuccinate (CHEMS), preferably cholesterol.

43. The pharmaceutical composition of any one of claims 34 to 42, wherein the composition comprises an anti- inflammatory agent, preferably wherein the anti-inflammatory agent comprises a steroid or a nonsteroidal anti-inflammatory drug (NSAID).

44. The pharmaceutical composition of any one of claims 34 to 43, wherein the composition comprises at least one RUNX1 inhibitor, wherein the RUNX1 inhibitor is preferably selected from a small molecule inhibitor of RUNX1 , an inhibitory nucleic acid (siRNA) of RUNX1 , or a nucleic acid encoding a RUNX1 inhibitor.

45. The pharmaceutical composition of claim 44, wherein the nucleic acid encoding a RUNX1 inhibitor encodes a CBFbeta-SMMHC fusion protein, for example a full length or truncated RUNX3 protein.

46. The pharmaceutical composition or the artificial nucleic acid of any one of the preceding claims, 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.

47. The pharmaceutical composition or the artificial nucleic acid of claim 68, wherein the administration is an ocular administration.

48. The pharmaceutical composition or the artificial nucleic acid of any one of the preceding claims, 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, 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 .

49. The pharmaceutical composition or the artificial nucleic acid of any one of the preceding claims, 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.

50. The pharmaceutical composition or the artificial nucleic acid of any one of the preceding claims, 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.

51. The pharmaceutical composition or the artificial nucleic acid of any one of the preceding claims, wherein ocular administration of the composition or the nucleic acid reduces or inhibits the cellular expression of RUNX1 , TGFbeta2, SMAD3, and/or COL1 A1 .

52. The pharmaceutical composition or the artificial nucleic acid of any one of the preceding claims, wherein ocular administration of the composition or the nucleic acid reduces or prevents pathological EMT, pathological cell proliferation, aberrant angiogenesis, aberrant neovascularization, degeneration, and/or fibrosis.

53. The pharmaceutical composition or the artificial nucleic acid of any one of claims 47 to 52, 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.

54. The pharmaceutical composition or the artificial nucleic acid of any one of claims 47 to 53, wherein the ocular administration is an intravitreal or intraoperative administration.

The pharmaceutical composition or the artificial nucleic acid of any one of claims 47 to 54, wherein the ocular administration is performed into a tamponade agent-filled human eye, preferably wherein the tamponade agent is a gas agent or a silicone agent.

55. A Kit or kit of parts comprising at least one artificial nucleic acid of any one of claims 1 to 33, and/or at least one pharmaceutical composition of any one of claims 34 to 55, optionally comprising a liquid vehicle for solubilising, and optionally comprising technical instructions providing information on administration and dosage of the components.

56. An artificial nucleic acid of any one of claims 1 to 33, or a pharmaceutical composition of any one of claims 34 to 55, or a kit or kit of parts of claim 56, for use as a medicament in treating or preventing a disease, disorder, or condition in a subject.

57. The artificial nucleic acid of any one of claims 1 to 33, or the pharmaceutical composition of any one of claims 34 to 55, or the kit or kit of parts of claim 56, for use as a medicament of claim 57, wherein the disease, disorder, or condition is associated with or caused by an overexpressed and/or an overactive RUNX1 transcription factor.

58. The artificial nucleic acid of any one of claims 1 to 33, or the pharmaceutical composition of any one of claims 34 to 55, or the kit or kit of parts of claim 56, for use as a medicament of claim 57, wherein the disease, disorder, or condition is associated with or caused by a downregulated and/or inhibited RUNX3.

59. The artificial nucleic acid of any one of claims 1 to 33, or the pharmaceutical composition of any one of claims 34 to 55, or the kit or kit of parts of claim 56, for use as a medicament of claim 57 or 58, 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 retinal pigment epithelial (RPE) cells in a subject.

60. The artificial nucleic acid of any one of claims 1 to 33, or the pharmaceutical composition of any one of claims 34 to 55, or the kit or kit of parts of claim 56, for use as a medicament of any one of claims 57 to 59, wherein the disease, disorder, or condition is an ocular disease, disorder, or condition 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, persistent hyperplastic primary vitreous (PHPV), thyroid eye disease, epiretinal membrane, small vessel disease, induction of epithelial cell differentiation, osteoarthritis, cancer or metastasis, ocular fibrosis, retinal degeneration, osteoporosis, epiretinal membranes (ERM) or proliferative vitreoretinopathy (PVR).

61. The artificial nucleic acid of any one of claims 1 to 33, or the pharmaceutical composition of any one of claims 34 to 55, or the kit or kit of parts of claim 56, for use as a medicament of claim 61 , wherein the ocular disease, disorder, or condition is epiretinal membranes (ERM) and/or PVR, preferably the prevention of ERM and/or PVR.

62. The artificial nucleic acid of any one of claims 1 to 33, or the pharmaceutical composition of any one of claims 34 to 55, or the kit or kit of parts of claim 56, for use as a medicament of claim 61 or 62, wherein cell proliferation and/or cell growth is reduced in eyes with ERM or PVR.

63. The artificial nucleic acid of any one of claims 1 to 33, or the pharmaceutical composition of any one of claims 34 to 55, or the kit or kit of parts of claim 56, for use as a medicament of any one of claims 57 to 63, 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.

64. The artificial nucleic acid of any one of claims 1 to 33, or the pharmaceutical composition of any one of claims 34 to 55, or the kit or kit of parts of claim 56, for use as a medicament of claim 64, wherein the retinal detachment disorder is selected from rhegmatogenous retinal detachment, exudative retinal detachment, or fractional retinal detachment.

65. The artificial nucleic acid of any one of claims 1 to 33, or the pharmaceutical composition of any one of claims 34 to 55, or the kit or kit of parts of claim 56, for use as a medicament of any one of claims 57 to 65, 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.

66. The artificial nucleic acid of any one of claims 1 to 33, or the pharmaceutical composition of any one of claims 34 to 55, or the kit or kit of parts of claim 56, for use as a medicament of claim 66, 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.

67. The artificial nucleic acid of any one of claims 1 to 33, or the pharmaceutical composition of any one of claims 34 to 55, or the kit or kit of parts of claim 56 for use as a medicament of any one of claims 57 to 67, 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.

68. 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 claims 1 to 33, or the pharmaceutical composition of any one of claims 34 to 55, or the kit or kit of parts of claim 56.

69. The method of treating or preventing a disease, disorder or condition of claim 69, wherein the method is further characterized by any of the features as defined in any one of claims 57 to 68.

70. A method of reducing the activity of a RUNX1 transcription factor in a cell or a subject, wherein the method comprises a) applying or administering an artificial nucleic acid comprising at least one coding sequence encoding at least one RUNX3 transcription factor or a fragment or variant thereof as defined in any one of claims 1 to 33; or b) applying or administering a pharmaceutical composition comprising the artificial nucleic acid comprising at least one coding sequence encoding at least one RUNX3 transcription factor or a fragment or variant thereof as defined in any one of claims 34 to 55; to a cell, tissue, or subject, wherein the RUNX3 transcription factor or a fragment or variant thereof is produced in the cell, tissue, or subject after administration or application to said cell, tissue, or subject.