Oligonucleotides encoding tissue inhibitor of metalloproteinase proteins for medical use in mammals

Abstract

The present invention relates to oligonucleotides encoding tissue inhibitors of metalloproteinases (TIMP) proteins, methods for treating diseases and conditions associated with deficiencies in extracellular matrix proteins, uses of said oligonucleotides in such treatments, and pharmaceutical compositions and medical devices comprising said oligonucleotides.

Description

[Technical Field]

[0001] The present invention relates to oligonucleotides encoding tissue inhibitors of metalloproteinases (TIMP) proteins, methods for treating diseases and conditions associated with deficiencies in extracellular matrix proteins, uses of said oligonucleotides in such treatments, and pharmaceutical compositions and medical devices comprising said oligonucleotides.

[0002] The present invention relates to the field of molecular medicine, in particular to the expression of proteins for therapeutic purposes, and more particularly to the treatment of cardiovascular diseases such as aneurysms in mammals. [Background technology]

[0003] Cardiovascular disease (CVD), including aneurysms, is a leading cause of death worldwide and an economic burden on healthcare systems. An aneurysm is an abnormal dilation of an artery due to a localized weakening of the vessel wall. Aneurysms can occur in the aorta (abdominal aortic aneurysm or thoracic aortic aneurysm), blood vessels supplying blood to the brain (cerebral aneurysm), and other parts of the body (peripheral aneurysm).

[0004] Aortic aneurysms can dissect or rupture with massive internal bleeding, resulting in an overall mortality rate of up to 90%. The most common aortic aneurysm is the abdominal aortic aneurysm (AAA). Precipitating factors include age, male gender, smoking, genetic predisposition, hypertension, and arteriosclerosis.

[0005] The arterial wall is composed of three layers. The innermost tunica intima is composed of an endothelial cell layer that contacts the blood and an underlying basement membrane. The tunica intima is surrounded by the tunica media, which contains smooth muscle cells (SMCs) and the extracellular matrix (ECM). The ECM contains elastic and collagen fibers. Elastic fibers provide elasticity and extensibility as elastin, while collagen fibers provide stability against tensile stress as type III collagen. The outermost tunica adventitia is composed of stable type I collagen and is responsible for binding to surrounding tissues.

[0006] The exact reason for the development of aneurysms is unknown. Aneurysms are associated with the degradation of the extracellular matrix (ECM), which weakens the aortic wall. Furthermore, ECM degradation products promote sterile inflammation, leading to the upregulation of smooth muscle cell apoptosis and proteolytic processes, which destabilize and destroy the aortic wall, further progressing the disease. Over time, blood pressure can cause the aorta to dilate, which can lead to aneurysm dissection and / or rupture, resulting in massive internal bleeding and potentially death.

[0007] One of the proteolytic processes is triggered by the production of matrix metalloproteinases (MMPs) by activated macrophages. MMPs are enzymes involved in the physiological turnover of ECM by degrading ECM components such as elastin and collagen. MMP activity is inhibited by the covalent binding of tissue inhibitors of metalloproteinases (TIMPs).

[0008] In abdominal aortic aneurysms (AAA), plasma concentrations and mRNA expression levels of MMP-9 (i.e., gelatinase B) are particularly elevated. TIMP-1, an inhibitor of MMP-9 and other MMPs, is a soluble protein secreted into the extracellular matrix (ECM) and is decreased in aortic aneurysms.

[0009] Treatment of aneurysms is largely limited to surgical treatment of patients with late-stage aneurysms, and there are no clinically relevant approved treatments or causative therapies.

[0010] Medical stabilization of aneurysms relies on the regular systemic administration of antihypertensive drugs. Their use is controversial because they do not repair the vessel wall and some antihypertensive drugs may promote aneurysm formation (Wilmink AB, Vardulaki KA, Hubbard CS, Day NE, Ashton HA, Scott AP, Quick CR. Are antihypertensive drugs associated with abdominal aortic aneurysms? J Vasc Surg. 2002 Oct;36(4):751-7). Furthermore, antihypertensive drugs have a wide variety of side effects. Therefore, there is no effective, specific pharmacologic therapy that can stabilize or cure aneurysms.

[0011] Therefore, monitoring the progression of aneurysms and surgically treating them with open aneurysm repair (OAR) or endovascular aneurysm repair (EVAR) in patients with advanced aneurysms are the only options to reduce the risk of rupture. However, OAR has a high incidence of surgical death and complications, and EVAR often requires repeated therapeutic interventions. Therefore, no efficient and safe surgical treatment exists.

[0012] Regarding gene therapy, it has been reported that retroviral transduction of TIMP-1 in rat smooth muscle cells, followed by seeding of these cells into rats with aortic xenografts from guinea pigs, successfully prevented aneurysmal dilation and rupture (Allaire, E., et al., Local overexpression of TIMP-1 prevents aortic aneurysm degeneration and rupture in a rat model. J Clin Invest, 1998. 102(7): pp. 1413-20). However, retroviral therapy has the disadvantages of mutagenicity and permanent integration into the host genome.

[0013] WO2013151671A1 discloses polynucleotides for the production of cosmetic proteins, one of which may be TIMP-1.

[0014] The present invention therefore aims to overcome or at least mitigate the disadvantages of the prior art. In particular, the present invention provides a medicament suitable for the improved preventive and / or curative treatment of cardiovascular diseases such as aneurysms and arteriosclerosis.

[0015] The present invention can completely solve these problems. Summary of the Invention [Means for solving the problem]

[0016] In one aspect of the present invention, the above-mentioned disadvantages are overcome by providing an oligonucleotide comprising a nucleotide sequence encoding a "tissue inhibitor of metalloproteinase (TIMP)" protein for use in the treatment of a disease or condition in a mammal.

[0017] Currently, four members of the TIMP protein family are known to exist. At least these four members are included within the scope of the present invention, but not excluding TIMPs that have not yet been discovered. These four TIMPs are TIMP-1 (corresponding to human Ensembl Gene-ID: ENSG00000102265), TIMP-2 (corresponding to human Ensembl Gene-ID: ENSG00000035862), TIMP-3 (corresponding to human Ensembl Gene-ID: ENSG00000100234), and TIMP-4 (corresponding to human Ensembl Gene-ID: ENSG00000157150).

[0018] The oligonucleotides of the invention can contain any of the naturally occurring nucleotides.

[0019] As used herein, the term "disease," as generally understood in the medical field, is interpreted as meaning any type of physiologically abnormal condition in a mammal being treated that may be caused by, for example, genetic, tumor, microorganism, bacteria, virus, or mental factors. The term "pathological condition" is also interpreted as meaning any type of physiologically abnormal condition that may be caused by, for example, physical damage to the mammalian body or aging. However, as generally understood, "pathological condition" may also refer to a disease resulting from such damage, and "disease" may also refer to a pathological condition. Therefore, the terms "disease" and "pathological condition" can be used interchangeably herein.

[0020] The oligonucleotides of the invention are suitable for use in mammals, preferably humans.

[0021] In another aspect of this embodiment, the disease or condition is a disease or condition associated with a deficiency of at least one extracellular matrix protein and is preferably selected from the group consisting of aneurysm, fibrosis, arteriosclerosis, aortic stenosis, emphysema, Williams-Beuren syndrome, congenital subaortic stenosis, myocardial infarction, stroke, rheumatoid arthritis, osteoarthritis and tumor metastasis, and more preferably selected from the group consisting of aortic aneurysm, cerebral aneurysm and peripheral aneurysm.

[0022] A disease or condition associated with a "deficiency of at least one extracellular matrix protein" is taken to mean any disease or condition that a mammal suffers from and that would benefit from halting or slowing the further degradation of at least one extracellular matrix (ECM) protein or increasing the abundance of at least one ECM protein in any tissue of that mammal.

[0023] By using the oligonucleotides of the present invention to treat diseases associated with a deficiency of at least one ECM protein, transient expression of TIMPs can be induced in at least one tissue of a mammal lacking at least one ECM protein, thereby inhibiting ECM degradation or restoring ECM in that tissue, potentially slowing progression of the disease and potentially curing the disease.

[0024] In a further aspect of this embodiment, the disease or condition is a disease or condition associated with an unphysiological ratio of MMP / TIMP activity.

[0025] As mentioned above, MMP is inhibited by TIMP.By enhancing MMP activity or reducing TIMP activity, MMP / TIMP ratio increases, resulting in ECM degradation.Therefore, non-physiological MMP / TIMP ratio is related to various diseases, such as cardiovascular disease, and is particularly related to aneurysm.The oligonucleotide of the present invention can be used to enhance the expression of TIMP protein in mammals, and thus has the advantage of reducing MMP / TIMP activity ratio.

[0026] The term "non-physiological" is taken to mean a situation in which a mammal may benefit from a reduction in the MMP / TIMP activity ratio.

[0027] In yet another aspect of this embodiment, the unphysiological MMP / TIMP activity ratio is associated with enhanced MMP activity.

[0028] In abdominal aortic aneurysms, MMP activity is increased, causing ECM degradation and resulting in disease progression. By using the present invention, the inventors have been able to advantageously reduce MMP activity in mammalian tissues.

[0029] In another aspect of this embodiment, the treatment is a preventative or curative treatment, and the treatment is preferably selected from the group consisting of preventing the formation or rupture of an aneurysm and healing an aneurysm, and more preferably the aneurysm is selected from the group consisting of an aortic aneurysm, a cerebral aneurysm, and a peripheral aneurysm.

[0030] The oligonucleotides of the present invention can be advantageously used for preventive treatment, because they can protect the ECM from MMP activity by reducing the MMP / TIMP ratio at a very early stage or before the onset of acute disease. On the other hand, curative treatment at an early stage of disease or at a later stage is also possible, which is beneficial to the health of the patient. Physicians can take advantage of the flexibility provided by the present invention to individually determine the need to start treatment for each patient.

[0031] In particular, with regard to aneurysms, it has been demonstrated that the oligonucleotides of the present invention can effectively reduce MMP activity, advantageously preventing or at least delaying weakening of the vascular wall, i.e., the formation and / or rupture of aneurysms. Advantageously, preventive treatment at an early stage can potentially heal aneurysms by inhibiting MMP degradation while allowing physiological ECM formation to restore the ECM.

[0032] Prior art aneurysm treatments (OAR and EVAR) require monitoring of disease progression, limiting medical intervention to late-stage aneurysms. Therefore, the preventative treatment enabled by the present invention significantly improves physicians' access to treatment while reducing patient risk.

[0033] In one embodiment of the present invention, the oligonucleotide of the present invention is an oligoribonucleotide, preferably an mRNA.

[0034] In recent years, the therapeutic application of synthetic mRNA has demonstrated promise due to several advantages over prior art technologies. First, synthetic mRNA can be easily produced by in vitro transcription (IVT). Second, synthetic mRNA does not integrate into the host genome and is physiologically degraded. Therefore, synthetic mRNA exists only transiently in cells, significantly reducing the risk of mutagenicity compared to viral vectors. Furthermore, other side effects associated with long-term protein overexpression can be avoided. Third, synthetic mRNA is smaller than plasmids or viral vectors, making it easier to deliver into cells.

[0035] As used herein, the terms "mRNA" and "synthetic mRNA" are used interchangeably. Furthermore, "synthetic" means that the mRNA is artificially produced, and preferably, the mRNA is artificially produced by in vitro transcription (IVT). There may or may not be structural or chemical differences between synthetic mRNA and mRNA.

[0036] In another aspect of this embodiment, the oligonucleotide of the invention comprises a 5' cap structure and / or a poly-A tail. The 5' cap structure is preferably a synthetic anti-reverse cap analog, more preferably 3'-O-Me-m7G(5')ppp(5')G. The poly-A tail is preferably a poly-A tail of at least about 70 adenine nucleotides, more preferably a poly-A tail of about 120 adenine nucleotides.

[0037] Such modifications mimic the natural structure of mammalian mRNA and thus have the advantage of conferring a degree of stability to the oligonucleotides of the invention, thereby reducing the rate of degradation of the oligonucleotides of the invention in cells to which they are delivered, thereby enabling translation of the oligonucleotides of the invention. Additionally, synthetic anti-reverse cap analogs have the advantage of improving the efficiency of the IVT reaction and the purity of the IVT reaction products.

[0038] The 5' cap structure may be naturally occurring. The 5' cap structure may also be synthetic or modified, and such cap structures are also referred to as "5' cap analogs." Thus, the term "5' cap structure" refers to any naturally occurring 5' cap structure used naturally by eukaryotic cells, as well as any non-naturally occurring synthetic / modified 5' cap structure or 5' cap analog that is suitable as a replacement for the naturally occurring 5' cap structure in terms of functionality and cytotoxicity.

[0039] While anti-reverse cap analogs are preferred, other 5' cap structures are also suitable. Examples of such cap structures include m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, and m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

[0040] In a further embodiment of the invention, at least one nucleotide is an analog of a natural nucleotide, and the substitution rate of one natural nucleotide with the analog is at least about 5%, preferably at least about 25%, and more preferably about 100%.

[0041] Natural, unmodified RNA is biologically unstable and immunogenic, limiting its bioavailability and potential for therapeutic applications. Furthermore, enzymes that degrade RNA are nearly ubiquitous, particularly in the extracellular space. Furthermore, the innate immune system, involving Toll-like receptors (TLRs), recognizes single-stranded RNA (TLR-7, TLR-8) and double-stranded RNA (TLR-3) to induce inflammatory immune responses.

[0042] This embodiment has the advantage of reducing the rate of degradation of the synthetic mRNA and preventing recognition of the synthetic mRNA by the immune system, thus avoiding an inflammatory response.

[0043] In this embodiment, the analogs of natural nucleotides account for a large proportion of the total nucleotides. "Natural nucleotide" refers to a nucleotide that contains a nucleobase that is naturally found in mammalian DNA or RNA. Specifically, naturally found nucleobases include adenine, guanine, cytosine, thymidine and uracil. "Analog" is understood to mean a nucleotide that reduces the degradation rate of the oligonucleotide of the present invention and / or reduces its immunogenicity, and has a chemical structure similar to that of natural nucleotides, allowing the nucleotide sequence encoded by the oligonucleotide of the present invention to be translated into the encoded protein by the translational machinery of a cell.

[0044] The term "majority" means that not all of a given naturally occurring nucleotide is necessarily substituted with an analog: at least about 5% of a given naturally occurring nucleotide is substituted with an analog, preferably about 25% of a given naturally occurring nucleotide is substituted with an analog, and more preferably about 100% of a given naturally occurring nucleotide is substituted with an analog.

[0045] In this embodiment, the analog is, for example, pseudouridine (Ψ), N 1 -Methylpseudouridine (me 1Ψ), 5-methylcytidine (5mC), phosphorothioate, phosphoramidate, peptide nucleotide, methylphosphonate, 7-deazaguanosine, 2-thiouridine, 5-methyluridine, 5-methyluridine-5'-triphosphate (m5U), 5-iodouridine-5'-triphosphate (I5U), 4-thiouridine-5'-triphosphate (S4U), 5-bromouridine-5'-triphosphate (Br5U), 2'-methyl-2'-deoxyuridine-5'-triphosphate (U2'm), 2'-amino-2'-deoxyuridine 2'-amino-2'-deoxycytidine-5'-triphosphate (C2'NH2), 2'-azido-2'-deoxyuridine-5'-triphosphate (U2'N3), 2'-fluoro-2'-deoxyuridine-5'-triphosphate (U2'F), inosine, 3-methylcytidine, 2-thiocytidine, 2'-methyl-2'-deoxycytidine-5'-triphosphate (C2'm), 2'-amino-2'-deoxycytidine-5'-triphosphate (C2'NH2), 2'-fluoro-2'-deoxycytidine-5'-triphosphate (C2'F), 5-iodocytidine-5'-triphosphate (I5 U), 5-bromocytidine-5'-triphosphate (Br5C), 2'-azido-2'-deoxycytidine-5'-triphosphate (C2'N3), 5-azauridine, 2-thio-5-azauridine, 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethylpseudouridine, 5-propynyluridine, 1-propynylpseudouridine, 5-taurinomethyluridine, 1-taurinomethylpseudouridine, 5-taurin 1-Taurinomethyl-2-thiouridine, 1-Taurinomethyl-4-thiouridine, 2-Thio-1-methylpseudouridine, 1-Methyl-1-deazapseudouridine, 2-Thio-1-methyl-1-deazapseudouridine, Dihydrouridine, Dihydropseudouridine, 2-Thiodihydrouridine, 2-Thiodihydropseudouridine, 2-Methoxyuridine, 2-Methoxy-4-thiouridine, 4-Methoxypseudouridine, 4-Methoxy-2-thiopseudouridine, 5-Azacytidine, Pseudoisocytidine, N 4 -acetylcytidine, 5-formylcytidine, N 4-methylcytidine, 5-hydroxymethylcytidine, 1-methylpseudoisocytidine, pyrrolocytidine, pyrrolopseudoisocytidine, 2-thio-5-methylcytidine, 4-thiopseudoisocytidine, 4-thio-1-methylpseudoisocytidine, 4-thio-1-methyl-1-deazapseudoisocytidine, 1-methyl-1-deazapseudoisocytidine, zebularine, 5-azazebularine, 5-methylzebularine, 5-aza-2-thiozebularine, 2-thiozebularine, 2-methoxycytidine, 2-methoxy-5-methylcytidine, 4-methoxypseudoisocytidine, 4-methoxy-1-methylpseudoisocytidine, 7-deazaadenine, 7-deaza-8-azaadenine, 1-methyladenosine, N 6 -methyladenosine, N 6 -Isopentenyl adenosine, N 6 -(cis-Hydroxyisopentenyl)-adenosine, 2-methylthio-N 6 -(cis-hydroxyisopentenyl)-adenosine, N 6 -Glycinylcarbamoyl adenosine, N 6 -Threonylcarbamoyladenosine, 2-methylthio-N 6 -Threonylcarbamoyladenosine, N 6 ,N 6 -dimethyladenosine, 7-methyladenine, 2-methylthioadenine, 2-methoxyadenine, 1-methylinosine, 7-deaza-8-azaguanosine, 6-thioguanosine, 6-thio-7-deazaguanosine, 6-thio-7-deaza-8-azaguanosine, 7-methylguanosine, 6-thio-7-methylguanosine, 7-methylinosine, 6-methoxyguanosine, 1-methylguanosine, N 2 -methylguanosine, N 2 ,N 2 -dimethylguanosine, 8-oxoguanosine, 7-methyl-8-oxoguanosine, 1-methyl-6-thioguanosine, and N 2 -methyl-6-thioguanosine, and combinations thereof.

[0046] In one aspect of this embodiment, the naturally occurring nucleotide uridine and / or uridine is replaced with a nucleotide analog, the naturally occurring nucleotide uridine and / or uridine is replaced with a nucleotide analog, the naturally occurring nucleotide uridine and / or uridine is replaced with a combination of pseudouridine and 5-methylcytidine, N ... 1 -methylpseudouridine and 5-methylcytidine replacing cytidine, and N replacing uridine 1 -methylpseudouridine or a combination thereof, and N substituting uridine is preferably 1 Most preferably, it is substituted with -methylpseudouridine.

[0047] Substitution of the above-mentioned natural nucleotides with such analogues has been found to be highly suitable for reducing the immunogenicity of the oligonucleotides of the invention, thereby allowing cell survival and expression of TIMPs during therapy.

[0048] Surprisingly, uridine was replaced with N without replacing cytidine. 1 Substitution with -methylpseudouridine results in the most efficient and highest TIMP expression. 1 It is highly preferred to only replace uridine with -methylpseudouridine.

[0049] Also in this embodiment, the substitution rate of one natural nucleotide with an analog is at least about 5%, preferably at least about 25%, and more preferably about 100%.

[0050] In another embodiment of the present invention, the TIMP protein is TIMP-1 protein, which is an inhibitor of various types of MMPs and therefore has the advantage of being able to efficiently reduce MMP activity.

[0051] In another embodiment of the invention, the TIMP-1 protein comprises the amino acid sequence of SEQ ID NO:1.

[0052] The use of this amino acid sequence of human TIMP-1 allows for accurate expression of TIMP-1 in human cells, which has the advantage that the oligonucleotides of the invention can be used for medical purposes targeting humans without the risk of immune response, cytotoxicity, or dysfunctional proteins.

[0053] In another embodiment of the present invention, the oligonucleotide of the present invention comprises the nucleotide sequence of SEQ ID NO:2.

[0054] The use of this nucleotide sequence of human TIMP-1 allows efficient translation and expression of TIMP-1 in human cells, which has the advantage that the oligonucleotides of the present invention can be used for medical purposes targeting humans. Furthermore, this sequence has low immunogenicity and high expression efficiency.

[0055] In another embodiment, the oligonucleotides of the invention are complexed with a polymer or lipid, preferably contained within a lipid nanoparticle or liposome, which has the advantages of further protecting the oligonucleotides of the invention from degradation, reducing immunogenicity, and facilitating delivery through improved cellular internalization.

[0056] In another aspect, the present invention relates to a pharmaceutical composition comprising an oligonucleotide according to any one of the preceding claims and a pharmaceutically acceptable carrier.

[0057] Those skilled in the art are familiar with the carrier suitable for the above-mentioned specific treatment.In addition, the pharmaceutically acceptable carrier used for synthetic mRNA is disclosed in, for example, Ouranidis A, et al., mRNA Therapeutic Modalities Design, Formulation and Manufacturing under Pharma 4.0 Principles.Biomedicines, 2021.Dec 27;10(1):50.The content of this document is incorporated herein by reference.

[0058] In one embodiment, the pharmaceutical composition of the present invention is adapted for local administration by injection into mammalian tissue or topical application onto mammalian tissue, and is preferably contained in a formulation or delivery form selected from the group consisting of a fluid, an implant, a balloon catheter, a vascular graft, a microneedle, a patch, and a stent.

[0059] The term "fluid" is understood to mean a material that undergoes continuous deformation when subjected to shear force. Fluids may be in the form of a gas, aerosol, or liquid. Unnecessary induction of TIMP expression in areas of the body that do not require ECM protection or repair can be harmful to the recipient mammal. Local administration is advantageous because it can specifically induce TIMP expression at the site of disease or pathology without unnecessarily affecting areas of the body that do not require ECM protection or repair.

[0060] Topical application of any kind is an added advantage because it is less invasive and therefore less stressful than other methods of administration.

[0061] The aforementioned formulation or delivery form in the form of a fluid is advantageous because the fluid allows the pharmaceutical composition of the present invention to be applied to almost any area of ​​the body by injection, inhalation, etc. In this regard, the use of microneedles as a delivery form allows for minimally invasive administration, and the use of patches as a delivery form allows for non-invasive administration.

[0062] The aforementioned implant-based formulations or delivery forms are advantageous because they allow repeated or sustained local application of the oligonucleotides of the invention to the desired tissue, optionally in an automated manner, without the need for chronic or repeated medical intervention other than for implant placement and, optionally, implant removal.

[0063] Formulations or delivery forms in the form of balloon catheters, artificial blood vessels or stents are advantageous because they allow the pharmaceutical compositions of the present invention to be applied to blood vessels and are widely used and approved for the treatment of aneurysms.

[0064] In one embodiment, the pharmaceutical compositions of the present invention are adapted for systemic administration, and are preferably contained in a formulation or delivery form selected from the group consisting of fluids and implants.

[0065] The aforementioned formulation or delivery forms in fluid form are advantageous because the fluid allows the application of the pharmaceutical compositions of the present invention to almost any area of ​​the body by injection, regardless of the size of the body area to which it is applied.

[0066] The aforementioned implant-based formulations or delivery forms are advantageous because they allow repeated or sustained local application of the oligonucleotides of the invention to the desired tissue, optionally in an automated manner, without the need for chronic or repeated medical intervention other than for implant placement and, optionally, implant removal.

[0067] In another embodiment, the pharmaceutical composition of the present invention comprises or is administered in combination with at least one antihypertensive agent, which may be advantageous, particularly in the treatment of acute and late stage aneurysms, to prevent the imminent risk of rupture or dissection.

[0068] Another aspect of the invention is a medical device comprising or coated with an oligonucleotide of the invention.

[0069] In one embodiment, the medical device of the present invention is configured for permanent or temporary placement within a mammalian body, and is preferably selected from the group consisting of a graft, a balloon catheter, a vascular graft, a microneedle, a patch, and a stent, which advantageously allows a physician to adjust the duration and extent of treatment according to the individual needs of the patient.

[0070] The features, properties and advantages described with respect to the oligonucleotides of the invention apply equally to the pharmaceutical compositions and medical devices of the invention.

[0071] Another aspect of the present invention is a method for treating a disease or condition associated with a deficiency in at least one extracellular matrix protein, comprising administering to a living mammal suspected of suffering from the condition an oligonucleotide and / or pharmaceutical composition and / or medical device of the present invention. This administration can be achieved by local or systemic application.

[0072] In one embodiment, the method of the present invention may include at least one repeated application of the oligonucleotide and / or pharmaceutical composition and / or medical device of the present invention. Repeated application of the oligonucleotide and / or pharmaceutical composition and / or medical device of the present invention allows for long-term or repeated inhibition of ECM degradation or ECM repair in at least one tissue to which they are applied. Such repeated application may be necessary because the expression of TIMPs by the oligonucleotide is temporary and non-permanent.

[0073] By convention, all nucleotide sequences in this disclosure are understood to be presented as DNA sequences, but the nucleotide sequences of this disclosure also include RNA sequences. In the RNA sequences corresponding to the DNA sequences described herein, all thymines (T, t) are replaced with uracils (U, u). In particular, for mRNA molecules, such substitutions are made, but the DNA sequences are also explicitly described herein. This means, for example, that the nucleotide sequence represented by SEQ ID NO: 4 (TTGGACCCTC GTACAGAAGC TAATACG), which is described as a DNA sequence, includes the RNA nucleotide sequence represented by UUGGACCCUC GUACAGAAGC UAAUACG. This also applies to the nucleotide sequences of other DNA molecules / RNA molecules of the present invention.

[0074] The features mentioned above and below may not only be used in the specific combinations described herein, but may also be used in other combinations or alone without departing from the scope of the invention.

[0075] The present invention will be described and explained in more detail with reference to the following examples and figures, but the present invention is not limited to these examples and figures. [Brief explanation of the drawings]

[0076] [Figure 1]Analysis of TIMP-1 protein expression after transfection of various cell lines with synthetic TIMP-1 mRNA is shown. 0.5 μg, 1.0 μg, or 1.5 μg of TIMP-1 mRNA was complexed with L2000 at a 1:1 ratio and transfected into 3 × 105 EA.hy926, NUFF, or HUVEC cells. After 4 h of incubation at 37°C and 5% CO2, the transfection complex was replaced with the appropriate cell culture medium for each cell line. After a further 24 h of incubation at 37°C and 5% CO2, the concentration of TIMP-1 protein in (A) the supernatant and (B) the cell lysate was measured using a human TIMP-1-specific ELISA. Cells treated with medium alone or medium containing L2000 served as controls. Results are shown as the mean + SD (n = 3). Statistical differences were determined by one-way analysis of variance followed by Tukey's multiple comparison test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0077] [Figure 2](A) and (B) show the application of synthetic hGLuc mRNA to ex vivo porcine and human aortic vessels. 1 μg of Cy3-labeled hGLuc mRNA was complexed with 1 μl of L2000 in 50 μl of Opti-MEM and injected intraluminally into porcine (A) and human (B) aortic vessels. After microinjection, the samples were fixed with 4% PFA and paraffin sections (2.5 μm) were prepared. Cell nuclei were stained with DAPI. White arrows indicate Cy3-labeled hGLuc mRNA in the tunica media and adventitia. DAPI (blue), Cy3-labeled hGLuc mRNA (red). I: intima, M: media, A: adventitia. (C) shows the detection of luciferase activity after microinjection of 1 μg of synthetic hGLuc mRNA with 1 μl of L2000 into ex vivo porcine aorta (left panel) and human aorta (right panel). After 24 or 48 hours of incubation at 37°C and 5% CO2 without changing the medium, cell supernatants were analyzed by luciferase assay. Controls included aortic vessels injected with medium alone or medium containing 1 μl of L2000. Statistical analysis was performed by two-way ANOVA followed by Bonferroni correction (**p ≤ 0.01). Results are presented as mean + SEM (pig: n = 4, human: n = 5).

[0078] [Figure 3] Detection of TIMP-1 levels in the supernatant of porcine aorta after microinjection of 3 μg of TIMP-1 mRNA. 3 μg of TIMP-1 mRNA was complexed with L2000 in 50 μl of Opti-MEM and injected into the aortic vessels of pigs. TIMP-1 levels in the supernatant were detected 24 and 48 hours after injection using TIMP ELISA. Results are shown as mean + SEM (n = 3). Statistical differences were determined by two-way ANOVA followed by Tukey's multiple comparison test (*p < 0.05).

[0079] [Figure 4]MMP activity was analyzed by in situ zymography after microinjection of porcine TIMP-1 mRNA into porcine aortas and incubation for 24 or 48 hours. Five micrograms of TIMP-1 mRNA was complexed with L2000 at a 1:1 ratio in 50 μl of Opti-MEM and injected ex vivo into porcine aortas from the intimal side. Aortic tissues were incubated at 37°C and 5% CO2 for 24 or 48 hours. Opti-MEM containing L2000 alone was injected as a control. After incubation, aortic tissues were fixed and 5 μm paraffin sections were prepared. MMP-9 activity was visualized by in situ zymography using DQ gelatin. To confirm enzymatic cleavage of gelatin by MMPs, control sections were blocked with EDTA and then mounted on a DQ substrate. Cell nuclei were stained with mounting medium containing DAPI. DQ gelatin: green; nuclei: blue. Magnification 20x (n=3).

[0080] [Figure 5] Human TIMP-1 mRNA was microinjected into human aortas and incubated for 24 or 48 hours before in situ zymography analysis of MMP activity. 5 μg of TIMP-1 mRNA was complexed with L2000 at a 1:1 ratio in 50 μl of Opti-MEM and injected ex vivo into human aortas from the intimal side. Aortic tissues were incubated at 37°C and 5% CO2 for 24 or 48 hours. Opti-MEM containing L2000 alone was injected as a control. After incubation, aortic tissues were fixed and sectioned into 5 μm paraffin. MMP-9 activity was visualized by in situ zymography using DQ gelatin. To confirm enzymatic cleavage of gelatin by MMPs, control sections were blocked with EDTA and then mounted on a DQ substrate. Cell nuclei were stained with mounting medium containing DAPI. DQ gelatin: green; nuclei: blue. Magnification 20x (n=3).

[0081] [Figure 6]The effect of nucleotide modifications on the expression efficiency of synthetic TIMP-1 mRNA in EA.hy926 cells was shown. 3 x 105 EA.hy926 cells were seeded and transfected with 1.5 μg of TIMP-1 mRNA complexed with 1.5 μl of Lipofectamine 2000 in OptiMEM for 4 hours at 37°C and 5% CO2. The transfection complex was then replaced with cell culture medium, and the cells were incubated at 37°C and 5% CO2. Control cells were treated with OptiMEM alone or OptiMEM containing L2000. After 24 hours, TIMP-1 concentrations in the harvested supernatants were measured using a human TIMP-1-specific ELISA. Results are shown as mean + SEM (n = 3). Statistical differences between mRNA treatment groups were determined by one-way analysis of variance followed by Bonferroni's multiple comparison test (*p < 0.05, ns: not significant).

[0082] [Figure 7] This figure shows the analysis of MMP activity using in situ zymography after adventitial microinjection of human TIMP-1 mRNA into porcine aortas. Five micrograms of TIMP-1 mRNA was complexed with L2000 at a 1:1 ratio in 50 μl of Opti-MEM and injected into the adventitial side. The aortic tissue was then incubated at 37°C and 5% CO2 for 24 or 48 hours. Opti-MEM containing L2000 was used as a control. After incubation, the aortic tissue was fixed and 5 μm paraffin sections were prepared. MMP-9 activity was visualized by in situ zymography using DQ gelatin. To confirm the enzymatic cleavage of gelatin by MMPs, control sections were blocked with EDTA and then mounted on a substrate. Cell nuclei were stained with mounting medium containing DAPI. DQ gelatin: green; nuclei: blue. Magnification: 20x (n=3). [Example]

[0083] 1. Materials and Methods 1.1 Cell culture EA.hy926 cells (ATCC) and human fibroblasts (NUFF, neonatal foreskin fibroblasts, AMS Biotechnology (Europe), Abingdon, UK) were cultured in high-glucose, L-glutamine-containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37°C and 5% CO2. Cells were then washed once with Dulbecco's phosphate-buffered saline (DPBS) and detached with 0.05% trypsin-EDTA. All cell culture reagents were obtained from Thermo Fisher Scientific (Waltham, MA, USA).

[0084] Human umbilical vein endothelial cells (HUVECs) (PromoCell, Heidelberg, Germany) were cultured in VascuLife EnGS endothelial cell medium (hydrocortisone-free) (Lifeline Cell Technology, Frederick, MD, USA) at 37°C and 5% CO2. Cells were then washed once with DPBS and detached using 0.04% trypsin / 0.03% EDTA and 0.1% BSA in 0.05% trypsin neutralizing solution (TNS) (both PromoCell, Heidelberg, Germany). Before seeding, cell culture flasks were coated with 0.1% gelatin (Fluka, Morristown, USA) in PBS for 15 min at room temperature (RT). The medium was changed every 3–4 days, and cells were passaged when they reached 80–90% confluence.

[0085] 1.2 mRNA synthesis Synthetic mRNA was produced by in vitro transcription (IVT) according to a previously reported method (Avci-Adali, M., et al., In vitro synthesis of modified mRNA for induction of protein expression in human cells. JoVE (Journal of Visualized Experiments), 2014(93): p. e51943). Plasmids containing the secreted humanized Gaussia luciferase coding sequence (hGLuc, SEQ ID NO: 3) and the human TIMP-1 coding sequence (hTIMP-1, SEQ ID NO: 2) were produced by Eurofins Genomics (Ebersberg, Germany). DNA templates containing the hGLuc and hTIMP-1 coding sequences were generated by PCR using these plasmids. Specifically, 50–100 ng of the plasmids were used with the HotStar HiFidelity Polymerase Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The forward primer (SEQ ID NO: 4) and reverse primer (SEQ ID NO: 5) were purchased from ELLA Biotech (Martinsried, Germany). PCR was performed at 94°C for 3 minutes, followed by 30 cycles of denaturation at 94°C for 45 seconds to generate single strands, primer hybridization at 60°C for 1 minute, and extension at 72°C for 1 minute, with a final extension at 72°C for 5 minutes. The amplified PCR products were purified using a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The concentration and purity of the resulting DNA were analyzed using a photometer, and the size of the amplified DNA fragments was controlled by 1% agarose gel electrophoresis.

[0086] In vitro transcription (IVT) was performed using the T7 MEGAscript Kit (Life Technologies, Darmstadt, Germany) according to the manufacturer's instructions. 1.5 μg of hGLuc or hTIMP-1 DNA template was used for IVT. For IVT, 2.5 mM 3'-O-Me-m7G(5')ppp(5')G RNA cap analog (ARCA, New England BioLabs, Frankfurt am Main, Germany) and the following nucleotides were incorporated: 1.875 mM GTP, 7.5 mM ATP, 7.5 mM 5-methyl-CTP (5mC), and 7.5 mM Ψ. This mRNA modification was designated Ψ / 5mC. To analyze the effect of this nucleotide modification on TIMP-1 protein expression, 7.5 mM N was added instead of Ψ. 1 -Methylpseudouridine (me 1 Ψ) and 7.5mM 5mC or (me 1 Ψ / 5mC), or 7.5mM N 1 -Methylpseudouridine (me 1 Ψ) and 7.5 mM CTP (me 1A separate TIMP-1 mRNA was generated using the Ψ / C primer. ATP, GTP, and CTP were used as supplied in the MEGAscript T7 Kit, and other nucleotides were purchased from TriLink BioTechnologies (San Diego, USA). 40 U of RiboLock RNase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA) was added to each IVT reaction and incubated at 37°C for 4 hours. To remove the DNA template, 1 μl of TurboDNase was added and incubated for an additional 15 minutes. The mRNA was then purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The mRNA was then dephosphorylated with 15 U of Antarctic phosphatase (New England BioLabs, Frankfurt am Main, Germany) for 30 minutes at 37°C and purified as described above. The concentration and purity of the mRNA were analyzed by spectrophotometer, and the size of the mRNA was confirmed by 1% agarose gel electrophoresis followed by staining with 1× GelRed (Biotium, Fremont, CA, USA) in 1× Tris-borate-EDTA (TBE) buffer for 1 h at room temperature (RT).

[0087] 1.3 Cy3 labeling of hGLuc mRNA To assess mRNA localization in the vascular wall, hGLuc mRNA was labeled with the fluorescent dye Cy3. To do this, Cy3 was linked to mRNA using copper-free click chemistry. During IVT, 5-azido-C3-UTP molecules (Jena Bioscience, Jena, Germany) were incorporated into mRNA, and then the 5-azido-C3-UTP molecules were labeled with Cy3. For this incorporation and labeling, IVT was performed using 25% 5-azido-C3-UTP (1.875 mM) and 75% Ψ (5.625 mM) as previously described in our previous study

[31] . After azido-mRNA purification, the azido-mRNA was coupled to DBCO-sulfo-Cy3 (Jena Bioscience) at a 5:1 ratio in a total volume of 40 μl of nuclease-free water for 1 h at 37°C. The labeled mRNA was purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and evaluated by 1% agarose gel electrophoresis.

[0088] 1.4 Transfection of TIMP-1 mRNA into cells 3×10 5HUVEC, NUFF, or EA.hy926 cells were seeded into each well of a 6-well plate and cultured overnight at 37°C and 5% CO2 in the appropriate cell culture medium. Prior to seeding, the cell culture plate was coated with 0.1% gelatin in PBS for 15 minutes. Cells were transfected with 0.5 μg, 1 μg, or 1.5 μg of synthetic mRNA complexed with Lipofectamine 2000 (L2000) at a 1:1 ratio in 1 ml of Opti-MEM (except for 0.5 μg mRNA, which required 1 μl of L2000). Opti-MEM alone or Opti-MEM plus 1.5 μl of Lipofectamine 2000 served as controls. To analyze the effect of modified nucleotides on TIMP-1 mRNA expression efficiency, 1.5 μg of each modified TIMP-1 mRNA variant was complexed with 1.5 μl of L2000 and transfected into EA.hy926 cells. Each transfection mixture was incubated at room temperature for 20 minutes. Before adding the transfection mixture to the cells, the cells were washed once with DPBS, and then the transfection complexes and cells were incubated at 37°C and 5% CO2 for 4 hours. The transfection mixture was then replaced with 2 ml of cell culture medium, and the cells were incubated at 37°C and 5% CO2 for an additional 24 hours. The cell culture supernatants were collected, snap-frozen in liquid nitrogen, and stored at -80°C until a TIMP-1 protein-specific ELISA was performed. Cells were then lysed, and intracellular TIMP-1 protein levels were detected by ELISA.

[0089] 1.5 Cell lysis after transfection of TIMP-1 mRNA After washing the cells twice with 1 ml of cold DPBS, 300 μl of cold 1x RIPA buffer containing a 1:100 dilution of Halt protease inhibitor cocktail (both from Thermo Fisher Scientific, Waltham, MA, USA) was added to the cells and incubated on ice for 5 minutes. The cells were then lysed by repeated pipetting, transferred to a reaction tube, and sonicated three times for 10 seconds with a 10-second pause. The cell lysate was then centrifuged at 13,000 rpm at 4°C for 25 minutes. The supernatant was collected, snap-frozen in liquid nitrogen, and stored at -80°C until TIMP-1 protein-specific ELISA was performed.

[0090] 1.6 Application of synthetic mRNA to vascular walls 1.6.1 Tissue preparation Porcine and human aortic tissue was placed in 0.9% NaCl solution immediately after harvest and used for microinjection the same day. Prior to microinjection, the tissue was cut into 0.5 × 0.5 cm strips and incubated for 30 minutes in an antibiotic solution consisting of DMEM (Thermo Fisher Scientific, Waltham, MA) containing 250 mg / mL gentamicin (Sigma-Aldrich, St. Louis, USA) and 1.25 mg / mL amphotericin B (Promocell, Heidelberg, Germany). The tissue was then washed with DPBS (Thermo Fisher Scientific, Waltham, MA).

[0091] 1.6.2 Injection of synthetic hGLuc mRNA or synthetic TIMP-1 mRNA To analyze synthetic mRNA-mediated protein expression in the aortic vascular wall, each synthetic mRNA was delivered ex vivo into the porcine and human aortic vascular wall by microinjection from the intimal side using a MicronJet 600 hollow microneedle (NanoPass Technologies, Ness Ziona, Israel). Specifically, 1 μg of hGLuc mRNA or 3 or 5 μg of TIMP-1 mRNA was complexed with Lipofectamine 2000 (L2000) at a 1:1 (μg:μl) ratio and incubated in 50 μl of Opti-MEM at room temperature for 20 minutes. The prepared mRNA solution was drawn up into a 1 ml (Luer-lock) syringe using a cannula, and the cannula was replaced with a microinjection needle, allowing injection from the luminal side of the vessel.

[0092] After injection, each sample was incubated for 5 minutes at room temperature, washed once with DPBS, and then incubated in 1 ml of EC medium in a 12-well plate at 37°C and 5% CO2 for 24–48 hours. Luciferase activity, TIMP-1 levels by ELISA, and exogenously produced TIMP-1 activity within the vessel wall were analyzed by in situ zymography. As a control, 50 μl of Opti-MEM and the corresponding volume of L2000 were microinjected into aortic tissue. For in situ zymography, tissue samples were incubated in 10x zinc fixative (formalin-free, BD Pharmingen, Becton Dickinson, Franklin Lakes, USA) at 4°C for 48 or 72 hours.

[0093] To assess mRNA localization after microinjection into the vessel wall, 1 μg of Cy3-labeled hGLuc-mRNA was complexed with 1 μl of L2000 in 50 μl of Opti-MEM for 20 minutes at room temperature. As a control, tissues treated with Opti-MEM and the corresponding amount of L2000 were used. After microinjection, tissues were washed once with DPBS and fixed overnight in 4% paraformaldehyde (PFA) at 4°C for histological analysis.

[0094] 1.6.3 Detection of luciferase activity After delivery of the synthetic hGLuc mRNA to the aortic vascular wall, the expression level of hGLuc in the tissue supernatant was measured by luciferase assay. Specifically, 40 μl of each supernatant was transferred in triplicate to a 96-well plate (Nunc Maxisorp, Thermo Fisher Scientific, Waltham, MA, USA) and diluted in DPBS (Ca) containing 20 μg / ml coelenterazine (Carl Roth, Karlsruhe, Germany). 2+ / Mg 2+ 100 μl of the solution (free of HCl) was automatically injected into each well, and luminescence was detected as relative light units (RLU) using a microplate reader Mithras LB 940 (Berthold Technologies, Bad Wildbad, Germany).

[0095] 1.7 Histological analysis 1.7.1 Detection of mRNA microinjected into the aortic vascular wall After fixation, each sample was washed twice with 2 ml of DPBS, transferred to an embedding cassette, and coated with 100% ethanol. Next, the samples were dehydrated and embedded in paraffin using an automated system at the Institute of Pathology, University Hospital Tübingen. 2.5 μm-thick tissue sections were then prepared. The tissue sections were deparaffinized using xylene four times for 5 minutes each, then rehydrated stepwise through an ethanol series: 99% ethanol twice for 1 minute, 96% ethanol twice for 1 minute, and 70% ethanol twice for 1 minute each. The sections were then washed twice with nuclease-free water and boiled in TBE buffer (pH 9) for 2 minutes. The slides were cooled under running water and washed three times with DPBS. Cy3 mRNA-injected samples were embedded in Fluoroshield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA). Xylene and ethanol were obtained from AnalaR NORMAPUR (VWR, Darmstadt, Germany). To detect Cy3-labeled mRNA, fluorescent images were acquired with an Axiovert135 microscope (Carl Zeiss, Oberkochen, Germany) and analyzed with AxioVision Rel 4.8 software.

[0096] 1.7.2 In situ zymography Tissues were fixed using a tissue embedding apparatus and automated tissue processor at the Institute of Pathology, University Hospital Tübingen, then transferred to embedding cassettes, dehydrated, and embedded. Tissues were then embedded in paraffin blocks and sectioned at 5 μm thickness using a Microm HM 355S microtome (Thermo Fisher Scientific, Waltham, MA, USA) onto Superfrost microscope slides (R. Langenbrinck, Emmendingen, Germany). Tissue sections were deparaffinized twice for 1 min each using xylene (100% xylene, isomer mixture, AnalaR NORMAPUR, VWR, Darmstadt, Germany) and then rehydrated in a descending series of ethanol (100%, 80%, 70%, 60%). After washing the slides with double-distilled water (ddH2O), staining was performed using the EnzChek Gelatinase / Collagenase Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). 250 μl of substrate solution consisting of 1 mg / ml fluorescently labeled DQ gelatin diluted 1:50 in reaction buffer (150 mM NaCl (NORMAPUR®, VWR International, Radnor, MA, USA), 5 mM CaCl2, 50 mM Tris-HCl, 0.2 mM sodium azide (all Sigma-Aldrich, Darmstadt, Germany); pH 7.6) was added to each section and incubated for 2 hours at 37°C in a humidified chamber. As a control, tissue sections were treated with 20 mM EDTA for 1 hour to inhibit the enzymatic activity of metalloproteinases. Slides were then incubated with the substrate solution for 2 hours at 37°C. Next, the tissue sections were washed three times with ddH2O for 1 minute each, fixed in 250 μl of 4% PFA in the dark for 10 minutes, and washed twice with DPBS for 5 minutes each. Finally, the tissue sections were mounted with Vectashield mounting medium containing DAPI (Vector Laboratories, Newark, AR, USA) to stain the cell nuclei.Images were acquired with an Axiovert135 fluorescence microscope (Carl Zeiss, Oberkochen, Germany) and analyzed with AxioVision Rel 4.8 program software.

[0097] 1.8 Human TIMP-1 ELISA After transfection with TIMP-1 mRNA and microinjection of mRNA into aortic tissue, TIMP-1 levels were detected in cell lysates and cell culture supernatants. TIMP-1 levels were detected using the Human TIMP-1 DuoSet ELISA (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Cell supernatants and cell lysates were diluted with DPBS containing 1% BSA (cell supernatant: 1:150–1:250, cell lysate: 1:50) before ELISA. Aortic tissue culture supernatants were not diluted. The absorbance of the samples was measured at 450 nm using a microplate reader (Eon Synergy 2, BioTek Instruments) with a correction wavelength of 540 nm.

[0098] 1.9 Statistical analysis Data are presented as mean + standard deviation (SD) or standard error of the mean (SEM). One-way or two-way analysis of variance (ANOVA) was performed followed by Tukey's multiple comparison test or Bonferroni's multiple comparison test. All statistical analyses were performed using GraphPad Prism version 9.3.1. Differences of p<0.05 were considered statistically significant.

[0099] 2. Results 2.1 Transfection of synthetic TIMP-1 mRNA into cells To analyze TIMP-1 protein production, 3 × 10 5EA.hy926 cells, NUFF cells, or HUVEC cells were transfected with 0.5 μg, 1.0 μg, or 1.5 μg of synthetic TIMP-1 mRNA. ELISA analysis revealed a significant increase in TIMP-1 levels in the supernatants of all cell types 24 h after TIMP-1 mRNA transfection (Figure 1A). Increasing the concentration of TIMP-1 mRNA to 1.5 μg significantly increased TIMP-1 protein expression in all cells except EA.hy926 cells. The amount of TIMP-1 detected was higher in the cell supernatant than in the cell lysate, indicating that TIMP-1 protein was secreted into the extracellular space after translation. Only EA.hy926 cells and NUFF cells transfected with 1.5 μg of TIMP-1 mRNA induced significantly higher intracellular TIMP-1 levels than cells treated with transfection reagent (L2000) alone (Figure 1B). In HUVEC, the amount of intracellular TIMP-1 was already significantly increased when 0.5 μg of TIMP-1 mRNA was transfected.

[0100] 2.2 Delivery of synthetic RNA into the aortic wall by microinjection To investigate the delivery of synthetic mRNA to the vascular wall, 1 μg of Cy3-labeled hGLuc mRNA was injected ex vivo into porcine and human aortas via the intraluminal site using a hollow microneedle. The localization of Cy3-labeled hGLuc mRNA was analyzed by fluorescence microscopy in cross-sections of multiple paraffin sections. The vascular, intimal, medial, and adventitia compartments were readily identified in these tissue sections. Cell nuclei were stained with DAPI (blue). Cy3-labeled hGLuc mRNA (red) was detected primarily in the adventitia of the porcine aortic wall (Figure 2A) and the human aortic wall (Figure 2B). To examine whether the administered hGLuc mRNA could be translated into protein, supernatants from ex vivo cultured aortas were collected 24 and 48 h after injection. Microinjection of hGLuc mRNA into human and porcine aortas significantly increased luciferase activity after 24 hours of incubation compared with control groups injected with Opti-MEM alone or Opti-MEM and L2000 (Figure 2C). Luciferase activity in the supernatant of porcine vessels measured after 48 hours of incubation was comparable to that measured after 24 hours, indicating that the majority of the injected mRNA was translated during the first 24 hours of incubation (Figure 2C, left panel). In human aortas, slightly higher luciferase activity was detected after 48 hours than after 24 hours. While human diseased vessels derived from aortic aneurysms may have reduced cellular activity compared with healthy vessels, significant amounts of expressed luciferase protein were detected after 24 or 48 hours of incubation following mRNA injection (Figure 2C, right panel).

[0101] Furthermore, we tested whether synthetic mRNA could be microinjected from the adventitial side in porcine aorta samples (data not shown). In this experiment, we obtained results similar to those obtained by injecting mRNA from the intimal side, and detected increased luciferase activity in aorta samples microinjected with hGLuc mRNA after 24 and 48 hours of incubation.

[0102] 2.3 TIMP-1 production in the porcine aortic wall after delivery of synthetic TIMP-1 mRNA To examine whether TIMP-1 mRNA administration increased TIMP-1 protein expression, supernatants from ex vivo cultured porcine aortic vessels were collected 24 and 48 hours after injection of 3 μg of TIMP-1 mRNA. After 48 hours of incubation, a significant increase in TIMP-1 levels was detected by ELISA analysis (Fig. 3).

[0103] 2.4 Detection of MMP-9 activity in porcine and human aortic walls after application of synthetic TIMP-1 mRNA Since we demonstrated that the desired protein could be expressed for up to 48 hours after synthetic mRNA injection into ex vivo aortic tissue using mRNA encoding hGLuc, we used the same ex vivo model to evaluate the functionality of the expressed TIMP-1 protein. In situ zymography using fluorescently labeled MMP substrate DQ gelatin can visualize and evaluate the proteolytic activity of MMP-9 in tissue sections. Enzymatic cleavage of DQ gelatin by active MMPs produces a green fluorescent signal (Hadler-Olsen, E., et al., Gelatin in situ zymography on fixed, paraffin-embedded tissue: zinc and ethanol fixation preserve enzyme activity. J Histochem Cytochem, 2010. 58(1): pp. 29-39). MMP-9 activity is enhanced by the addition of zinc, which is required for catalytic activity. 2+Because EDTA binding to ions inhibits MMP-9, tissue sections incubated with EDTA were used as a positive control for in situ zymography. TIMP-1 covalently binds to MMP-9 and inhibits the cleavage of DQ gelatin. Based on this, the inhibitory activity of TIMP-1 protein produced after microinjection of TIMP-1 mRNA into the vessel wall was investigated in porcine and human blood vessels. To conduct this study, 5 μg of human synthetic TIMP-1 mRNA was injected ex vivo into the intimal side of porcine and human aortic vessels and incubated for 24 or 48 hours. After fixation, paraffin sections were used to assess MMP-9 activity by in situ zymography. The cleavage efficiency of DQ gelatin by MMP-9 was measured and compared between TIMP-1 mRNA-treated and untreated groups. At 24 and 48 hours after injection of TIMP-1 mRNA from the intimal (Fig. 4) or adventitial (Fig. 7) side, the DQ gelatin signal detected in the porcine vessel wall was significantly lower than that in vessels treated with L2000 alone. As expected, the lowest MMP-9 activity was detected in vessel sections treated with EDTA.

[0104] Similar results were obtained in human aortic tissue after TIMP-1 mRNA injection and incubation for 24 or 48 hours (Figure 5). In this experiment, we observed a significant decrease in the DQ gelatin signal 24 and 48 hours after injection of synthetic TIMP-1 mRNA compared to the L2000 control. This result confirmed the inhibitory function of the produced TIMP-1 protein. It was able to inhibit and reduce proteolytic MMP-9 activity in the mRNA-treated tissue.

[0105] 2.5 Analysis of translation efficiency of TIMP-1 mRNA with various nucleotide modifications To improve the protein expression efficiency of mRNA, various nucleotide modifications were incorporated into mRNA and tested. In the previous analysis, Ψ / 5mC was used as the nucleotide modification. Ψ is defined as me 1 By substituting Ψ, TIMP-1 mRNA is1 Ψ / 5mC or me 1 1.5 μg of each mRNA variant was complexed with L2000 and 3 × 10 5 After 24 hours, the supernatant was analyzed for protein translation efficiency by TIMP-1-specific ELISA (Fig. 6). 1 Incorporation of Ψ alone significantly improved protein expression efficiency, and further modification with 5mC did not improve translation.

[0106] 3. Conclusion The present inventors have provided oligonucleotides that can treat cardiovascular diseases such as aneurysms and arteriosclerosis with low incidence of complications and high efficiency.

Claims

1. An oligonucleotide comprising a nucleotide sequence encoding a tissue inhibitor of metalloproteinase (TIMP) protein for use in the treatment of a disease or condition in a mammal.

2. The oligonucleotide of claim 1, wherein the disease or pathological condition is a disease or pathological condition associated with a deficiency of at least one extracellular matrix protein, preferably the disease or pathological condition is selected from the group consisting of aneurysm, fibrosis, arteriosclerosis, aortic stenosis, emphysema, Williams-Beuren syndrome, congenital subaortic stenosis, myocardial infarction, stroke, rheumatoid arthritis, osteoarthritis and tumor metastasis, more preferably the disease or pathological condition is selected from the group consisting of aortic aneurysm, cerebral aneurysm and peripheral aneurysm.

3. 10. The oligonucleotide of any one of the preceding claims, wherein the disease or condition is a disease or condition associated with an unphysiological MMP / TIMP activity ratio, preferably wherein the unphysiological MMP / TIMP activity ratio is associated with increased MMP activity.

4. 10. The oligonucleotide of any one of the preceding claims, wherein the treatment is a prophylactic or therapeutic treatment, preferably the treatment is selected from the group consisting of preventing the formation or rupture of an aneurysm and healing an aneurysm, more preferably the aneurysm is selected from the group consisting of an aortic aneurysm, a cerebral aneurysm and a peripheral aneurysm.

5. the oligonucleotide is an oligoribonucleotide, preferably an mRNA; Preferably, the oligoribonucleotide is a 5' cap structure, preferably a synthetic anti-reverse cap analog, more preferably 3'-O-Me-m7G(5')ppp(5')G, and / or a polyA tail, preferably a polyA tail of at least about 70 adenine nucleotides, more preferably a polyA tail of about 120 adenine nucleotides; 10. The oligonucleotide of any one of the preceding claims, comprising:

6. 10. The oligonucleotide of claim 1, wherein at least one nucleotide is an analog of a natural nucleotide, and the substitution rate of one natural nucleotide by the analog is at least about 5%, preferably at least about 25%, and more preferably about 100%.

7. The natural nucleotide uridine and / or uridine is substituted with a nucleotide analog, and preferably, the natural nucleotide uridine and / or uridine is substituted with a combination of pseudouridine substituting uridine and 5-methylcytidine substituting cytidine, N-methylcytidine substituting uridine, or N-methylcytidine substituting uridine. 1 -methylpseudouridine and 5-methylcytidine replacing cytidine, and N replacing uridine 1 -methylpseudouridine, most preferably N-methyl-uridine, 1 The oligonucleotide of claim 6, wherein the substituted uridine is -methylpseudouridine.

8. 10. The oligonucleotide according to any one of the preceding claims, wherein the TIMP protein is a TIMP-1 protein, preferably wherein said TIMP-1 protein comprises the amino acid sequence of SEQ ID NO:

1.

9. 10. The oligonucleotide of any one of the preceding claims, comprising the nucleotide sequence of SEQ ID NO:

2.

10. 10. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide is complexed with a polymer or a lipid, preferably the oligonucleotide is contained within a lipid nanoparticle or a liposome.

11. 10. A pharmaceutical composition comprising an oligonucleotide according to any one of the preceding claims and a pharmaceutically acceptable carrier.

12. 12. The pharmaceutical composition of claim 11, adapted for local administration by injection into mammalian tissue or topical application onto mammalian tissue, preferably contained in a formulation or delivery form selected from the group consisting of a fluid, an implant, a balloon catheter, a vascular graft, a microneedle, a patch, and a stent.

13. 12. The pharmaceutical composition of claim 11, adapted for systemic administration, preferably contained in a formulation or delivery form selected from the group consisting of fluids and implants.

14. The pharmaceutical composition according to any one of claims 11 to 13, which comprises at least one antihypertensive drug or is administered in combination with at least one antihypertensive drug.

15. A medical device comprising or coated with the oligonucleotide of any one of claims 1 to 10, preferably configured for permanent or temporary placement in the body of a mammal, more preferably selected from the group consisting of a graft, a balloon catheter, an artificial blood vessel, a microneedle, a patch and a stent.

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