Codon-optimized oligonucleotides for directing de novo synthesis of elastin in mammals

Abstract

The present invention relates to oligonucleotides for inducing the de novo synthesis of elastin in mammals, methods for treating diseases and conditions associated with a lack of tissue elasticity, the use of said oligonucleotides in such treatments, and pharmaceutical and cosmetic compositions containing such oligonucleotides.

Description

[Technical Field]

[0001] The present invention relates to oligonucleotides for inducing the de novo synthesis of elastin in mammals, methods for treating diseases and conditions associated with a lack of tissue elasticity, the use of said oligonucleotides in such treatments, and pharmaceutical and cosmetic compositions containing such oligonucleotides.

[0002] The present invention relates to the field of molecular medicine, in particular to the expression of proteins for therapeutic or cosmetic purposes, and more particularly to the induction of de novo synthesis of desired extracellular matrix proteins in mammals. [Background technology]

[0003] Elastin is a component of the extracellular matrix (ECM) of vertebrates and provides tissues with elasticity and flexibility. Elastin fibers are formed by covalent cross-linking of lysine residues in tropoelastin (TE), the precursor of elastin. During development, cells such as smooth muscle cells, fibroblasts, and endothelial cells secrete soluble monomers of TE, which are then assembled by the enzyme lysyl oxidase within the ECM to form elastin fibers, which are insoluble polymers with high stability. This process of elastin fiber formation is referred to herein as "elastic fiber formation."

[0004] The majority of TE expression is seen during fetal life and the early postnatal years. Elastin synthesis declines after adolescence and ceases in adulthood. Elastin has a half-life of approximately 74 years, but age-related degradation, disease, or injury can result in a loss of elastin fibers, resulting in a loss of tissue elasticity and flexibility, and a loss of integrity and functionality.

[0005] Elasticity, or elastin, is crucial for various organs, including the lungs, heart, skin, and blood vessels, especially the aorta. For example, in various genetic disorders, such as Williams-Beuren syndrome (WBS) and skin laxity, elastic fiber formation is impaired, resulting in skin laxity and vascular disorders such as supravalvular aortic stenosis. Damage to dermal elastin fibers due to injury, disease, sunburn, and age-related degradation leads to irreversible loss of skin elasticity. Furthermore, loss of elastin in the dermis after severe burns leads to serious physical damage, including scarring, wound contraction, and loss of skin extensibility. Therefore, regeneration of elastin fibers plays a crucial role in wound healing, scar formation, and the restoration of skin function and elasticity.

[0006] Given this situation, there has been growing interest in inducing de novo elastin synthesis. Various strategies have been used to restore skin elasticity by inducing elastic fiber formation. The most well-known are, for example, the use of viral vectors (Xiong, J., et al., Elastic fibers reconstructed using adenovirus-mediated expression of tropoelastin and tested in the elastase model of abdominal aortic aneurysm in rats. J Vasc Surg, 2008. 48(4): pp. 965-73) and synthetic mRNA (German Patent Publication No. 102013005361(A1); Lescan, M., et al., De Novo Synthesis of Elastin by Exogenous Delivery of Synthetic Modified mRNA into Skin and Elastin-Deficient Cells. Mol Ther Nucleic Acids, 2018. 11: pp. 475-484).

[0007] Therapeutic applications of synthetic mRNA have demonstrated great promise and have attracted considerable interest due to several advantages. 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 with 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.

[0008] German Patent Publication No. 102013005361 (A1) discloses synthetic mRNAs containing nucleotide analogs encoding elastic fiber proteins, including elastin, but the enhancement of elastin expression is limited, requiring the application of large amounts of mRNA, at least 5 μg.

[0009] Lescan et al. (2018) reported that synthetic TE mRNA transfected various types of human cells and ex vivo porcine skin intradermally injected with synthetic TE mRNA via microinjection increased elastin synthesis.

[0010] The challenges of using RNA for therapeutic applications include its biological instability and immunogenicity, which limit its bioavailability and potential applications. Furthermore, enzymes that degrade RNA are nearly ubiquitous, particularly in the extracellular space. Furthermore, the innate immune system, mediated by Toll-like receptors (TLRs), recognizes single-stranded RNA (TLR-7, TLR-8) and double-stranded RNA (TLR-3) to induce inflammatory immune responses.

[0011] Therefore, an object of the present invention is to provide oligonucleotides that overcome or at least mitigate the drawbacks of the prior art. More specifically, the present invention provides oligonucleotides that can induce de novo synthesis of elastin in mammalian cells and tissues by improving the expression efficiency of synthetic mRNA encoding tropoelastin (TE). The present invention completely solves these problems. Summary of the Invention [Means for solving the problem]

[0012] In one aspect of the present invention, the above disadvantages are overcome by providing an oligonucleotide comprising a nucleotide sequence encoding a tropoelastin (TE) protein, wherein the nucleotide sequence is codon-optimized for expression in mammalian cells.

[0013] As commonly practiced in the art, "codon optimization" herein refers to replacing a codon with at least one synonymous codon with a codon predicted to achieve higher expression efficiency. The prediction of which codon will result in higher expression efficiency depends on various factors, the most common of which are the GC content of the codon and the overall sequence, and the species of organism. Codon optimization can be parameterized as the codon adaptation index (CAI) (Sharp, PM and WH Li, "The codon adaptation index—a measure of directional synonymous codon usage bias, and its potential applications." Nucleic Acids Res, 1987. 15(3): pp. 1281-95). The CAI is the geometric mean value of the proportion of all synonymous codons that encode a particular amino acid that are identical to one of the synonymous codons and that are most frequently used to encode this particular amino acid in a reference gene set.

[0014] As demonstrated by the present inventors, optimizing the codons of synthetic mRNA encoding a TE can favorably affect its expression efficiency and stability, thereby increasing protein expression levels (Presnyak, V., et al., Codon optimality is a major determinant of mRNA stability. Cell, 2015. 160(6): pp. 1111-24). This finding was particularly surprising and unexpected with respect to TEs for the reasons described below.

[0015] One of the most important parameters in the design of synthetic mRNA is typically the GC content, which should be designed to be neither too low to prevent degradation and a decrease in expression efficiency, nor too high to prevent the formation of secondary structures within the RNA molecule that limit access to the translational machinery.

[0016] Codon-optimized synthetic mRNAs have been disclosed in studies on the expression of proteins encoded by wild-type nucleotide sequences that do not have a high GC content. U.S. Patent Publication No. 10,898,584 (B2) discloses synthetic mRNAs with significantly increased GC content. Codon-optimized synthetic mRNAs have also been disclosed in association with non-fibrous, non-structural, and non-ECM proteins, such as interferon (IFN)-α and erythropoietin (EPO) (Kariko, K., et al., Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin. Mol Ther, 2012. 20(5): pp. 948-53; and Hochmann, S., et al., Evaluation of modified Interferon alpha mRNA constructs for the treatment of non-melanoma skin cancer. Sci Rep, 2018. 8(1): pp. 12954).

[0017] According to the prior art, increasing the GC content is necessary to improve the expression efficiency of synthetic mRNA. Because the wild-type sequence of human TE already has a high GC content of 64.3%, it has been thought that improving the expression efficiency of TE proteins is impossible. In other words, 76% of the total amino acids in human TE proteins are composed of glycine (29%), alanine (22%), valine (13%), and proline (12%). Since these four amino acids all have GC-rich codons, optimizing the GC content of codons corresponding to at least 76% of the total amino acids is impossible. For the remaining 24%, GC-rich synonymous codons do not exist, or even if such synonymous codons are available, they may be difficult to use for expression in humans or other mammals.

[0018] Surprisingly, the present inventors have succeeded in optimizing the codons of the human wild-type nucleotide coding sequences of naturally GC-rich TEs, rather than being bound by the conventional wisdom of increasing the GC content, thereby improving expression efficiency, which also has the advantage of providing more flexibility in creating such optimized sequences.

[0019] In one embodiment of the present invention, the oligonucleotide of the present invention is an oligoribonucleotide, preferably an mRNA.

[0020] By selecting ribonucleic acid as the oligonucleotide of the present invention, it is possible to avoid the permanent introduction of the oligonucleotide of the present invention into the cell genome, thereby greatly reducing the risk of mutagenicity.In addition, since the half-life of elastin is very long, the transient existence of mRNA in cells is advantageous in that it allows these cells to return to the physiological state of not producing elastin.Therefore, it is possible to achieve the desired result of depositing additional elastin fibers in ECM with high specificity, without permanently changing the behavior of the cell into which the oligonucleotide of the present invention is introduced.

[0021] 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.

[0022] In another aspect of this embodiment, the oligonucleotide of the invention comprises a 5' cap structure and / or a poly-A tail, preferably selected from the group consisting of 3'-O-Me-m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, and m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, and the poly-A tail preferably consists of at least about 70 adenine nucleotides, more preferably about 120 adenine nucleotides. The 5' cap structure may be natural, synthetic, or modified. Thus, the term "5' cap structure" refers to any natural 5' cap structure naturally used by eukaryotic cells, as well as any non-natural, synthetic, or modified 5' cap structure that is suitable as a replacement for the natural 5' cap structure in terms of functionality and cytotoxicity.

[0023] Such modifications mimic the natural structure of mammalian mRNA and therefore have the advantage of conferring a degree of stability to the oligonucleotides of the present invention, thereby reducing the rate of degradation of the oligonucleotides of the present invention in cells to which they are delivered, and enabling induction of TE synthesis by the oligonucleotides of the present invention.

[0024] The nucleotides contained in the oligonucleotides of the present invention may be natural nucleotides, i.e., unmodified nucleotides. However, in another embodiment of the present invention, at least one of the nucleotides contained in the oligonucleotides of the present invention is an analog of a natural nucleotide. This has the advantage of further reducing the degradation rate of the synthetic mRNA and preventing recognition of the synthetic mRNA by the immune system, thereby avoiding inflammatory reactions. In this embodiment, the analogs of natural nucleotides account for a large proportion of the total nucleotides. "Natural nucleotide" refers to a nucleotide containing a nucleobase that is naturally found in mammalian DNA or RNA. Specifically, naturally occurring nucleobases include adenine, guanine, cytosine, thymidine, and uracil. "Analog" is understood to mean a nucleotide that reduces the degradation rate of the oligonucleotides of the present invention and / or reduces their immunogenicity, and that contains a chemical structure similar to that of natural nucleotides, thereby allowing the nucleotide sequence encoded by the oligonucleotides of the present invention to be translated into the encoded protein by the cell's translational machinery.

[0025] The term "majority" means that not all naturally occurring nucleotides of a given type are necessarily substituted with analogs, but rather that at least 5% of naturally occurring nucleotides of a given type are substituted with analogs, preferably 25% or more of naturally occurring nucleotides of a given type are substituted with analogs, and more preferably 100% of naturally occurring nucleotides of a given type are substituted with analogs.

[0026] In this embodiment, the analogue may be, for example, pseudouridine, N 1-methylpseudouridine, 5-methylcytidine, phosphorothioate, phosphoramidate, peptide nucleotide, methylphosphonate, 7-deazaguanosine, 2-thiouridine, 5-methyluridine, 5-methyluridine-5'-triphosphate (m5U), 5-iodouridine-5'-triphosphate (15U), 4-thiouridine-5'-triphosphate (S4U), 5-bromouridine-5'-triphosphate (Br5U), 2'-methyl-2'-deoxyuridine-5'-triphosphate (U2'm), 2'-amino-2'-deoxyuridine-5'-triphosphate (U2'NH2), 2'-azido-2'-deoxyuridine-5' 2'-amino-2'-deoxycytidine-5'-triphosphate (C2'NH2), 2'-fluoro-2'-deoxycytidine-5'-triphosphate (C2'F), 5-iodocytidine-5'-triphosphate (U), 5-bromocytidine-5'-triphosphate (Br), or 2'-azido-2'-deoxycytidine-5'-triphosphate (C2'N), and may be pseudouridine, N 1 Preferably, the analogue is selected from the group consisting of 5-methylpseudouridine and 5-methylcytidine, which have been found to be highly suitable for reducing the immunogenicity of the oligonucleotides of the invention while increasing the expression efficiency of the oligonucleotides of the invention.

[0027] In another embodiment of the invention, the TE protein comprises the amino acid sequence of SEQ ID NO:1.

[0028] The use of this human TE sequence allows the TE to be expressed in human cells, which has the advantage that the oligonucleotides of the present invention can be used in medical or cosmetic applications targeted at humans.

[0029] In another embodiment of the invention, the oligonucleotide of the invention comprises the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0030] These sequences are codon-optimized variants of the wild-type sequence and offer the advantages of low immunogenicity and high expression efficiency.

[0031] In the following description, the term "expression efficiency" is interpreted as the amount of elastin protein synthesized by a mammalian cell or mammalian tissue in a certain period of time, expressed as a ratio to the amount of applied oligonucleotide. In this specification, the term "applied" is interpreted as any method that can be used to introduce DNA or RNA into cells or that includes introducing DNA or RNA into cells.

[0032] In another embodiment of the present invention, the oligonucleotide of the present invention can be used in the treatment of diseases and pathologies related to tissue inelasticity deficiency.This use has the advantage that it can transiently induce the de novo synthesis of elastin fiber in the ECM of mammalian tissue, thereby improving tissue elasticity.Preferably, this mammal is human, preferably, mammal is human or domestic mammal, more preferably, mammal is human.

[0033] 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, which 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.

[0034] A disease or condition associated with "deficient elasticity" is understood to mean any disease or condition that a mammal suffers from that would benefit from improved tissue elasticity. In this context, "elasticity" refers to the ability of tissue to resist strain and to return to its original size and shape when the strain is removed.

[0035] In this embodiment, the treatment is preferably selected from the group consisting of treatment for genetic defects in elastin synthesis, treatment for arteriosclerosis, treatment for aortic stenosis, treatment for aortic aneurysms and / or cerebral aneurysms, treatment for chronic obstructive pulmonary disease (COPD), treatment for eye diseases (e.g., AMD), treatment for aortic regurgitation, treatment for cutis laxa, treatment for Williams-Beuren syndrome, treatment for skin laxity, treatment for ligament disorders, treatment for congenital subaortic stenosis (SVAS), treatment for scar tissue, and treatment for scarless wound healing. Mammals suffering from such diseases or conditions can benefit from improved tissue elasticity.

[0036] In another aspect, the present invention relates to a pharmaceutical composition comprising an oligonucleotide of the present invention and a pharmaceutically acceptable carrier.

[0037] Those skilled in the art are familiar with the carrier suitable for 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.

[0038] In one embodiment, the pharmaceutical composition of the present invention is configured for systemic administration to a mammal, preferably via a parenteral route, more preferably via intravenous injection. Systemic administration may be via any oral or parenteral route, preferably via a parenteral route, more preferably via intravenous injection. Systemic administration has the advantage of enabling treatment of the entire body, and may be particularly advantageous in treating diseases or conditions associated with insufficient elasticity of vascular tissue.

[0039] 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 cream, a gel, a solution, a paste, a spray, a plaster, a microneedle, a medical bandage, a face mask, an implant, and a stent.

[0040] Unwanted deposition of elastin fibers in areas of the body where they are not required can be harmful to the mammal receiving the treatment, but local administration is advantageous because it can specifically induce de novo synthesis of elastin at the site of disease or pathology without unnecessarily affecting areas of the body where they are not required.

[0041] Topical application of any kind is an added advantage because it is less invasive and therefore less stressful than other methods of administration.

[0042] The above formulations or delivery forms in the form of creams, gels, solutions, pastes, sprays or plasters are advantageous as they allow the application of the pharmaceutical compositions of the present invention to any area of ​​the body, regardless of the size of that area.

[0043] Formulations or delivery forms in the form of plasters or microneedles have the advantage that the pharmaceutical composition of the present invention can be applied to a very small, predetermined area of ​​the body where application of the pharmaceutical composition of the present invention is required, and the pharmaceutical composition can be exposed to such an area of ​​the body for a long period of time, and this area of ​​the body can usually be protected by covering it with a fabric so as not to interfere with the application of the pharmaceutical composition in daily life.

[0044] Formulations or delivery forms in the form of bandages or face masks are advantageous in that they allow for uniform and prolonged distribution of the pharmaceutical compositions of the present invention over the face or other large body areas, for example, areas of the body that have been burned.

[0045] Advantageously, the pharmaceutical compositions of the present invention can be applied to blood vessels and / or local tissue areas by utilizing a formulation or delivery form in the form of an implant or stent.

[0046] In another aspect, the present invention relates to a cosmetic composition comprising the oligonucleotide of the present invention for use in enhancing the elasticity of human tissue, preferably for use in treating or preventing wrinkles formed in human skin, which can induce de novo synthesis of elastin fibers in human skin, thereby restoring or maintaining sufficient elasticity of the skin and preventing wrinkles, etc.

[0047] In another aspect, the present invention relates to a cosmetic method for inducing de novo synthesis of elastin in mammalian tissue, the method comprising the step of topically applying a cosmetic composition of the present invention by injection into mammalian tissue or by topical application onto mammalian tissue.

[0048] In one embodiment of the present invention, when carrying out the cosmetic method of the present invention, topical application is repeated at least once. Repeated topical application of a cosmetic composition containing the oligonucleotide of the present invention can cause elastin fibers to accumulate in tissues, and such repeated administration may be necessary or desirable because the induction of de novo synthesis of elastin fibers by the oligonucleotide is transient and non-permanent.

[0049] In one embodiment of the present invention, the cosmetic composition of the present invention is comprised in a formulation or delivery form selected from the group consisting of a cream, a gel, a liquid, a paste, a spray, a plaster, a microneedle, a medical bandage, and a face mask.

[0050] Formulations or delivery forms in the form of creams, gels, liquids, pastes, sprays, microneedles or plasters are advantageous because they allow the cosmetic composition of the present invention to be applied to any area of ​​the body, regardless of the size of that area.

[0051] A formulation or delivery form in the form of a plaster has the advantage that the cosmetic composition of the present invention can be applied to a very small, specific area of ​​the body where application of the cosmetic composition of the present invention is required, the cosmetic composition can be exposed to this area of ​​the body for a long period of time, and this area of ​​the body can usually be protected by covering it with a fabric so as not to interfere with application of the cosmetic composition in daily life.

[0052] Formulations or delivery forms in the form of bandages or face masks are advantageous in that they allow for uniform and prolonged distribution of the cosmetic compositions of the present invention over the face or other large body areas, for example, areas of the body that are scarred.

[0053] The features, properties and advantages described with respect to the oligonucleotides of the invention apply equally to the pharmaceutical and cosmetic compositions of the invention.

[0054] Another aspect of the present invention is a method for treating diseases or conditions associated with insufficient tissue elasticity.The method comprises administering the oligonucleotide and / or pharmaceutical composition of the present invention to an organism suspected of suffering from the above-mentioned condition.This administration can be carried out by local administration, such as injection into the desired tissue of a mammal or by external application on the desired tissue of a mammal, or by systemic administration.

[0055] In one embodiment, the method of the present invention may comprise at least one repeated administration of the oligonucleotide and / or pharmaceutical composition of the present invention. Repeated administration of a pharmaceutical composition containing the oligonucleotide of the present invention can result in the accumulation of elastin fibers in tissues, and such repeated administration may be necessary or desirable because the induction of de novo elastin fiber synthesis by the oligonucleotide is transient and non-permanent.

[0056] In another embodiment, the treatment methods of the present invention include, prior to administering the oligonucleotides and / or pharmaceutical compositions of the present invention, identifying one or more body areas suitable for administration of the oligonucleotides and / or pharmaceutical compositions of the present invention and in which the patient desires a change in appearance, and optionally obtaining approval of whether the patient desires a change in medical necessity.

[0057] 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.

[0058] 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]

[0059] [Figure 1] The time course of elastin production is shown. The effect of various TE mRNA variants on elastin synthesis was tested by elastin ELISA. 3 x 105 EA.hy926 cells were transfected with 2.5 μg of each TE mRNA complexed with 4 μl of Lipofectamine 2000 in OptiMEM at 37°C and 5% CO2 for 4 hours. The transfection complex was then replaced with cell culture medium, and the cells were incubated at 37°C and 5% CO2 without further medium changes. Elastin concentrations were measured in the cell supernatants after 24, 48, and 72 hours. As controls, cells were treated with Lipofectamine 2000 ("L2000") or OptiMEM alone ("medium"). Elastin concentrations were measured in the cell supernatants after 24, 48, and 72 hours. Results are shown as mean + SEM (n = 3).

[0060] [Figure 2]Analysis of elastin synthesis after delivery of each TE mRNA variant into cells is shown. The effect of various TE mRNA variants on elastin synthesis was tested by elastin ELISA. 3 x 105 EA.hy926 cells were transfected with 2.5 μg of each TE mRNA complexed with 4 μl of Lipofectamine 2000 in OptiMEM at 37°C and 5% CO2 for 4 hours. The transfection complex was then replaced with cell culture medium, and the cells were incubated at 37°C and 5% CO2 for 48 hours. As a control, cells were treated with Lipofectamine 2000 ("L2000") alone. Elastin concentrations were measured in the supernatants of cells transfected with (A) unmodified TE mRNA variants, (B) Ψ / m5C-modified TE mRNA variants, (C) me1Ψ / m5C-modified TE mRNA variants, or (D) me1Ψ / C-modified TE mRNA variants. Results are shown as mean + SEM (n = 3). Statistical differences were determined by one-way ANOVA followed by Bonferroni's multiple comparison test (*p<0.05, **p<0.01, ****p<0.0001); += statistically different from L2000 control (+p<0.05, ++p<0.01, +++p<0.001, ++++p<0.0001).

[0061] [Figure 3]The effect of each TE sequence variant on cell viability is shown. 3 x 105 EA.hy926 cells were transfected in OptiMEM with 2.5 µg of each TE mRNA variant complexed with 4 µl of Lipofectamine 2000 at 37°C and 5% CO2 for 4 hours. The transfection complex was then replaced with cell culture medium, and the cells were incubated at 37°C and 5% CO2. Cell viability was analyzed using the Presto Blue assay 24 hours after transfection with (A) unmodified TE mRNA variant, (B) Ψ / m5C TE mRNA variant, (C) me1Ψ / m5C TE mRNA variant, or (D) me1Ψ / C TE mRNA variant. The viability of cells treated with OptiMEM ("medium") was considered 100%. Results are shown as mean + SEM (n = 3). Statistical differences were determined by one-way analysis of variance followed by Bonferroni's multiple comparison test. (ns = not significant; # = statistically different from medium control (#p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001); + = statistically different from L2000 control (+p<0.05, ++p<0.01, +++p<0.001).

[0062] [Figure 4]The effect of various nucleotide modifications in each TE mRNA variant on elastin synthesis is shown. 3 x 105 EA.hy926 cells were transfected in OptiMEM with 2.5 µg of each TE mRNA complexed with 4 µl of Lipofectamine 2000 for 4 hours at 37°C and 5% CO2. The transfection complex was then replaced with cell culture medium, and the cells were incubated for 48 hours at 37°C and 5% CO2. As a control, cells were treated with Lipofectamine 2000 ("L2000") alone. Elastin concentrations were measured in the supernatants of cells transfected with (A) TE mRNA variant 1, (B) TE mRNA variant 3, (C) TE mRNA variant 4, (D) TE mRNA variant 14, or (E) native TE mRNA variant. Results are shown as mean + SEM (n = 3). Statistical differences were determined by one-way analysis of variance followed by Bonferroni's multiple comparison test (*p<0.05, **p<0.01, ****p<0.0001). +=Statistical differences compared to L2000 control (+p<0.05, ++p<0.01, +++p<0.001, ++++p<0.0001).

[0063] [Figure 5]The effect of various nucleotide modifications in each TE mRNA variant on cell viability is shown. Cells were transfected in OptiMEM with 2.5 μg of each TE mRNA complexed with 4 μl of Lipofectamine 2000. Cell viability was analyzed using the Presto Blue assay 24 hours after transfection with (A) TE mRNA variant 1, (B) TE mRNA variant 3, (C) TE mRNA variant 4, (D) TE mRNA variant 14, or (E) native TE mRNA variant. The viability of cells treated with OptiMEM ("medium") was considered 100%. Results are shown as +SEM (n=3). Statistical differences were determined by one-way ANOVA followed by Bonferroni's comparison test. (*p<0.05, **p<0.01, ***p<0.001, ns=not significant; #=statistically different from medium control; +=statistically different from L2000 control (+p<0.05, ++p<0.01, +++p<0.001, ++++p<0.0001).

[0064] [Figure 6] Analysis of TE mRNA presence in EA.hy926 cells after transfection with each TE mRNA variant is shown. The effect of various TE mRNA sequence variants and nucleotide modifications on mRNA degradation was examined by qRT-PCR. To perform this study, 3 x 10 cells were transfected with 2.5 μg of each TE mRNA complexed with 4 μl of Lipofectamine 2000 in OptiMEM at 37°C and 5% CO2 for 4 hours. The transfection complex was then replaced with cell culture medium, and the cells were incubated at 37°C and 5% CO2 for 2 hours, after which RNA was isolated. The amount of total TE mRNA was measured by qPCR. Results are shown as mean ± SEM (n = 3).

[0065] [Figure 7]Representative photographs of pig skin after intradermal injection of each TE mRNA variant are shown. 9 x 10 μl of lactated Ringer's buffer containing 3 μg, 10 μg, or 30 μg of each TE mRNA variant, or 9 x 10 μl of lactated Ringer's buffer without TE mRNA variant, was injected into a designated skin area of ​​1 x 1 cm, and each injection site was marked with a tattoo pen. The injection sites for (A) the unmodified TE mRNA variant and (B) the me1Ψ / C-modified TE mRNA variant are shown 48 hours later. No visible skin inflammation or redness was observed.

[0066] [Figure 8]Analysis of elastin expression by ElaNIR staining after intradermal delivery of each TE mRNA variant into pig skin in vivo is shown. Each selected TE mRNA variant was prepared in lactated Ringer's (RL) buffer alone. Using a BD Micro-Fine™ insulin syringe, 9 x 10 μl of lactated Ringer's buffer containing each TE mRNA variant was injected into a designated skin area, and each injection site was marked with a tattoo pen. As a control, buffer alone without added mRNA was injected. In addition, biopsies of untreated skin were taken at the end of the experiment. (A-C): TE mRNA variant 1, TE mRNA variant 3, TE mRNA variant 4, TE mRNA variant 14, or native TE mRNA, either unmodified or modified with me1Ψ / C, was injected intradermally into pig skin in 90 μl of lactated Ringer's buffer containing 3 μg, 10 μg, or 30 μg of each TE mRNA variant. Each mRNA was administered intradermally in five replicates in two pigs. Forty-eight hours after intradermal administration, the pigs were euthanized, and skin biopsies were stained with ElaNIR to detect elastin content in the skin using IVIS. D) In ​​four or five replicates, 90 μl of lactated Ringer's buffer containing 30 μg of TE_mCherry mRNA was injected intradermally into the pig's skin. Forty-eight hours after intradermal administration, the pigs were euthanized, and the skin biopsies were fixed with 4% PFA. The mCherry fluorescent signal was measured using IVIS. Fluorescence intensity was quantified as mean radiant efficiency [p / s / cm² / sr] / [μW / cm²] and normalized to the corresponding buffer-only control. Results are shown as the mean + SD. Statistical differences were determined by one-way ANOVA followed by Dunnett's multiple comparison test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). E) Fluorescence microscopy analysis of paraffin sections of pig skin biopsies 48 hours after intradermal application of 30 μg of TE_mCherry mRNA. Untreated skin biopsies served as negative controls. Arrows indicate TE-mCherry produced in the skin. BF: bright field; DAPI: blue; mCherry: red. Scale bar: 100 μm.

[0067] [Figure 9] Detection of TE production after transfection with TE_mCherry is shown. 3 x 105 EA.hy926 cells were transfected in OptiMEM with 2.5 µg of mCherry mRNA or TE_mCherry mRNA complexed with 4 µl of Lipofectamine 2000 (L2000) at 37°C and 5% CO2. After 4 hours, the transfection complex was replaced with cell culture medium, and the cells were incubated at 37°C and 5% CO2 for 24 hours. TE concentrations in the cell culture supernatant were then measured by ELISA. As controls, cells were treated with Lipofectamine 2000 ("L2000") or OptiMEM alone ("medium"). Results are shown as mean + SEM (n = 3). Statistical differences were determined by one-way analysis of variance followed by Tukey's comparison test (**p < 0.01).

[0068] [Figure 10] Representative images of ElaNIR-stained pig skin samples detected by IVIS after intradermal in vivo injection of each TE mRNA variant are shown. Skin biopsies 48 hours after injection of the TE mRNA variant 14_me1Ψ / C are shown, along with the corresponding lactate Ringer's buffer only control and untreated control groups. Biopsies were collected from the injection site using a biopsy trephine. An untreated skin area served as a control. Biopsies were stained with 20 μM ElaNIR for 30 minutes. Photographs containing fluorescence heat maps were taken to show fluorescence intensity and distribution area. Fluorescence emission in a given region of interest (ROI) was normalized to photons / s / cm² / sr and expressed as mean radiant efficiency [p / s / cm² / sr] / [μW / cm²]. w = with ElaNIR staining; wo = without ElaNIR staining.

[0069] [Figure 11]Analysis of cell viability and immune markers after injection of each TE mRNA variant into a human skin model is shown. (A) 30 μg of each of TE mRNA variant 14_me1Ψ / C, TE mRNA variant 14_unmod, TE mRNA variant native_me1Ψ / C, or TE mRNA variant native_Ψ / m5C in 90 μl (9 × 10 μl) of lactated Ringer's (RL) buffer solution was injected into a human Phenion® full-thickness (FT) skin model. Each skin sample was incubated on filter paper in growth medium at 37°C and 5% CO2 for 24 hours according to the manufacturer's instructions. Skin samples treated with lactated Ringer's buffer alone and untreated skin samples served as controls. (B) Cell viability in each skin sample was measured by MTT assay 24 hours after injection. Cell viability in untreated skin samples was considered 100%. Results are shown as + SEM (n = 3). (C) Immune activation was analyzed by qRT-PCR 24 hours after injection of each TE mRNA variant. RNA was isolated from untreated skin models or skin models injected with lactated Ringer's buffer alone as controls. Gene expression levels were normalized to the expression level of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as x-fold induction relative to untreated skin model samples. Results are shown as mean + SEM (n = 3). Statistical differences were determined using Friedman's test followed by Dunn's comparison test. [Example]

[0070] 1. Materials and Methods 1.1 In vitro synthesis of tropoelastin (TE) mRNA variants We selected tropoelastin (TE) mRNA variant coding sequences (CDS) based on GC content and human codon adaptation index (CAI). The expression efficiency of four differentially modified TE mRNA variants was compared with that of human native elastin mRNA. A total of five TE sequence candidates were selected for in vitro analysis (Table 1).

[0071] TE mRNA variants were synthesized by in vitro transcription (IVT) according to the method described by Lescan et al. (2018). DNA encoding TEs was amplified using pcDNA 3.3 or pUC57 plasmids containing various codon-optimized sequences of human TEs. Each plasmid was prepared by Aldevron (Fargo, ND, USA). PCR was performed using the HotStar HiFidelity Polymerase Kit (Qiagen, Hilden, Germany) with 0.7 mM each of the forward primer (5'-TTGGACCCTCGTACAGAAGCTAATACG-3' (SEQ ID NO: 6)) and the reverse primer (5'-T120-CTTCCTACTCAGGCTTTATTCAAAGACCA-3' (SEQ ID NO: 7)). A poly-T tail consisting of 120 thymidines (T) was added to the plasmid insert during the amplification process. Primers were purchased from ELLA Biotech (Martinsried, Germany). PCR was performed using a cycling protocol consisting of an initial activation at 94°C for 3 min, followed by 30 cycles of denaturation at 94°C for 45 s, annealing at 60°C for 1 min, and extension at 72°C for 1 min. After a final extension at 72°C for 5 min, the amplified PCR products were purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.

[0072] Next, 1.5 μg of each PCR product was in vitro transcribed using the MEGAscript T7 Kit (Life Technologies, Darmstadt, Germany) according to the manufacturer's instructions. Various mRNA variants were obtained (Table 1). To generate unmodified mRNA variants, 1.875 mM GTP, 7.5 mM ATP, 7.5 mM CTP, and UTP were used. Modified mRNA variants were generated by substituting 7.5 mM Ψ or me instead of UTP. 1The Ψ was used, and 7.5 mM m5CTP (m5C) was used instead of CTP. CTP and UTP were used as provided in the MEGAscript T7 Kit. Other nucleotides were purchased from TriLink BioTechnologies (San Diego, USA). Each in vitro transcription reaction was supplemented with 2.5 mM 3'-O-Me-m7G(5')ppp(5')G RNA cap analog (New England BioLabs, Frankfurt am Main, Germany) and 40 U RiboLock RNase inhibitor (Thermo Scientific, Waltham, MA, USA). After 4 hours of incubation at 37°C, 1 μl of TurboDNase was added to remove the DNA template. After further incubation at 37°C for 15 minutes, mRNA was purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and dephosphorylated with 15 U Antarctic phosphatase (New England Biolabs, Frankfurt am Main, Germany) for 30 minutes at 37°C. The mRNA was then purified using the RNeasy Mini Kit. The concentrations of DNA and mRNA products were measured using a BioPhotometer (Eppendorf, Hamburg, Germany). The purity and quality of the amplified DNA and synthetic mRNA were analyzed by staining with GelRed (Biotium, Fremont, CA, USA) after 1% agarose gel electrophoresis (1 hour, 100 V) in 1x Tris-borate-EDTA (TBE) buffer.

[0073] [Table 1]

[0074] 1.2 Cell culture EA.hy926 cells (ATCC, Manassas, VA, USA) were cultured in high-glucose, L-glutamine-containing Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) at 37°C and 5% CO2. Cells were passaged when they reached 80% confluence. To passage, cells were washed with Dulbecco's phosphate-buffered saline (DPBS) and detached with 0.05% trypsin-EDTA. Cell culture medium was changed every 3–4 days. All cell culture reagents were obtained from Fisher Scientific.

[0075] 1.3 Transfection of TE mRNA into cells For transfection of EA.hy926 cells, 3 × 10 cells were transfected into 2 ml of cell culture medium in each well of a 6-well plate. 5 EA.hy926 cells were seeded and cultured for 24 hours at 37°C and 5% CO2. Lipoplexes were generated by complexing 2.5 μg of TE mRNA with 4 μl of Lipofectamine 2000 in 1 ml of OptiMEM I reduced serum-free medium for 20 minutes at room temperature (RT). EA.hy926 cells were washed once with DPBS and incubated with lipoplexes for 4 hours at 37°C and 5% CO2. The transfection medium was then replaced with 1 ml of cell culture medium, and the cells were incubated for 24 to 72 hours at 37°C and 5% CO2. As controls, cells were incubated in OptiMEM ("medium") alone or in OptiMEM supplemented with 4 μl of Lipofectamine 2000 ("L2000"). All reagents were obtained from Thermo Fisher Scientific. After 24 and 72 hours of culture, elastin expression in the supernatant was detected by ELISA.

[0076] 1.4 Elastin ELISA Supernatants were collected 24, 48, and 72 hours after transfection and centrifuged at 3,000 g for 10 minutes at room temperature. 750 μl of supernatant was then transferred to a new low-protein-binding tube, snap-frozen in liquid nitrogen, and stored at -80°C until analysis. Elastin concentrations in the collected supernatants were measured using a human elastin ELISA kit (Biozol and Cloud-Clone, Eching, Germany) according to the manufacturer's instructions. Supernatants from cells transfected with TE mRNA variants containing modified nucleosides were diluted 1:50 with DPBS. All other supernatants were used undiluted.

[0077] 1.5 Detection of cell viability by Presto Blue assay The effect of different mRNA variants on cell viability was analyzed by Presto Blue assay. 5 EA.hy926 cells were seeded into 6-well plates and cultured at 37°C and 5% CO2 for 24 hours. Next, 2.5 μg of TE mRNA complexed with 4 μl of Lipofectamine 2000 in OptiMEM was transfected for 4 hours at 37°C and 5% CO2. After 4 hours, the transfection medium was replaced with cell culture medium, and the cells were incubated for 24 hours at 37°C and 5% CO2. As controls, cells were treated with Lipofectamine 2000 ("L2000") or OptiMEM ("medium") alone. After 24 hours, the cells were washed once with DPBS, and 500 μl of Presto Blue reaction solution (Invitrogen, Carlsbad, CA, USA) diluted 1:10 in cell culture medium was added to each well and incubated for 1.5 hours at 37°C. 100 μl of each sample was measured in triplicate using a multimode microplate reader (Mithras LB 940; Berthold Technologies) at an excitation wavelength of 530 nm and an emission wavelength of 600 nm.

[0078] 1.6 Analysis of the presence or absence of TE mRNA in EA.hy926 cells EA.hy926 cells were transfected with 2.5 μg of each TE mRNA, and 48 and 72 hours later, the amount of TE mRNA in the EA.hy926 cells was detected.

[0079] 1.7 Isolation of synthesized RNA and cDNA Cells were washed with 1 ml of DPBS, detached with 0.05% trypsin-EDTA, and centrifuged at 1,000 x g for 5 minutes at room temperature. Next, cells were washed once with DPBS and centrifuged at 1,000 x g for 5 minutes at room temperature. Cell pellets were snap-frozen in liquid nitrogen and stored at -80°C until detection of intracellular synthetic TE mRNA was performed. RNA was isolated using Trizol reagent according to a standard protocol. Briefly, 1 ml of Trizol (Invitrogen, Carlsbad, CA, USA) was added to the frozen cell pellet and vortexed until the cells were completely lysed. Next, 0.2 ml of chloroform was added and the mixture was centrifuged at 12,000 x g for 15 minutes at 2-8°C. The aqueous phase was transferred to a new tube and 0.5 ml of 2-propanol was added. The mixture was mixed and incubated at room temperature for 15 minutes, followed by centrifugation at 12,000 x g for 10 minutes at 2-8°C. The RNA precipitate formed a pellet, which was washed with 95% EtOH, dried, and then dissolved in RNAse-free water.

[0080] cDNA was synthesized by transcribing 1 μg of RNA using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions and stored at −20°C until use.

[0081] 1.8 Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) To measure TE mRNA levels by qRT-PCR, a standard curve was prepared using cDNA standards with known TE mRNA content. A 100-fold serial dilution series of four template concentrations was used to generate a standard curve starting from 3 ng, and all reactions were performed in duplicate. After target detection in each standard sample, a standard curve was plotted as Cq values ​​versus the logarithm of the template concentration. The TE mRNA concentration in each sample was adjusted to fit within this standard curve, and this standard curve was used to measure the amount of TE mRNA in cell pellets or skin biopsies. The amount of TE mRNA was expressed as the amount of TE mRNA (ng) relative to the amount of total RNA used for cDNA synthesis.

[0082] 1.9 In vivo studies in pigs 1.9.1 Ethics statement This study was conducted in accordance with the recommendations for the care and use of laboratory animals from the Federation of European Laboratory Animal Science Associations (FELASA) and the American Association for Laboratory Animal Science (AALAS). All experiments were approved by the Institutional Animal Care and Use Committee and conformed to Austrian law (BMBWF-68.205 / 0088-V / 3b / 2019).

[0083] 1.9.2 Animals and experimental setup Six domestic pigs (Sus scrofa domestica), 12 weeks old and weighing approximately 30 kg, were obtained from a specific-pathogen-free local breeding facility (Gutshof Medau / Schweineanlage, Berndorf A-2560). Animal experiments were performed at the University of Veterinary Medicine Vienna, and the pigs were housed in the pig stables of the Vienna Veterinary University Hospital. Experiments began after a one-week acclimatization period, and each experiment lasted 48 hours. Clinical examinations of the pigs were performed daily 48 hours before the start of the experiment and continued until the end of the experiment.

[0084] A total of six pigs were used to test 11 mRNA variants. Each mRNA was administered in parallel to two pigs, with triplicate administrations per pig. Administration of the various mRNA variants with different nucleotide modifications was performed by marking the application sites with a permanent marker. Euthanasia was performed under anesthesia with an intramuscular injection of ketamine hydrochloride (Narketan®, 10 mg / kg body weight) and azaperone (Stresnil®, 1.3 mg / kg body weight). Biopsies of the marked sites were taken postmortem after intracardiac injection of T61® (1 ml / 10 kg body weight).

[0085] 1.9.3 In vivo application of TE mRNA The efficiency of elastin protein expression in vivo after intradermal application to pig skin was compared between all unmodified TE mRNA variants and me 1 Ψ / C TE mRNA variants were evaluated. Furthermore, to identify newly produced exogenously expressed TE proteins in the skin, TE variants expressing mCherry-tagged TE (TE_mCherry) were also injected. Each mRNA was dissolved in lactated Ringer's (RL) buffer (Fresenius Kabi, Austria) at concentrations of 3 μg, 10 μg, or 30 μg in a total volume of 90 μl. For intradermal application of TE_mCherry mRNA, 30 μg was used. Intradermal injections were performed using a BD Micro-Fine TM The injections were performed using an insulin syringe (BD, Franklin Lakes, NJ, USA) by injecting 9 x 10 μl into a 1 x 1 cm defined skin area. As a control, only lactated Ringer's buffer without added mRNA was injected. The pigs were euthanized 48 h after injection, and biopsies were taken from all injection sites using a 10 mm biopsy trephine. Additionally, biopsies of untreated skin were taken at the end of the experiment. Each biopsy was snap-frozen in liquid nitrogen and stored at -80°C until analysis of elastin content using the elastin-specific ElaNIR stain.

[0086] 1.9.4 Staining and detection of skin biopsy samples with ElaNIR The fluorescent dye ElaNIR was used to specifically stain elastin fibers in the skin. ElaNIR is described in detail in Su, D., et al., "Seeing Elastin: A Near-Infrared Zwitterionic Fluorescent Probe for In Vivo Elastin Imaging." Chem, 2018. 4(5): pp. 1128-1138. To perform this staining, 1 μmol of ElaNIR was dissolved in 1 ml of DMSO (Sigma-Aldrich, St. Louis, MO, USA). Skin biopsies were incubated overnight at 4°C with 750 μl of DPBS containing 10% DMSO and 20 μM ElaNIR. The samples were then washed six times with DPBS at room temperature for 30 minutes. Near-infrared fluorescent signals (excitation: 745 nm, emission: 800 nm) were detected using an in vivo imaging system (IVIS Spectrum, PerkinElmer). Images were analyzed with Living Image version 4.4 software (PerkinElmer). Fluorescence intensity in a given region of interest (ROI) was calculated as mean radiant efficiency [p / s / cm ] after subtracting background signal. 2 / sr] / [μW / cm 2 ]. Data were normalized to the corresponding control.

[0087] 1.9.5 Histological analysis of TE_mCherry mRNA-injected skin biopsy samples Skin biopsy samples were collected, preserved in 70% ethanol (PanReac AppliChem ITW Reagents, Darmstadt, Germany), transferred to embedding cassettes, and fixed overnight at 4°C in 4% paraformaldehyde (PFA, Merck, Darmstadt, Germany). The samples were then dehydrated, infiltrated with paraffin using an automated tissue processor, and embedded in paraffin blocks using a tissue embedding device. The paraffin blocks were sectioned into 5-μm-thick sections using a microtome (Thermo Fisher Scientific), mounted on Superfrost microscope slides (R. Langenbrinck, Emmendingen, Germany), and allowed to dry overnight at room temperature in the dark. Paraffin sections were deparaffinized twice in 100% xylene (PanReac AppliChem ITW Reagents; Darmstadt, Germany) for 2 minutes each, then rehydrated in a descending series of ethanol (100%, 80%, 70%, and 60%) for 2 minutes each and washed in deionized distilled water for 1 minute. Cell nuclei were stained using Vectashield mounting medium containing the fluorescent dye DAPI (Vector Laboratories, Burlingame, CA, USA). Fluorescent images were acquired using an Axiovert 135 fluorescence microscope (Zeiss) and analyzed using AxioVision Rel 4.8 software.

[0088] 1.10 Analysis of potential cytotoxicity and immune activation in a human skin model 1.10.1 Application of TE mRNA in a human skin model The human Phenion® full-thickness (FT) skin model (Henkel, Düsseldorf, Germany) was used to analyze whether each synthetic TE mRNA variant exhibited potential toxic and immunogenic effects after intradermal application. All components for culturing the FT skin model were purchased from Henkel. The skin model was placed in an air-liquid interface culture system in a Petri dish and incubated in air-liquid interface (ALI) medium at 37°C and 5% CO2 for 24 hours. Next, 30 μg of each TE mRNA variant 14_me was administered. 1 Ψ / C, TE mRNA variant 14_unmod, TE mRNA variant native_me 190 μl of lactated Ringer's buffer solution containing Ψ / C or TE mRNA variant native_Ψ / m5C, or 90 μl of lactated Ringer's buffer solution without TE mRNA variants was added to a BD Micro-Fine TM The FT skin models were injected with insulin syringes (9 × 10 μl). The skin models were then cultured for another 24 hours at 37°C and 5% CO2. Untreated FT skin models or FT skin models injected with lactated Ringer's buffer alone were used as controls. Six skin models were used for each treatment, three of which were used for immune activation analysis and the remaining three for cytotoxicity analysis.

[0089] 1.10.2 In vitro skin toxicity analysis After injection of each TE mRNA variant or control into FT skin models, skin toxicity was analyzed by MTT assay according to OECD Test Guideline No. 439. 24 hours after injection, all skin samples were washed eight times with 600 μL of DPBS and placed in each well of a 24-well plate containing 1 ml of DPBS containing 0.5 mg / ml MTT reaction solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO, USA). The samples were then incubated at 37°C and 5% CO2 for 3 hours. Each skin model was then dried and transferred to a 24-well plate containing 1 ml of 2-propanol (VWR International; Radnor, USA) in each well. The samples were then incubated overnight at 4°C with shaking to elute formazan from each skin model. The skin model was removed from each well, and the eluted formazan was diluted 1:1 with 1 ml of 2-propanol. From each sample, 200 μl was transferred to each well of a 96-well plate, and the absorbance was measured at 540 nm using a microplate reader (Mithras, Bad Wildbad, Germany).

[0090] 1.10.3 RNA isolation from skin models RNA isolation was performed 24 hours after injection of each mRNA into the Phenion FT skin model using the RNeasy Mini Kit (Qiagen, Hilden, Germany). To perform RNA isolation, half of the skin tissue was cut into eight pieces and placed in 350 μl of RTL buffer supplemented with 10 μl / ml β-mercaptoethanol buffer (Sigma, St. Louis, MO, USA). The pieces were then incubated at room temperature and 300 rpm in a thermomixer for 35 minutes. The resulting tissue homogenate was then mixed with 500 μl of RNAse-free water and 10 μl of proteinase K (both from Qiagen), incubated at 55°C for 40 minutes, and centrifuged at 8,000 × g for 30 seconds. The supernatant was collected in a new reaction tube and gently mixed with 0.5 volumes of 100% ethanol. Next, 700 μl of the lysate was transferred to an RNeasy Mini Kit spin column and centrifuged at 8,000 × g for 15 seconds, and the flow-through was discarded. The remaining tissue lysate was applied to the RNeasy Mini Kit spin column and the above procedure was repeated. The spin column was washed with 350 μl of RW1 buffer and centrifuged at 8,000 × g for 15 seconds. To remove DNA, 80 μl of DNAse was prepared from the RNase-free DNase Set (Qiagen) according to the manufacturer's instructions, added to each spin column, and incubated for 15 minutes. Next, 350 μl of RW1 buffer was added, and each spin column was centrifuged at 8,000 × g for 15 seconds. After discarding the flow-through, the spin column was washed twice with 500 μl of RPE buffer and centrifuged at 8,000 × g for 30 seconds. The RPE buffer was removed, and the spin column was centrifuged at maximum speed for 4 minutes. RNA was eluted with 50 μl of RNase-free water and centrifuged at 8,000 × g for 1 minute. The isolated RNA was snap frozen in liquid nitrogen and stored at -80°C.

[0091] 1.10.4 qRT-PCR Immune activation in tissues was investigated by analyzing the expression of IL-6, IL-8, CXCL-10, and IFN-β as markers using qRT-PCR. Total RNA was isolated as described above, and 900 ng of the isolated RNA was analyzed using iScript PCR.TM cDNA synthesis was performed using a cDNA Synthesis Kit (Bio-Rad) at 25°C for 5 minutes, 42°C for 30 minutes, and 85°C for 5 minutes. qRT-PCR of 1:10 diluted cDNA was performed using iQ SYBR Green Supermix (Bio-Rad) according to the manufacturer's instructions. Reactions were performed in triplicate on an iCycler iQ Real-Time PCR Detection System (Bio-Rad). Primers used for specific amplification of CXCL-10 (forward primer: aagtggcatt caaggagtac c (SEQ ID NO: 10), reverse primer: acgtggacaa aattggcttg c (SEQ ID NO: 11)), primers used for specific amplification of IFN-β (forward primer: tacctgaagg ccaaggagta cag (SEQ ID NO: 12), reverse primer: cggaggtaac ctgtaagtct gttaa (SEQ ID NO: 13)), primers used for specific amplification of IL-6 (forward primer: cacacagaca gccactcacc tc (SEQ ID NO: 14), reverse primer: ctgccagtgc ctctttgctg (SEQ ID NO: 15)), primers used for specific amplification of IL-8 (forward primer: gacttccaag ctggccgtg (SEQ ID NO: 16), reverse primer: ctccttggca The primers used for specific amplification of GAPDH (forward primer: tcaacagcga cacccactcc (SEQ ID NO: 18), reverse primer: tgaggtccac caccctgttg (SEQ ID NO: 19)) were purchased from Ella Biotech (Martinsried, Germany). Expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control and for normalization of each expression level. Each result is shown as the expression level relative to the control mRNA level of the untreated sample.

[0092] 1.11 Statistics Data are presented as mean ± SEM. Statistical analysis of the data was performed using GraphPad Prism version 9.0.1. For repeated measurements, one-way analysis of variance was performed, followed by Bonferroni or Tukey's multiple comparison test, or Friedman's test, followed by Dunn's comparison test. Differences were considered statistically significant when P<0.05.

[0093] 2. Results 2.1 In silico optimization of TE mRNA sequences The coding sequences (CDS) of TEs were selected taking into account GC content and the human codon adaptation index (CAI). The CAI value ranges from 0 to 1. The CAI defines the relative fitness of a gene's codon usage compared to that of highly expressed genes. A higher CAI value indicates a higher proportion of the most frequently used codons, which in this case indicates an optimal fit for the human translational machinery and results in higher expression levels.

[0094] In this study, we selected four codon-optimized TE mRNA variants from a large number of TE sequences and tested their expression by comparing them with that of native human TE mRNA (Table 2). Elastin is a particularly challenging protein to optimize because 76% of its amino acids are composed of only glycine (29%), alanine (22%), valine (13%), and proline (12%). These four amino acids all have highly GC-rich codons, resulting in a very high overall GC content in TE mRNA.

[0095] To analyze the effects of various codon-optimized TE mRNA variants (Table 2) and various types of nucleotide modifications on expression efficiency, we used unmodified cytidine and unmodified uridine nucleotides (CTP / UTP), pseudouridine and 5-methylcytidine (Ψ / m5C), N 1 -Methylpseudouridine (me 1 Ψ) and 5-methylcytidine (me 1 Ψ / mC), or N 1-Methylpseudouridine and cytidine (me 1 We generated five TE mRNA variants (Table 1) with different Ψ / C.

[0096] [Table 2]

[0097] 2.2 Codon optimization of TE mRNA significantly impacts expression efficiency without affecting cell viability in vitro To analyze the effect of TE codon sequence variation on elastin expression and cell viability, 2.5 μg of TE mRNA complexed with 4 μl of Lipofectamine 2000 was cultured in OptiMEM at 3 × 10 5 TE mRNA variant 14 was transfected into EA.hy926 cells. Supernatants were collected at 24, 48, and 72 hours, and elastin concentrations were detected by ELISA. Increased elastin production was detected as early as 24 hours, but the maximum amount of elastin was quantitatively measured 48 hours after transfection (Figure 1). Cells treated with Lipofectamine 2000 ("L2000") alone were used as a control. Regardless of the nucleotide modification, TE mRNA variant 14 induced the highest elastin expression (Figure 2). The amount of elastin detected in the supernatant after transfection of cells with unmodified TE mRNA ("unmod") was very low (Figure 2A), possibly due to the high cytotoxicity of unmodified mRNA in vitro (Figure 3A). TE mRNA variant 3 was transfected with either Ψ / m5C (Figure 2B) or me. 1 Modification with Ψ / m5C (Figure 2B) resulted in significantly higher elastin expression levels compared to the control ("L2000"). The highest protein expression was observed in the TE mRNA variant 3. 1 TE mRNA variant 14 was detected when modified with Ψ / m5C (Figure 2B), me 1 Ψ / m5C (Fig. 2B), me 1 Ψ / m5C (Figure 2C), or me 1When modified with Ψ / C (Figure 2D), the expression level of elastin was significantly increased, and me 1 The highest expression level was obtained when the mRNA was modified with Ψ / m5C (Fig. 2C). 1 Both Ψ / C-modified natural mRNAs (Fig. 2D) showed significantly higher elastin expression levels than the control, and me 1 The modification with Ψ / C showed the highest elastin expression level.

[0098] The effect of each mRNA variant on cell viability was assessed using Presto Blue 24 hours after transfection. TM The effect of the modified mRNA on cell viability was investigated using an assay (Figure 3). As a control, cells treated with OptiMEM ("medium") or Lipofectamine 2000 ("L2000") were used. Regardless of the type of sequence variant, unmodified mRNA showed higher cytotoxicity to cells compared to their nucleotide-modified variants (Figure 3A). Surprisingly, when the same nucleotide modifications were used, differences in nucleic acid sequence did not affect cell viability. 1 The highest cell viability, up to 79%, was observed when cells were transfected with Ψ / m5C-modified TE mRNA variants (Fig. 3C), followed by me 1 High cell viability was observed when cells were transfected with Ψ / C-modified TE mRNA variants and Ψ / m5C-modified TE mRNA variants (FIGS. 3B and 3D).

[0099] 2.3 Nucleotide modifications in codon-optimized TE mRNA variants strongly modulate in vitro expression efficiency and reduce cytotoxicity The modification of each TE mRNA variant with modified nucleotides had a strong effect on the translation of each TE mRNA variant, as shown by the amount of elastin protein produced in Figure 4. In particular, me 1 Ψ / m5C nucleotide or me 1 The incorporation of the Ψ / C nucleotide alone had a beneficial effect on the expression level of elastin protein. In TE mRNA variant 1 (Fig. 4A) and TE mRNA variant 4 (Fig. 4C), me1 Simply incorporating Ψ / C into mRNA significantly increased the expression level of elastin compared to the control ("L2000"). The significant increase in elastin expression was due to the Ψ / m5C modification, me 1 Ψ / C modification or me 1 This was also observed in TE mRNA variant 3, which incorporates the Ψ / m5C modification (Figure 4B). 1 Ψ / m5C or me 1 When we engineered natural TE mRNA variants and TE mRNA variant 14 incorporating Ψ / C, the expression of elastin protein significantly increased compared to the control (Figure 4D). 1 The incorporation of the Ψ / C modification resulted in the highest translation efficiency in TE variants 1, 4, and the natural TE variants. In contrast, TE variants 3 and 14 showed no significant difference in me 1 Incorporation of Ψ / m5C resulted in the highest expression of elastin. These data indicate that in addition to codon optimization, modification of each TE mRNA variant with modified nucleotides has a strong effect on translation.

[0100] Furthermore, we analyzed the effect of nucleotide modifications in TE mRNA variants on cell viability (Figure 5). Both TE mRNA sequence variants (Figure 5A-E) showed that me 1 Ψ / m5CTP or me 1 The use of Ψ resulted in the highest cell viability compared to TE mRNA variants with unmodified nucleotides or TE mRNA variants with the Ψ / m5C modification. Overall, me 1 The highest cell viability was observed in cells transfected with TE mRNA containing the Ψ / m5C nucleotide modification. 1 Ψ / m5CTP or me 1Incorporation of Ψ improved cell viability by up to 30%. The presence of the transfected nucleotide-modified TE mRNA could be detected in cells up to 72 hours after transfection (Figure 6). No differences in mRNA degradation were detected between the different mRNA variants or nucleotide modifications.

[0101] In summary, the highest elastin expression was detected in TE mRNA variant 14. Nucleotide modifications in each TE mRNA variant increased the protein expression level, and 14_me 1 Ψ / m5C or 14_me 1 The highest protein expression was obtained after transfection of Ψ / C. Surprisingly, codon optimization did not affect cell viability, but nucleotide modifications did. The highest cell viability was observed with me 1 This was observed in the Ψ / m5C modification. The ranking of all 20 mRNA variants tested in vitro is shown in Table 3. High protein expression is desirable, but toxicity should be reduced as much as possible.

[0102] [Table 3]

[0103] 2.4 In vivo administration of TE mRNA to pig skin significantly increases elastin expression me 1 TE mRNA sequence variants with Ψ nucleotide modifications exhibited reduced cytotoxicity and increased protein expression efficiency, and mRNA stability was demonstrated as assessed by mRNA degradation analysis in cells. 1 The natural TE mRNA variants, TE mRNA variant 1, TE mRNA variant 3, TE mRNA variant 4, and TE mRNA variant 14, modified with Ψ / C, and me 1The Ψ / C-modified native TE_mCherry was selected and screened in pig skin to analyze the in vivo protein expression efficiency.

[0104] No skin inflammation was observed at the injection site 48 hours after injection (Figure 7). Forty-eight hours after intradermal injection of each TE mRNA, de novo elastin synthesis was measured in pig skin. The elastin content of the whole skin biopsy samples was measured using elastin-specific ElaNIR staining. The ElaNIR-specific fluorescent signal was then measured using IVIS (Figures 8A, 8B, 8C, and 8D).

[0105] Injection of 10 μg or 30 μg of unmodified TE mRNA variant 14 (Figures 8A and 8B) resulted in significantly higher elastin expression compared with injection of lactated Ringer's buffer alone. 1 For Ψ / C-modified TE mRNA variant 14, only 3 μg of 14_me 1 The intradermal application of Ψ / C alone significantly increased the expression of elastin, and 10 μg or 30 μg of 14_me 1 Intradermal injection of Ψ / C also increased elastin expression (Figure 8B). Representative images of ElaNIR-stained pig skin samples detected by IVIS 48 hours after intradermal injection of each TE mRNA variant in vivo are shown in Figure 10. Furthermore, 30 μg of native_me 1 Injection of Ψ / C also significantly increased the amount of elastin in the skin (Fig. 8A).

[0106] To facilitate the distinction between endogenous and de novo synthesis of elastin expressed after TE mRNA administration, we designed a TE mRNA construct containing an N-terminal mCherry tag sequence and administered it in vivo. When 2.5 mg of TE-mCherry mRNA was transfected into EA.hy926 cells, TE protein production was confirmed (Figure 9).

[0107] After injection of 30 μg of TE_mCherry mRNA, expression of mCherry-tagged elastin protein was detectable in skin biopsies, showing a significant increase in fluorescence intensity compared to untreated skin controls (Figure 8D). Fluorescence microscopy of thin sections of biopsies showed that the majority of mCherry-tagged elastin was distributed in the dermis near resident cells (nuclei stained with DAPI) (Figure 8E).

[0108] 2.5 No skin toxicity or immune activation was detected after cutaneous administration of each TE mRNA variant. After intradermal application of each TE mRNA variant to the human Phenion FT skin model, we analyzed whether these TE mRNA variants might exhibit toxic and immunogenic effects in the skin. The TE mRNA variant that showed the highest protein expression efficiency in vivo, i.e., 14_me 1 Ψ / C, 14_unmod or native_me 1 Ψ / C or native_Ψ / m5C were injected into skin models (Figure 11A). Similar to the in vivo experiments, 30 μg of each TE mRNA in 90 μl of lactated Ringer's buffer was injected. Skin models injected with lactated Ringer's buffer alone and untreated skin models were used as controls. 24 hours after injection, cell viability in each skin model was measured using an MTT assay. Injection of various TE mRNA variants did not negatively affect cell viability (Figure 11B). Analysis of immune activation markers by qRT-PCR yielded similar results. 24 hours after injection, no significant increases in the expression of immune activation markers IL-6, IL-8, CXCL-10, and IFN-β were detected in the TE mRNA-treated groups compared to the control group, nor were they detected in comparisons between the various TE mRNA groups (Figure 11C). The low immunogenicity of TE mRNA is advantageous for its use in the pharmaceutical or cosmetic compositions described herein. 3. Conclusion The present inventors provide oligonucleotides that allow for the de novo synthesis of elastin in mammalian cells and mammalian tissues with enhanced expression efficiency.

Claims

1. 1. An oligonucleotide comprising a nucleotide sequence encoding a tropoelastin protein, said nucleotide sequence being codon-optimized for expression in a mammalian cell.

2. The oligonucleotide of claim 1, which is an oligoribonucleotide, preferably an mRNA.

3. The mRNA a 5' cap structure, preferably 3'-O-Me-m7G(5')ppp(5')G, and / or A poly-A tail, preferably a poly-A tail of at least about 70 adenine nucleotides, more preferably a poly-A tail of about 120 adenine nucleotides. The oligonucleotide of claim 2, comprising:

4. The oligonucleotide of any one of claims 1 to 3, wherein at least one of the nucleotides is an analogue of a natural nucleotide.

5. The oligonucleotide of claim 4, wherein the analog is selected from the group consisting of pseudouridine, N-methylpseudouridine, and 5-methylcytosine.

6. 6. The oligonucleotide of claim 1, wherein the tropoelastin protein comprises the amino acid sequence of SEQ ID NO:

1.

7. The oligonucleotide of any one of claims 1 to 6, comprising the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:

5.

8. An oligonucleotide according to any one of claims 1 to 7 for use in the treatment of diseases and conditions associated with tissue insufficiency.

9. The oligonucleotide of claim 8, wherein the treatment is selected from the group consisting of treatment of genetic defects in elastin synthesis, treatment of arteriosclerosis, treatment of aortic stenosis, treatment of aortic aneurysm and / or cerebral aneurysm, treatment of chronic obstructive pulmonary disease (COPD), treatment of eye diseases, treatment of age-related macular degeneration (AMD), treatment of aortic regurgitation, treatment of cutis laxa, treatment of Williams-Beuren syndrome, treatment of skin laxity, treatment of ligament disorders, treatment of congenital subaortic stenosis (SVAS), treatment of scar tissue, and treatment of scarless wound healing.

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

11. 11. The pharmaceutical composition of claim 10, adapted for systemic administration to a mammal, preferably wherein said systemic administration is via a parenteral route of administration, more preferably via intravenous injection.

12. 11. The pharmaceutical composition of claim 10, 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 cream, a gel, a liquid, a paste, a spray, a plaster, a microneedle, a medical bandage, a face mask, an implant, and a stent.

13. A cosmetic composition comprising an oligonucleotide according to any one of claims 1 to 7 for use in increasing the elasticity of human tissue, preferably for use in the treatment or prevention of wrinkles formed on human skin.

14. A cosmetic method for inducing de novo synthesis of elastin in mammalian tissue, comprising topically applying the cosmetic composition of claim 13 by injection into mammalian tissue or topical application onto mammalian tissue.

15. The cosmetic method according to claim 13, wherein the topical application is repeated at least once, and preferably the cosmetic composition is contained in a formulation or delivery form selected from the group consisting of a cream, a gel, a liquid, a paste, a spray, a plaster, a microneedle, a medical bandage, and a face mask.

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