Klotho mRNA

Abstract

The present invention provides Klotho messenger RNA (mRNA) having a 5' CAP region, a 5' untranslated region (5'-UTR), a coding region encoding a Klotho polypeptide, a 3' untranslated region (3'-UTR), and a polyadenosine tail (poly-A tail), wherein the Klotho polypeptide comprises the KL1 domain of human Klotho, preferably the coding region encoding the Klotho polypeptide comprises an RNA sequence having at least 80% sequence identity with SEQ ID NO: 1, and preferably the coding region encoding the Klotho polypeptide has at least 54% GC content.

Description

[Technical Field]

[0001] This invention relates to Klotho, particularly Klotho mRNA, and the field of formulations, kits, and uses relating thereto. [Background technology]

[0002] Klotho is an enzyme encoded by the KL gene in humans. Klotho has three subfamilies: α-Klotho, β-Klotho, and γ-Klotho. Where a subfamily is not specified, the term "Klotho" typically refers to the α-Klotho subfamily, as α-Klotho was discovered before the other subfamily members. Therefore, "Klotho" and "α-Klotho" are used interchangeably herein.

[0003] Klotho is described in more detail, for example, in International Publication No. 2019 / 113373: two transcripts have been identified arising from a single Klotho gene by alternative RNA splicing. The first transcript encodes Klotho isoform 1—a full-length 1,012-amino acid single-pass transmembrane protein, which has a short cytoplasmic tail (human residues 1003-1012), a transmembrane (TM) domain (human residues 982-1002), and an extracellular region or domain (human residues 1-981) containing two (mostly) homologous (internal repeat) domains (called KL1 (human residues 56-506, 450 residues long) and KL2 (human residues 515-953, 438 residues long), which share 20-40% amino acid sequence homology with β-glucosidase, but may lack similar levels of glucosidase catalytic activity), and a signal peptide (SP) domain (human residues 1-33; also called the "signal sequence" or "SS"). The extracellular regions containing the SP, KL1, and KL2 domains (human residues 1-981) can be enzymatically cleaved by α / β-secretase and released into the circulation as a 130 kDa circulating protein called soluble Klotho (or sKlotho, s-Klotho, alpha-soluble Klotho, etc.). The extracellular regions can also be cleaved into separate 68 kDa proteins (KL1+SS) and 64 kDa proteins (KL2).

[0004] The second transcript is a splicing variant of alpha-Klotho mRNA, encoding a second isoform of the Klotho protein, primarily corresponding to the KL1 domain. The internal splice donor site is thought to be located in exon 3 of the Klotho gene. The resulting alternatively spliced ​​transcript contains a 50 bp insertion after exon 3 and has an in-frame translation stop codon at its terminus. The expressed protein product is secreted into the circulatory system and is called secreted Klotho (or Klotho isoform 2), which differs from the canonical sequence of isoform 1 by amino acid residues 535-549 and has a deletion of amino acid residues 550-1012.

[0005] Klotho was first described in 1997 as a repressor of aging. Klotho knockout mice exhibit a severe aging phenotype at 4-5 weeks of age and die within 9 weeks. Mice that overexpress Klotho from birth live up to 30% longer than their wild-type littermates.

[0006] The Klotho gene is highly expressed in the liver, kidneys, and brain. Its anti-aging benefits are primarily related to the inhibition of intracellular glucose uptake (i.e., inhibition of the interaction between insulin and the IGF-1 receptor). Furthermore, Klotho has positive effects on neuroprotection and neurogenesis, stimulation of autophagy, regulation of oxidative stress, growth factor signaling, ion homeostasis, and organ protection.

[0007] Several studies suggest that serum Klotho levels are high in children and young adults and decrease by 30-40% with age. Children also have better insulin uptake. Blood Klotho levels falling below a certain threshold appear to correlate with AD / dementia, heart disease, and reduced lifespan. A gradual decline in serum Klotho levels provides an opportunity for replenishment. Furthermore, the difference in Klotho levels between healthy and diseased states is very small. Therefore, even a slight increase in Klotho levels may be sufficient to exceed the threshold. Thus, Klotho can be considered a promising anti-aging compound.

[0008] In addition to its anti-aging effects, Klotho deficiency can be seen in various diseases. In particular, increased Klotho levels have been shown to be beneficial for kidney, cardiovascular, brain, and lung diseases, as outlined in Prud'homme et al., 2022, table 1 and 3 (Prud'homme et al. “Pathobiology of the Klotho Antiaging Protein and Therapeutic Considerations.” Frontiers in Aging 3 (2022)). Therefore, Klotho also acts as a tumor suppressor in various types of tumors (Sachdeva, et al. “Klotho and the treatment of human malignancies.” Cancers 12.6 (2020): 1665).

[0009] Given the potential benefits of Klotho, strategies are being pursued to increase the expression level of Klotho in individuals and / or to administer Klotho, e.g., recombinant s-Klotho. Such strategies and uses of Klotho are described, for example, in International Publication Nos. 2019 / 113373, 2022211420, 2022008971, 2020163017, 2020106997, 2020106351, 2020039425, 2019140250, and other publications. This information is contained in International Publication No. 2019113373, International Publication No. 2019113061, International Publication No. 2018098375, International Publication No. 2017210607, International Publication No. 2016127097, International Publication No. 2016088059, International Publication No. 2014152993, International Publication No. 2011084452, and International Publication No. 2008135993.

[0010] However, despite the promising mechanisms to which the Klotho protein has been shown to be involved, no approved treatment has yet appeared on the market in the approximately 25 years that Klotho has been discussed as a promising target by large and small pharmaceutical companies. [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] The objective of this invention is to address this need. [Means for solving the problem]

[0012] Accordingly, the present invention provides Klotho mRNA, which is a Klotho messenger RNA (mRNA) having a 5' CAP region, a 5' untranslated region (5'-UTR), a coding region encoding a Klotho polypeptide, a 3' untranslated region (3'-UTR), and a polyadenosine tail (poly-A tail), wherein the Klotho polypeptide comprises the KL1 domain of human Klotho, preferably the coding region encoding the Klotho polypeptide comprises an RNA sequence having at least 80% sequence identity with SEQ ID NO: 1, and preferably the coding region encoding the Klotho polypeptide has at least 54% GC content.

[0013] One of the main reasons pharmaceutical companies have failed to realize the potential of Klotho proteins is that players in this field have so far focused on the application of exogenously produced Klotho proteins, such as those derived from E. coli or cell cultures. These types of proteins miss at least two characteristics of how Klotho proteins are endogenously expressed by cells in the body: (1) Being fully humanized, fully individualized, and fully personalized; (2) Body positioning and delivery strategies for Klotho.

[0014] Klotho mRNA is not expressed solely by direct transcription from DNA in parental cells, such as kidney cells. Klotho mRNA is also most likely packaged in exosomes (see Sahu, “Regulation of aged skeletal muscle regeneration by circulating extracellular vesicles.” Nature Aging 1.12 (2021): 1148-1161). These then budded off from the cell, spread into the bloodstream, and reach other target tissues and cells, including brain cells. Further drawbacks / challenges at the protein level may also be considered, such as the short half-life of the protein itself, its intracellular availability, lack of targeting, lack of specificity for intravenous administration, and its inability to be administered orally. Therefore, despite the current availability of methods for administering recombinant Klotho and increasing the expression levels of endogenous Klotho, novel and improved alternatives are needed to leverage the benefits of this protein. In particular, novel and improved methods, and perhaps more importantly, novel formats including exosomes, are desired to deliver Klotho's benefit portfolio to individuals in order to positively impact aging and / or leverage therapeutic outcomes.

[0015] mRNA has emerged as a revolutionary tool in the field of medicine due to its ability to transmit genetic instructions to cells and direct the production of specific proteins. While the use of mRNA-based vaccines has attracted attention in recent years, mRNA-based therapeutics also show great potential for treating a variety of symptoms and diseases. The basic principle of exogenous mRNA-based therapeutics is to introduce such mRNA molecules into cells, which can then instruct the cells to produce therapeutic proteins that would otherwise be deficient, dysfunctional, or insufficiently expressed in the body.

[0016] The success of such mRNAs, particularly mRNA-based therapies, largely depends on the level of mRNA expression in the body, as this level determines the amount of protein produced. Several common approaches to improving mRNA expression levels include modifying the 5' and 3' untranslated regions (UTRs) of the mRNA, as well as / or optimizing the formulation and delivery methods. Taken together, however, it is often the sequence of the individual mRNA itself that determines the expression level achieved.

[0017] General methods for preparing and using mRNA molecules for such methods are described, for example, in International Publication No. 2013 / 151736. International Publication No. 2013 / 151736 describes numerous different examples of such mRNA. Among them, Example 125 in paragraph number [0001618] refers to the intravenous administration of Klotho mRNA to CD1 mice. The RNA sequence is included as Sequence ID No. 196 in the sequence listing of International Publication No. 2013 / 151736. Analysis of this sequence appears to encode Klotho isoform 2. Example 125 of International Publication No. 2013 / 151736 describes that the mRNA was chemically modified in various ways and administered in complex with lipofectamine 2000. However, International Publication No. 2013 / 151736 does not state that the RNA sequence itself was optimized in any way.

[0018] While the use of mRNA as a therapeutic agent holds great potential for treating various diseases, its application to specific cases can be challenging. The primary reason hindering the development of effective Klotho mRNA so far has likely been insufficient expression levels. Low expression levels are a significant challenge in the development of mRNA-based therapeutics. mRNA molecules transmit instructions for protein synthesis, and their expression level determines the amount of protein produced within the cell. Insufficient expression levels result in insufficient therapeutic efficacy and limit clinical usefulness. If expression levels are too low, the therapeutic effect may not be adequate, leading to treatment failure. Therefore, improving mRNA expression levels is necessary to enable the development of more effective therapeutics and other applications.

[0019] RNA sequences are crucial determinants of protein expression levels, and modifying them can significantly impact the effectiveness of mRNA-based therapeutics. With the help of codon optimization algorithms, it's possible to modify RNA sequences without altering the protein sequence. These algorithms typically take into account various factors such as codon usage bias, amino acid frequency, and mRNA secondary structure. Many of these codon optimization algorithms are available as software or online, providing researchers with easy access to optimize the sequence of their desired mRNA.

[0020] The inventors of the present invention set out to obtain mRNA for the expression of Klotho in order to achieve a high expression level. In the first experiment, soluble Klotho, that is, the one corresponding to amino acids M1 to S981 of Klotho isoform 1, was selected. These were starting from the sequence of native human Klotho isoform 1 according to the NCBI reference sequence: NM_004795.4. As described in more detail in the examples, the sequence encoding soluble Klotho, that is, amino acids M1 to S981, was extracted (SEQ ID NO: 17). This sequence is 2946 nt in length and can be divided into a signal peptide (amino acids 1 to 33; nt 1 to 99), KL1 and KL2 domains including the adjacent linker region (amino acids E34 to S981; nt 100 to 2946). The part encoding the signal peptide was kept constant, and the latter part of the sequence corresponding to nt 100 to 2946 was optimized using four different codon optimization algorithms provided through the websites of different gene synthesis providers: - "GENEius algorithm" provided by Eurofins Genomics Germany GmbH (https: / / eurofinsgenomics.eu / en / gene-synthesis-molecular-biology / gene-synthesis / geneius / ): resulting in SEQ ID NO: 25 - "IDT Codon Optimization Tool" provided by Integrated DNA Technologies, Inc. (https: / / eu.idtdna.com / pages / tools / codon-optimization-tool): resulting in SEQ ID NO: 26 - "ExpOptimizer" provided by NovoPro Bioscience, Inc. (https: / / www.novoprolabs.com / tools / codon-optimization): resulting in SEQ ID NO: 27 - "Twist Codon Optimization Tool" provided by Twist Bioscience (https: / / www.twistbioscience.com / twist-ordering-platform): SEQ ID NO: 28

[0021] The expression levels obtained with four commercially available optimized sequence variants showed quite a large variation (see Examples and Drawings). Therefore, the inventors decided to test whether it was possible to achieve even higher expression levels. When the inventors compared the optimized sequences with the native human Klotho sequence (SEQ ID NO: 17), they finally noticed the surprising fact that all the optimized sequences had a significantly low GC content (see Table 1). Further investigation revealed that the expression levels seemed to follow the GC content: the sequence obtained from the "GENEius algorithm" (SEQ ID NO: 25) had the highest GC content (52.6%) and also the highest expression level (see Examples and Drawings). The second best result was followed by the sequence obtained from "ExpOptimizer" (SEQ ID NO: 27; GC content 51.1%), the sequence from the "IDT Codon Optimization Tool" (SEQ ID NO: 26; GC content: 49.9%), and finally the sequence from the "Twist Codon Optimization Tool" (SEQ ID NO: 28; GC content: 48.7%).

[0022] Therefore, the inventors came up with the hypothesis that a high GC content is beneficial for Klotho expression. The inventors tested this hypothesis with several mRNA variants having different GC contents, namely: - Variant "GC62" (SEQ ID NO: 18); GC content: 61.6% - Variant "GC60" (SEQ ID NO: 19); GC content: 60.1% - Variant "GC57" (SEQ ID NO: 22); GC content: 57.4% - Variant "GC55" (SEQ ID NO: 23); GC content: 54.9% - Mutant "GC53" (SEQ ID NO: 24); GC content: 52.8% The test was conducted by fabricating the following.

[0023] Surprisingly, a direct relationship was indeed found between GC content and expression levels. Expression levels continued to increase from the mutant "GC53" to the mutant "GC62," even exceeding the GC content of native Klotho.

[0024] The table below summarizes the sequences tested and their GC content.

[0025] [Table 1]

[0026] Furthermore, the inventors investigated the sequence identity of various sequences. As a result, they found that mutants that were closer in sequence identity to the best mutant, "GC62," had higher expression levels. Interestingly, this trend also applied to four sequences obtained from commercially available sequence optimization tools. The best sequence obtained from the "GENEius algorithm" (SEQ ID NO: 25) showed the highest sequence identity to "GC62" (80.6%); the second best sequence was obtained from "ExpOptimizer" (SEQ ID NO: 27; 79.6% sequence identity to "GC62"); the third best sequence was obtained from "IDT Codon Optimization Tool" (SEQ ID NO: 26; 77.3% sequence identity to "GC62"); and finally, the result from "Twist Codon Optimization Tool" was the lowest (SEQ ID NO: 28; 76.4% sequence identity to "GC62"). All custom mutants had higher sequence identity to "GC62" than those obtained from commercially available sequence optimization tools. In this case as well, the expression level increased continuously with increasing sequence identity to "GC62," starting with mutant "GC53" (SEQ ID NO: 24; 81.6% sequence identity to "GC62") and progressing to "GC60" (SEQ ID NO: 19; 92.0% sequence identity to "GC62"). Thus, a direct relationship was found between sequence identity to "GC62" and the expression level. The sequence identity between all the mutants tested is summarized in the table below.

[0027] [Table 2]

[0028] Interestingly, it should be noted that the four sequences obtained from commercially available sequence optimization tools do not share high sequence identity with one another. As is clear from the table above, the sequence identity of each pair of the four sequences is below 79%. Thus, despite the four sequences being optimized in clearly different ways, none of them come close to the preferred variant "GC 62," with the closest being the one obtained from the "GENEius algorithm" (sequence number 25), which has 80.6% sequence identity with "GC62."

[0029] In addition to the sequence variant encoding soluble Klotho (amino acids M1 to S981) described above, the inventors also designed a corresponding sequence variant encoding human Klotho isoform 2 (amino acids M1 to H549). As mentioned above, human Klotho isoform 2 shares amino acids M1 to V534 with Klotho isoform 1. Therefore, the inventors designed an isoform 2 "GC62" variant (sequence number 41) in which nucleotides 1 to 1602 (encoding M1 to V534) are identical to nucleotides 1 to 1602 of the isoform 1 "GC62" variant (sequence number 18). Only nucleotides 1603 to 1650 (encoding D535 to H549) of isoform 2 variant sequence number 41 differ from isoform 1 variant sequence number 18. The GC content of isoform 2 variant sequence number 41 is similar to that of isoform 1 variant sequence number 18, i.e., 62.8%.

[0030] It is beneficial to compare preferred isoform 2 variants prepared in the context of the present invention with the Klotho mRNA sequence disclosed in the aforementioned International Publication No. 2013 / 151736. Sequence ID No. 192 of International Publication No. 2013 / 151736 has 1793 nucleotides and contains an open reading frame of 1650 nucleotides that is thought to encode human Klotho isoform 2. Accordingly, the inventors compared this sequence with Sequence ID No. 41, a superior "GC62" variant prepared in the context of the invention. Surprisingly, the sequence used in International Publication No. 2013 / 151736 also has a low GC content (53.8% for Sequence ID No. 192 of International Publication No. 2013 / 151736; compared to 62.8% for Sequence ID No. 41 of this application). Furthermore, Sequence ID No. 192 of International Publication No. 2013 / 151736 has only 75.7% sequence identity with Sequence ID No. 41 of this application. Therefore, the sequence variants produced in the context of the invention are advantageous over the mRNA sequence disclosed in International Publication No. 2013 / 151736 for the same reasons as described above for the commercially optimized variants of the mRNA encoding Klotho isoform 1. Specifically, they have a higher GC content and higher sequence identity than the best "GC62" variants produced in the context of the invention.

[0031] All embodiments of the present invention are described together in the following detailed description, and all preferred embodiments are similarly relevant to all embodiments and aspects. All detailed descriptions, for example, of mRNA and compositions, relate to preferred embodiments of all aspects of the present invention, for example, compositions for use, use and methods. All embodiments can be combined with one another unless otherwise stated.

[0032] Human Klotho isoform 1 (UniProt Q9UEF7-1) has the following sequence; SEQ ID NO: 49: [ka] It has.

[0033] The domains with different sequences are highlighted above and are as follows: - Italicized and underlined: Signal peptide ("SP") (M1 to A33) - Bold and underlined: KL1 domains (L56 to F506) - Bold italics: KL2 domains (L515 to G952) - Underlined: Transmembrane ("TM") domain (L982 to Y1002) - Italics: Cytoplasmic tail ("CT") (Y1003 to K1012) - No emphasis: Interdomain linking region ("lr") The human Klotho isoform 2 (UniProt Q9UEF7-2) is sequenced as follows; Sequence ID 50: [ka] It has.

[0034] The domains with different sequences are highlighted above and are as follows: - Italicized and underlined: Signal peptide ("SP") (M1 to A33) - Bold and underlined: KL1 domains (L56 to F506) - Bold italics: Sequence corresponding to the KL2 domain of isoform 1 (L515 to V534) - Underlined: Alternative sequence (S535 to H549) - No emphasis: Interdomain linking region ("lr")

[0035] As shown in the sequences above, in this specification, all sequences between different domains are referred to as “linking regions” (lr). Which sequence a given “lr” refers to depends on the adjacent domain. For example, when this application refers to the portion “KL1-lr-KL2” of Klotho isoform 1, this corresponds to amino acids L56 to G952, and in this example, “lr” refers to P507 to P514. Similarly, “lr-KL1-lr-KL2-lr” refers to E34 to S981, where the first “lr” is E34 to G55, the second “lr” is P507 to P514, and the third “lr” is F953 to S981.

[0036] In the sequence of Klotho isoform 2, the portion from P507 to H549 (i.e., the C-terminal portion of the protein following the KL1 domain) is referred to herein as the “terminal region” (“Terminal”). Thus, as used herein and as shown above, “Terminal” corresponds to P507 to H549 and includes lr(P507 to P514), “the sequence corresponding to the KL2 domain of isoform 1” (L515 to V534), and “alternative sequence” (S535 to H549).

[0037] All domains and regions of sequences referred to herein, such as KL1, KL2, SP, TM, CT, lr, terminal domains and regions, may refer to sequences of sequence numbers 49 and 50 shown above, or their variants, in particular variants having altered or improved properties as described below herein. These domains and regions preferably refer to sequences of sequence numbers 49 and 50 above.

[0038] In one embodiment, the present invention relates to Klotho messenger RNA (mRNA) having a 5' CAP region, a 5' untranslated region (5'-UTR), a coding region encoding a Klotho polypeptide, a 3' untranslated region (3'-UTR), and a polyadenosine tail (poly-A tail), wherein the Klotho polypeptide comprises the KL1 domain of human Klotho, and the present invention relates to Klotho mRNA.

[0039] It is known in the art that the KL1 domain of human Klotho exerts beneficial effects independently of other parts of the Klotho protein. This has been reported, for example, in Gupta et al. ("KL1 domain of longevity factor Klotho mimics the metabolome of cognitive stimulation and enhances cognition in young and aging mice." Journal of Neuroscience 42.19 (2022): 4016-4025). Therefore, if a Klotho polypeptide contains at least the KL1 domain of human Klotho, it can exert important beneficial effects.

[0040] In preferred embodiments, the Klotho polypeptide further comprises the signal peptide and / or KL2 domain of human Klotho. Preferably, the Klotho polypeptide comprises at least the KL1 and KL2 domains of human Klotho. Particularly preferred is that the Klotho polypeptide comprises the signal peptide, KL1 domain and KL2 domain of human Klotho.

[0041] In another preferred embodiment, the Klotho polypeptide further comprises the transmembrane (TM) domain of human Klotho. Preferably, the Klotho polypeptide further comprises the cytoplasmic tail (CT) of human Klotho. The presence of the TM domain, preferably the CT domain, offers the advantage that a membrane-anchored version of the Klotho protein can be delivered to the individual. This is a significant advantage over the recombinant protein administration strategies previously used for Klotho. When recombinant proteins are administered, only the soluble version of Klotho is available, thus providing access only to the soluble side of Klotho signaling. In contrast, by using mRNA, the membrane-anchored version of Klotho can be delivered, allowing access to the entire Klotho signaling domain.

[0042] As used herein, the terms “KL1 domain,” “KL2 domain,” and “signal peptide” in relation to human Klotho preferably refer to the KL1, KL2, and SP sequences relating to human Klotho isoform 1 (UniProt Q9UEF7-1; SEQ ID NO: 49) and human Klotho isoform 2 (UniProt Q9UEF7-2; SEQ ID NO: 50), or any variant thereof, particularly any of the variants described below. Preferably, the signal peptide is SEQ ID NO: 51, the KL1 domain is SEQ ID NO: 52, and the KL2 domain is SEQ ID NO: 54.

[0043] A functional variant of Klotho called "KL-VS" is found in certain segments of the human population (see, for example, Arking et al. “Association between a functional variant of the KLOTHO gene and high-density lipoprotein cholesterol, blood pressure, stroke, and longevity” Circulation Research 96.4 (2005): 412-418; Arking et al. “Association of human aging with a functional variant of Klotho” PNAS 99.2 (2002): 856-861). This variant contains two mutations in the KL1 domain, namely F325V and C370S. This variant has been associated with a reduction in overall mortality and other positive effects. Therefore, in a preferred embodiment, the KL1 domain of human Klotho is the VS variant. Preferably, the KL1 domain is Sequence ID No. 53.

[0044] Any other suitable variant of the SP, KL1 domain and / or KL2 domain of human Klotho may be advantageously used in the context of the present invention. Preferably, the Klotho polypeptide is a native Klotho protein, in particular a native human Klotho protein, in particular a native human Klotho isoform 1 or a native human Klotho isoform 2, in particular SEQ ID NO: 49 or 50.

[0045] It is more preferable that the Klotho polypeptide is a Klotho protein, protein fragment, and / or protein variant as described in International Publication No. 2019 / 113373, which is incorporated herein by reference. The Klotho polypeptide may comprise all or a subset of amino acid residues 1-1012, 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, or 131-549 of human Klotho isoform 1 or 2. Some embodiments may comprise a protein having one or more amino acid mutations compared to human Klotho isoform 1. Typically, the protein may comprise a human C370 variant. For example, the protein may comprise a C370S modification, thereby comprising S370. In some embodiments, the protein may comprise something other than human F352 or F352V. In at least one embodiment, the protein may include a C370S modification, thereby excluding the F352V variant and preferably including S370 containing F352. The protein may include variants other than H193 or H193R. All other standard amino acid substitutions at amino acid residues (or positions) 193, 352, and / or 370 of human Klotho isoform 1 are contemplated and expressly disclosed herein.

[0046] Some embodiments may involve a mutation at amino acid residue 45 of human Klotho isoform 1. The residue at position 45 may be valine (Val; V), phenylalanine (Phe; F), or another amino acid.

[0047] In some embodiments, the Klotho polypeptide may include a signal peptide or a signal sequence. For example, the protein may include a native Klotho signal sequence. The protein may include a non-native signal sequence. In some embodiments, the signal sequence may be an N-terminal signal sequence and / or upstream (or N-terminal) of the Klotho protein sequence. In other embodiments, the signal sequence may be located at the C-terminus or elsewhere. Preferably, the signal sequence is the SP of human Klotho shown in SEQ ID NO: 51.

[0048] Substituting phenylalanine for the valine at position 45 of human Klotho (V45F) has been reported to have advantages. For example, V45F soluble Klotho, and its fragments or fusion proteins, are expressed at higher levels than native wild-type V45 protein in CHO cells and HEK-293 cells, as reported in International Publication 2019 / 113373. Accordingly, some embodiments of this disclosure include Klotho polypeptides having the V45F substitution.

[0049] The mRNA used in the present invention contains (at least) five essential elements, all of which are known and available to those skilled in the art (in order from 5' to 3'): the 5' CAP region, the 5' untranslated region (5'-UTR), the coding region, the 3' untranslated region (3'-UTR), and the polyadenosine tail (poly-A tail).

[0050] The coding region, naturally, codes for a (human) Klotho polypeptide, and the other components may be a (native) Klotho UTR, or preferably another UTR. Particularly preferred UTRs according to the present invention are UTRs that improve the properties of the mRNA molecule according to the present invention, namely, UTRs that improve the above properties by facilitating better and / or longer and / or more effective translation of mRNA to the Klotho polypeptide at the administration site.

[0051] The "CAP region" ("5'CAP") refers to the structure found at the 5' end of an mRNA molecule, and generally consists of a guanosine nucleotide attached to the mRNA via a specific 5'-5' triphosphate linkage. This guanosine nucleotide is methylated at position 7 directly after capping in vivo by methyltransferase ("7-methylguanylate cap" ("m7G", "CAP-0")). Further modifications may involve methylation of the 2' hydroxyl groups of the first two ribose sugars at the 5' end of the mRNA (i.e., "CAP1" and "CAP2"): "CAP1" has a methylated 2'-hydroxyl group on the first ribose sugar, while "CAP2" has methylated 2'-hydroxyl groups on both the first two ribose sugars. Chemically, the 5' cap is similar to the 3' end of the RNA molecule (the carbon on the 5' side of the cap ribose is bonded, while the carbon on the 3' side is not). This results in significant resistance to 5' exonucleases and therefore in vivo stability. CAP analogs, including monomethylated CAP analogs (mCAP), anti-reverse CAP analogs (ARCA CAP), m7G(5')ppp(5')A RNA CAP structural analogs, G(5')ppp(5')A RNA CAP structural analogs, and G(5')ppp(5')G RNA CAP structural analogs, may also be used for mRNA generation according to the present invention.

[0052] The term "(5'- or 3'-)UTR" refers to the well-established concept of the untranslated region of mRNA in molecular genetics. There is one UTR on each side of the coding sequence on the mRNA strand. The 5'-side UTR is the 5'-UTR (or leader sequence), and the 3'-side UTR is the 3'-UTR (or trailer sequence). The 5'-UTR is located upstream of the coding sequence. Within the 5'-UTR is a sequence recognized by ribosomes, which bind to the ribosome and initiate translation. The mechanism of translation initiation differs between prokaryotes and eukaryotes. The 3'-UTR is found immediately after the translation stop codon. The 3'-UTR plays an important role not only in translation termination but also in post-transcriptional gene expression. The UTRs used in this invention typically provide beneficial stability and expression (translation) characteristics to the mRNA molecule according to the present invention.

[0053] A "poly(A) tail" is a naturally occurring part of mRNA consisting of multiple adenosine monophosphates and containing only adenine bases. This process, called "polyadenylation," is part of the process of generating mature messenger RNA (mRNA) for translation during gene expression. The natural process of polyadenylation begins when gene transcription is completed. The outermost 3' segment of the newly produced premRNA is first cleaved by a series of proteins, which then synthesize the poly(A) tail at the 3' end of the RNA. In some genes, these proteins attach the poly(A) tail to one of several possible sites. Thus, polyadenylation, like alternative splicing, can generate two or more transcripts from a single gene (alternative polyadenylation). The poly(A) tail is important for mRNA nuclear export, translation, and stability. Therefore, in this invention, the primary considerations for sufficient polyadenylation of the mRNA molecule according to the present invention are translational properties and stability. During the process of protein production, the tail shortens over time, and when it becomes sufficiently short, the mRNA is enzymatically degraded. The poly(A) tail according to the present invention is provided in a manner currently used and applied in the art of administering mRNA molecules in human therapy. For example, the poly(A) tail may be at least 60 adenosine monophosphate lengths. According to a preferred embodiment, the poly(A) tail is at least 100 adenosine monophosphate lengths, and in particular at least 120 adenosine monophosphate lengths. This enables excellent stability and protein production, but in terms of other characteristics, the action and activity of the mRNA molecule according to the present invention can also be modulated by the characteristics of the poly(A) tail.

[0054] As described in more detail above and as shown in the examples herein, it has been surprisingly found that in the context of the present invention, there is a direct relationship between the GC content of Klotho mRNA and the expression level of Klotho polypeptide. As used herein, the “GC content” of a particular mRNA or a particular portion of a particular mRNA refers to the sum of G and C bases relative to the total number of bases in the mRNA or the portion of the mRNA, and is expressed as a percentage. Thus, the GC content can be calculated as 100%*(C+G) / (A+C+G+U), where “C”, “G”, “A”, and “U” refer to the number of C-, G-, A-, and U- bases, respectively. In relation to the nucleoside variants disclosed below herein, it is important to note that the specific preferred variants described above do not affect the GC content; that is, the variants are conserved in this respect (for example, the cytidine variant is still counted as cytidine for the calculation of the GC content).

[0055] Therefore, in a preferred embodiment of the Klotho mRNA of the present invention, the coding region encoding the Klotho polypeptide has a GC content of at least 50%, preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, and more preferably at least 63%. It is even more preferable that the coding region encoding the Klotho polypeptide has a GC content of 50% to 74%, preferably 51% to 73%, more preferably 52% to 72%, more preferably 53% to 71%, more preferably 54% to 70%, more preferably 55% to 69%, more preferably 56% to 68%, more preferably 57% to 67%, more preferably 58% to 66%, more preferably 59% to 65%, more preferably 60% to 64%, and more preferably 61% to 63%.

[0056] In a preferred embodiment, the Klotho mRNA has a GC content of at least 50%, preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, and more preferably at least 63%. It is even more preferable that the Klotho mRNA has a GC content of 50% to 74%, preferably 51% to 73%, more preferably 52% to 72%, more preferably 53% to 71%, more preferably 54% to 70%, more preferably 55% to 69%, more preferably 56% to 68%, more preferably 57% to 67%, more preferably 58% to 66%, more preferably 59% to 65%, more preferably 60% to 64%, and more preferably 61% to 63%.

[0057] As described in more detail above, during the process of the present invention, it was found that the closer the Klotho mRNA has in sequence identity to the "GC62" variant produced during the process of the present invention, the higher its expression level. The sequence of the GC62 variant of the KL1 domain of native human Klotho, as shown in SEQ ID NO: 52, is shown in SEQ ID NO: 1. Therefore, in a preferred embodiment, the coding region encoding the Klotho polypeptide contains an RNA sequence having at least 80% sequence identity to SEQ ID NO: 1. Preferably, the sequence identity is at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8%. Preferably, the RNA sequence is SEQ ID NO: 1 or 3, particularly SEQ ID NO: 1.

[0058] In a preferred embodiment, the code region encoding the Klotho polypeptide includes SEQ ID NOs: 1, 2, 3, or 4, preferably SEQ ID NO: 1.

[0059] Sequence IDs 1 and 2 encode the KL1 domain of native human Klotho by Sequence ID 52, while Sequence IDs 3 and 4 encode the KL1 domain of the aforementioned VS variant by Sequence ID 53.

[0060] The sequence of the GC62 variant of the domain KL1-lr-KL2 of native human Klotho isoform 1 (amino acids L56 to G952 of SEQ ID NO: 49) is shown in SEQ ID NO: 9. Therefore, in a further preferred embodiment, the coding region encoding the Klotho polypeptide comprises an RNA sequence having sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% with respect to SEQ ID NO: 9 or 11, particularly SEQ ID NO: 9.

[0061] In a preferred embodiment, the coding region encoding the Klotho polypeptide includes SEQ ID NOs: 9, 10, 11, or 12, preferably SEQ ID NO: 9.

[0062] The sequence of the GC62 variant of the domain lr-KL1-lr-KL2-lr of native human Klotho isoform 1 (amino acids E34 to S981 of SEQ ID NO: 49) is shown in SEQ ID NO: 13. This sequence encodes the entire sequence of soluble Klotho isoform 1 except for the N-terminal SP. It should be noted that in the experiments mentioned above and described in more detail in the following examples and drawings, the beneficial effects of increased GC content and sequence identity for the "GC62" variant (SEQ ID NO: 18) were observed independently of the sequence of the signal peptide used. In fact, in comparative studies comparing four variants obtained from commercially available codon optimization algorithms (GENEius algorithm, IDT Codon Optimization Tool, ExpOptimizer, Twist Codon Optimization Tool), and the variants "GC53," "GC55," "GC57," "GC60," and "GC62," the same SP sequence was used, and only the remaining portion of the mRNA construct (corresponding to the domain lr-KL1-lr-KL2-lr; nucleotides 100-2946) was optimized. Therefore, the advantageous effect of high sequence similarity with the "GC62" variant was independent of the specific SP sequence used.Therefore, in a further preferred embodiment, the coding region encoding the Klotho polypeptide includes an RNA sequence having at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% sequence identity with respect to SEQ ID NO: 13 or 15, particularly SEQ ID NO: 13.

[0063] In a preferred embodiment, the code region encoding the Klotho polypeptide includes SEQ ID NOs: 13, 14, 15, or 16, preferably SEQ ID NO: 13.

[0064] The sequence of the GC62 variant of the domain lr-KL1-lr-KL2-lr-TM-CT of native human Klotho isoform 1 (amino acids E34 to K1012 in SEQ ID NO: 49) is shown in SEQ ID NO: 64. This sequence encodes the entire sequence of Klotho isoform 1 except for the N-terminal SP, but includes the transmembrane domain and cytoplasmic tail. This is particularly advantageous because, by combining it with any suitable signal peptide (native or non-native), it becomes possible to deliver a membrane-anchored version of the Klotho protein, making it possible to utilize not only the soluble portion of Klotho signaling but its entire region. Therefore, in a further preferred embodiment, the coding region encoding the Klotho polypeptide comprises an RNA sequence having at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% sequence identity with respect to SEQ ID NO: 64 or 66, particularly SEQ ID NO: 64.

[0065] In a preferred embodiment, the coding region encoding the Klotho polypeptide includes SEQ ID NOs. 64, 65, 66, or 67, preferably SEQ ID NO. 64.

[0066] The sequence of the GC62 variant of fully soluble native human Klotho isoform 1 (amino acids M1 to S981 in SEQ ID NO: 49; corresponding to SEQ ID NO: 57) is shown in SEQ ID NO: 18. This sequence was also used in the context of the comparative experiments mentioned above and is described in more detail in the following examples and drawings. Therefore, in a further preferred embodiment, the coding region encoding the Klotho polypeptide comprises an RNA sequence having at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% sequence identity with respect to SEQ ID NO: 18 or 20, particularly SEQ ID NO: 18.

[0067] In a more preferred embodiment, the coding region encoding the Klotho polypeptide includes SEQ ID NOs: 18, 19, 20, or 21, preferably SEQ ID NO: 18.

[0068] The sequence of the GC62 variant of the fully native human Klotho isoform 1 (encoding the entire amino acid sequence of SEQ ID NO: 49) is shown in SEQ ID NO: 68. As explained above with respect to SEQ ID NO: 64, this sequence also has the advantage of being able to deliver a membrane-anchored version of the Klotho protein, making it possible to utilize not only the soluble portion of the Klotho signaling pathway but its entire region. Therefore, in a more preferred embodiment, the coding region encoding the Klotho polypeptide includes an RNA sequence having at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% sequence identity with respect to SEQ ID NO: 68 or 70, particularly SEQ ID NO: 68.

[0069] In a preferred embodiment, the coding region encoding the Klotho polypeptide includes SEQ ID NOs. 68, 69, 70, or 71, preferably SEQ ID NO. 64.

[0070] The sequence of the GC62 variant of the KL1 terminal domain of native human Klotho isoform 2 (amino acids L56 to H549 of SEQ ID NO: 50) is shown in SEQ ID NO: 33. Therefore, in a preferred embodiment, the coding region encoding the Klotho polypeptide comprises an RNA sequence having at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% sequence identity with respect to SEQ ID NO: 33, preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8%, wherein the RNA sequence is preferably SEQ ID NO: 33 or 35, particularly SEQ ID NO: 33.

[0071] In a more preferred embodiment, the code region encoding the Klotho polypeptide includes SEQ ID NOs: 33, 34, 35, or 36, preferably SEQ ID NO: 33.

[0072] The sequence of the GC62 variant of the lr-KL1 terminal domain of native human Klotho isoform 2 (amino acids E34 to H549 in SEQ ID NO: 50) is shown in SEQ ID NO: 33. This sequence encodes the entire sequence of soluble Klotho isoform 2 except for the N-terminal SP. Therefore, in a more preferred embodiment, the coding region encoding the Klotho polypeptide comprises an RNA sequence having at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% sequence identity with respect to SEQ ID NO: 37 or 39, particularly SEQ ID NO: 37.

[0073] In a more preferred embodiment, the coding region encoding the Klotho polypeptide includes SEQ ID NOs: 37, 38, 39, or 40, preferably SEQ ID NO: 37.

[0074] The sequence of the GC62 variant of fully soluble native human Klotho isoform 2 (amino acid sequence SEQ ID NO: 50) is shown in SEQ ID NO: 41. Therefore, in a more preferred embodiment, the coding region encoding the Klotho polypeptide comprises an RNA sequence having at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% sequence identity with respect to SEQ ID NO: 41, preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8%, wherein the RNA sequence is preferably SEQ ID NO: 41 or 43, particularly SEQ ID NO: 41.

[0075] In a preferred embodiment, the coding region encoding the Klotho polypeptide includes SEQ ID NOs: 41, 42, 43, or 44, preferably SEQ ID NO: 41.

[0076] In a particularly preferred embodiment, the coding region encoding the Klotho polypeptide has sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably 100% with respect to sequence number 5. It is particularly preferred that the coding region encoding the Klotho polypeptide is sequence number 5, 6, 7, or 8.

[0077] In a further particularly preferred embodiment, the coding region encoding the Klotho polypeptide has sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably 100% with respect to sequence number 18. It is particularly preferred that the coding region encoding the Klotho polypeptide is sequence number 18, 19, 20, or 21. In a further embodiment, the Klotho polypeptide is sequence number 22, 23, or 24.

[0078] In a particularly preferred embodiment, the coding region encoding the Klotho polypeptide has sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably 100% with respect to sequence number 68, 69, 70, or 71.

[0079] In a particularly preferred embodiment, the coding region encoding the Klotho polypeptide has sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably 100% with respect to sequence number 41, 42, 43, or 44.

[0080] In the context of this invention, as described above, it is preferable that the Klotho polypeptide comprises the signal peptide, KL1 domain and / or KL2 domain of human Klotho.

[0081] In a preferred embodiment, the KL1 domain of the Klotho polypeptide has sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 99.5% with respect to SEQ ID NO: 52, and preferably the KL1 domain of the Klotho polypeptide is SEQ ID NO: 52 or 53, particularly SEQ ID NO: 52.

[0082] In a further preferred embodiment, the KL2 domain of the Klotho polypeptide has sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 99.5% with respect to SEQ ID NO: 54, and preferably the KL2 domain of the Klotho polypeptide is SEQ ID NO: 54.

[0083] In a further preferred embodiment, the signal peptide of the Klotho polypeptide has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 99.5% sequence identity with respect to SEQ ID NO: 51, in particular, the signal peptide of the Klotho polypeptide is SEQ ID NO: 51.

[0084] A combination of the above embodiments relating to the SP, KL1 domain, and KL2 domain is particularly preferred. For example, it is preferred that the Klotho polypeptide comprises a KL1 domain of the Klotho polypeptide having at least 80% sequence identity to SEQ ID NO: 52, a KL2 domain of the Klotho polypeptide having at least 80% sequence identity to SEQ ID NO: 54, and / or a signal peptide of the Klotho polypeptide having at least 80% sequence identity to SEQ ID NO: 51.

[0085] In a further preferred embodiment, the Klotho polypeptide has sequence identity of at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably 100% with respect to SEQ ID NO: 55. It is particularly preferred that the Klotho polypeptide is SEQ ID NO: 55 or 56, preferably 55.

[0086] In a further preferred embodiment, the Klotho polypeptide has sequence identity of at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably 100% with respect to SEQ ID NO: 57. It is particularly preferred that the Klotho polypeptide is SEQ ID NO: 57 or 58, preferably 57.

[0087] In a further preferred embodiment, the Klotho polypeptide has sequence identity of at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably 100% with respect to SEQ ID NO: 49. It is particularly preferred that the Klotho polypeptide is SEQ ID NO: 49 or 76, preferably 49.

[0088] In a further preferred embodiment, the Klotho polypeptide has sequence identity of at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably 100% with respect to SEQ ID NO: 59. It is particularly preferred that the Klotho polypeptide is SEQ ID NO: 59 or 60, preferably 59.

[0089] In the context of the present invention, several specific sequences for 5'-UTR and 3'-UTR have been found to be particularly advantageous for achieving particularly high expression levels.

[0090] Specifically, for the 5'-UTR, the sequence of SEQ ID NO: 61 was found to be advantageous. Therefore, in a preferred embodiment of Klotho mRNA, the 5'-UTR has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably 100% sequence identity with respect to SEQ ID NO: 61. It is particularly preferred that the 5'-UTR is SEQ ID NO: 61.

[0091] Similarly, for the 3'-UTR, the sequence of sequence number 62 was found to be particularly advantageous. Therefore, in a preferred embodiment, the 3'-UTR has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably 100% sequence identity with respect to sequence number 62. It is particularly preferred that the 3'-UTR is sequence number 62.

[0092] According to a preferred embodiment of the present invention, the mRNA of the present invention comprises one or more stabilizing sequences in the 5'-UTR and / or 3'-UTR (preferably in the 3'UTR) that can increase the intracellular half-life of the mRNA. These stabilizing sequences may exhibit 100% sequence homology to naturally occurring sequences present in viruses, bacteria, and eukaryotic cells, but may be partially or completely synthetic. Examples of such stabilizing sequences are described in Nucleic Acids Res. 2010; 38 (Database issue): D75-D80. UTRdb and UTRsite (RELEASE 2010): a collection of sequences and regulatory motifs of the untranslated regions of eukaryotic mRNAs and under http: / / utrdb.ba.itb.cnr.it / .

[0093] In one embodiment of Klotho mRNA, the 5'-UTR or 3'-UTR or 5'-UTR and 3'-UTR correspond to native human Klotho mRNA, preferably the native human Klotho mRNA described in NCBI Reference Sequence:NM_004795.4. Alternatively, in another preferred embodiment, the 5'-UTR or 3'-UTR or 5'-UTR and 3'-UTR are different from native human Klotho mRNA.

[0094] Further examples of stabilizing sequences that may be used in the present invention include, for example, the untranslated sequence (UTR) of the β-globin gene of Homo sapiens or Xenopus laevis.

[0095] Another example of a stabilizing sequence is described by Holcik et al. (Proc. Natl. Acad. Sci. USA 1997, 94: 2410 to 2414), with the general formula: (C / U)CCAN x CCC(U / A)Py x UC(C / U)CC(Sequence ID 63) It has, This is found, for example, in the 3'UTR of alpha(1)-collagen or alpha-globin, and highly stable mRNA encoding ALOX15 or tyrosine hydroxylase (where "x" is (N) x and Py x Independently in each case, integers from 0 to 10, preferably integers from 0 to 5 (Holcik et al., 1997), particularly 0, 1, 2, 4 and / or 5).

[0096] Such stabilizing sequences can be used individually or in combination with each other to stabilize the mRNA of the present invention, as well as in combination with other stabilizing sequences known to those skilled in the art. For example, the stabilizing effect of the human β-globin 3'-UTR sequence is further enhanced by arranging two human β-globin 3'-UTRs in a head-to-tail direction.

[0097] Therefore, a preferred embodiment of the Klotho mRNA according to the present invention is an mRNA molecule in which the 5'-UTR or 3'-UTR or 5'-UTR and 3'-UTR are different from native Klotho mRNA, preferably the 5'-UTR or 3'-UTR or 5'-UTR and 3'-UTR are at least one stabilizing sequence, preferably of the general formula (C / U)CCAN. x CCC(U / A)Py x This mRNA molecule contains a stabilizing sequence with UC(C / U)CC (SEQ ID NO: 38).

[0098] Preferably, the 5'-UTR and / or 3'-UTR are 5'-UTR and / or 3'-UTR of human mRNA different from Klotho, and are preferably selected from alphaglobin, betaglobin, albumin, lipoxygenase, ALOX15, alpha(1) collagen, tyrosine hydroxylase, ribosomal protein 32L, eukaryotic elongation factor 1a (EEF1A1), 5'-UTR elements present in orthopoxviruses, and mixtures thereof, in particular selected from alphaglobin, betaglobin, alpha(1) collagen, and mixtures thereof.

[0099] Therefore, the present invention preferably relates to mRNA containing one or more stabilizing sequences in the 3'-UTR that can increase the half-life of mRNA in the cytoplasm. These stabilizing sequences may show 100% sequence homology to naturally occurring sequences found in viruses, bacteria, and eukaryotic cells, but may also be partially or completely synthesized sequences. An example of a stabilizing sequence that may be used in the present invention is, for example, the untranslated sequence (UTR) of the β-globin gene of Homo sapiens or African clawed frog. As already mentioned, another example of a stabilizing sequence is the general formula (C / U)CCAN x CCC(U / A)Py xThe UC(C / U)CC is present in the 3'-UTR of highly stable mRNA encoding alpha-globin, alpha-(1)-collagen, 15-lipoxygenase, or tyrosine hydroxylase (see Holcik et al., Proc. Natl. Acad. Sci. USA 1997, 94: 2410 to 2414). Such stabilizing sequences can be used individually or in combination with each other to stabilize the modified mRNA of the present invention, as well as in combination with other stabilizing sequences known to those skilled in the art.

[0100] Another preferred embodiment of the present invention is a 5'-TOP-UTR derived from ribosomal protein 32L followed by a stabilizing sequence derived from albumin-3'-UTR.

[0101] Therefore, a preferred embodiment of the Klotho mRNA according to the present invention is an mRNA molecule containing a tract of multiple adenosine monophosphates at the 3' end of the 3'-UTR. This so-called polyadenosine (polyA) tail consists of at least 60 adenosine monophosphates, preferably at least 100, and most preferably at least 120.

[0102] In some cases, it may be desirable to destabilize mRNA to limit the duration of protein production. This effect can be achieved by incorporating destabilizing elemental sequences (DSEs), such as AU-rich elements, into the 3'-UTR, which rapidly degrades the mRNA and shortens the protein expression period.

[0103] In certain embodiments, it may be desirable to provide mRNA that does not contain destabilizing sequence elements (DSEs) in the 3' and / or 5' UTR, but there may be other embodiments in which the presence or introduction of such DSEs is advantageous. Generally, "DSE" refers to a sequence that reduces the half-life of a transcript, for example, in cells and / or organisms, for example, in the body of a human patient, according to the present invention. Thus, a DSE includes a sequence of nucleotides that reduces the intracellular half-life of an RNA transcript.

[0104] DSE sequences are found in short-lived mRNAs such as c-fos, c-jun, c-myc, GM-CSF, IL-3, TNF-alpha, IL-2, IL-6, IL-8, IL-10, urokinase, bcl-2, SGL T1 (Na(+)-bound glucose transporter), Cox-2 (cyclooxygenase 2), PAI-2 (plasminogen activator inhibitor type 2), beta(1)-adrenergic receptor, or GAP43 (5'-UTR and 3'-UTR).

[0105] Further DSEs are AU-rich elements (ARE) and / or U-rich elements (URE), including single copies, tandem copies, multiple copies, or duplicate copies of the nonomer UUAUUUA(U / A)(U / A) (where U / A is either A or U) and / or the pentamer AUUUA and / or the tetramer AUUU. Further DSEs are described in Nucleic Acids Res. 2010; 38 (Database issue): D75-D80. UTRdb and UTRsite (RELEASE 2010): a collection of sequences and regulatory motifs of the untranslated regions of eukaryotic mRNAs and under http: / / utrdb.ba.itb.cnr.it / .

[0106] Therefore, it is also preferable that the 5'-UTR or 3'-UTR or 5'-UTR and 3'-UTR contain at least one destabilizing sequence element (DSE), preferably an AU-rich element (ARE) and / or a U-rich element (URE), in particular a single copy, tandem copy, multiple copies, or duplicate copies of a nonamer UUAUUUA(U / A)(U / A), such as a pentamer AUUUA and / or a tetramer AUUU (where the term "U / A" means either A or U).

[0107] These stabilizing and destabilizing elements can be used alone or in combination to target a predetermined duration of protein production and to personalize the treatment of the present invention for localized skin malnutrition, particularly atrophic skin conditions, the severity of affected skin, and / or specific patient groups.

[0108] General concepts for improving mRNA-based therapies (see, for example, Sahin et al., Nat. Rev. Drug Disc. 2014. 13(10): 759-780) are also applicable to the present invention.

[0109] In most cases, the use of canonical nucleotides alone is preferred, but there may be certain occasions in which the Klotho mRNA according to the present invention contains other monophosphate nucleosides other than cytidine (C), uridine (U), adenosine (A), or guanosine (G) residues (canonical nucleotides). There are (further) numerous naturally occurring analogues of these monophosphate nucleosides and synthetic variants of these mRNA residues. Embodiments of such modifications can be found, for example, in International Publication No. 2014 / 153052.

[0110] According to one embodiment, in the Klotho mRNA of the present invention, - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all cytidine residues are replaced with 5-methylcytidine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all cytidine residues are replaced with 2-amino-2-deoxycytidine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all cytidine residues are replaced with 2-fluoro-2-deoxycytidine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all cytidine residues are replaced with 2-thiocytidine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all cytidine residues are replaced with 5-iodocytidine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all uridine residues are replaced with pseudouridine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all uridine residues are replaced with 1-methyl-pseudridine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all uridine residues are replaced with 2-thiouridine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all uridine residues are replaced with 5-methyluridine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all adenosine residues are replaced with N6-methyladenosine residues.

[0111] A specific embodiment is Klotho mRNA, and in Klotho mRNA, - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all cytidine residues are replaced with 5-methylcytidine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all uridine residues are replaced with pseudouridine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all uridine residues are replaced with 2-thiouridine residues.

[0112] In a further preferred embodiment of Klotho mRNA, the coding region has a codon adaptation index (CAI) of at least 0.70, preferably at least 0.75, more preferably at least 0.80, more preferably at least 0.85, more preferably at least 0.86, more preferably at least 0.87, more preferably at least 0.88, more preferably at least 0.89, more preferably at least 0.90, more preferably at least 0.91, more preferably at least 0.92, more preferably at least 0.93, more preferably at least 0.94, more preferably at least 0.95, more preferably at least 0.96, more preferably at least 0.97, more preferably at least 0.98, more preferably at least 0.99, and more preferably 1.00. Surprisingly, however, lowering the CAI may also be effective in improving the expression level. Therefore, it is preferable for the coding region to have a codon adaptation index (CAI) of less than 1.00, preferably less than 0.99, more preferably less than 0.98, more preferably less than 0.97, more preferably less than 0.96, and more preferably less than 0.95.

[0113] CAI is a measure of the relative fitness of a gene's codon use relative to the codon use of a highly expressed gene. The relative fitness (w) of each codon is the ratio of its use to the most frequently used codon for the same amino acid. The CAI index is defined as the geometric mean of these relative fitness values. Non-synonymous codons and stop codons (dependent on the genetic code) are excluded. CAI values ​​range from 0 to 1, with higher values ​​indicating a higher proportion of the most abundant codon (Sharp et al., Nucleic Acids Res. 15 (1987): 1281-1295, Jansen et al., Nucleic Acids Res. 31 (2003): 2242-2251).

[0114] In a particularly preferred embodiment of the Klotho mRNA, the Klotho mRNA has sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably 100% with respect to SEQ ID NO: 29, 30, 31, or 32, preferably SEQ ID NO: 29.

[0115] In a further particularly preferred embodiment of the Klotho mRNA, the Klotho mRNA has sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably 100% with respect to SEQ ID NO: 72, 73, 74, or 75, preferably SEQ ID NO: 72.

[0116] In a further particularly preferred embodiment of the Klotho mRNA, the Klotho mRNA has sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably 100% with respect to SEQ ID NO: 45, 46, 47, or 48, preferably SEQ ID NO: 45.

[0117] In the course of this invention, it was found that minor modifications to the above sequences could make them more useful and manageable. More specifically, it was found that by replacing a small number of nucleotides, restriction enzyme recognition sequences could be removed, making these sequences easier to handle for molecular cloning. Therefore, based on sequence numbers 1-21, 29-48, and 64-75 of the above sequences, modified sequences containing up to five modified bases were prepared to remove a specific restriction site. These modifications did not significantly affect the expression levels achieved in the construct. The modified sequences and their corresponding underlying sequences are shown in the table below:

[0118] [Table 3]

[0119] An overview of these sequences is also shown in Table 3B below.

[0120] All embodiments described herein, including the original SEQ ID NOs: 1-21, 29-48, and 64-75, are also preferred with respect to modified sequences 79-131. For example, if, as specified herein, the coding region encoding the Klotho polypeptide contains an RNA sequence having at least 80% sequence identity to SEQ ID NO: 1, it is equally preferred that the coding region encoding the Klotho polypeptide also contains an RNA sequence having at least 80% sequence identity to SEQ ID NO: 79. Similarly, if the coding region encoding the Klotho polypeptide contains SEQ ID NO: 13, it is equally preferred that the coding region also contains SEQ ID NO: 91.

[0121] In another embodiment, the present invention relates to a pharmaceutical formulation comprising Klotho mRNA as described in any one of the above embodiments.

[0122] Appropriate formulations for mRNA therapeutics are readily available in the art (see, for example, Sahin et al., 2014; International Publication No. 2014 / 153052 (paragraphs 122-136)).

[0123] Accordingly, the present invention also relates to pharmaceutical formulations comprising Klotho mRNA according to the present invention. The formulations of the present invention preferably contain mRNA in a pharmaceutically acceptable environment, for example, together with appropriate components (excipients, carriers, buffers, auxiliary substances (e.g., stabilizers), etc.) that are typically provided in mRNA therapeutics.

[0124] The mRNA formulation according to the present invention preferably contains a suitable carrier. Suitable carriers include polymer-based carriers, such as cationic polymers containing linear and branched PEIs and vilomers, lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, cationic amphiphilic lipids, such as SAINT®-lipids, natural and synthetic exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticles, calcium phosphate nanoparticles, calcium phosphate nanoparticles, silicon dioxide nanoparticles, nanocrystalline particles, semiconductor nanoparticles, dried powders, poly(D-arginine), nanodendrimers, starch-based delivery systems, micelles, emulsions, sol-gels, niosomes, plasmids, viruses, calcium phosphate nucleotides, aptamers, peptides, peptide conjugates, small molecular weight targeted conjugates, polylactic acid-coglycolic acid (PLGA) polymers, and other vector tags. Bionanopapillaries and assemblies of other viral capsid proteins may also be used as suitable carriers. (Hum. Gene Ther. 2008 Sep;19(9):887-95).

[0125] Preferred carriers include cationic polymers containing linear and branched PEIs and vilomers, lipid nanoparticles and liposomes, transfersomes, and nanoparticles containing calcium phosphate nanoparticles (i.e., naked RNA is administered after precipitation with CaCl2).

[0126] A preferred embodiment of the present invention relates to the use of uncomplexed mRNA, i.e., uncomplexed mRNA in a suitable aqueous buffer, preferably a physiological glucose-buffered aqueous solution (physiological). For example, 1×HEPES buffer; 1×phosphate buffer; sodium citrate buffer; sodium acetate buffer; Ringer's lactate solution; preferably in combination with glucose (e.g., 5% glucose); physiological solutions may be applied.

[0127] Preferably, the present invention applies liposomes, particularly DOTAP, DOTMA, Dotap-DOPE, DOTAP-DSPE, Dotap-DSPE-PEG, Dotap-DOPE-PEG, Dotap-DSPE-PEG-Na-Cholate, Dotap-DOPE-PEG-Na-Cholate, DOTAP having a cationic amphiphilic polymer (CAM) as a complex, and liposomes based on combinations thereof.

[0128] In preferred embodiments of the pharmaceutical formulation of the present invention, the formulation comprises further mRNA, the further mRNA having further 5' CAP region, further 5'-UTR, further coding region encoding further polypeptide, further 3'-UTR and further poly-A tail.

[0129] Preferably, further polypeptides, when expressed in the cells of an organism, provide further beneficial effects. It is particularly preferable that the further polypeptides have an additive, preferably synergistic, effect with the Klotho polypeptide. Preferably, the further polypeptides are anti-aging peptides or anti-aging proteins, i.e., any peptide or protein known to those skilled in the art to have anti-aging effects. Alternatively, or in addition, it is preferable that the further polypeptides have therapeutic effects, preferably therapeutic effects related to any of the therapeutic indications described herein.

[0130] In preferred embodiments, further polypeptides are bactericidal / permeability-enhancing protein family B, member 4 (BPIFB4) protein, or its isoforms or variants, preferably longevity-associated variants of BPIFB4 (LAV-BPIFB4). Preferably, BPIFB4 is as described in UniProt Entry P59827-1 or P59827-2, particularly P59827-1 (sequence version 2). Preferably, the LAV- variant is as described in UniProt Entry P59827-1 or P59827-2, particularly P59827-1 (sequence version 2), with the following amino acids substituted: Ile229Val, Asn281Thr, Leu488Phe, and Ile494Thr.

[0131] In a preferred embodiment, BPIFB4 has at least one, preferably at least two, more preferably at least three mutations selected from Ile229Val, Asn281Thr, Leu488Phe, and Ile494Thr; preferably, BPIFB4 has all of the named mutations.

[0132] In a further preferred embodiment, the further polypeptide is the BPIFB4 protein or its isoforms or variants described in International Publication No. 2014 / 102343 or International Publication No. 2019 / 034723 (both incorporated herein by reference).

[0133] In a further preferred embodiment, the additional polypeptide is the protein described in Bin-Jumah et al. “Genes and longevity of lifespan.” International Journal of Molecular Sciences 23.3 (2022): 1499. In particular, further polypeptides include apolipoprotein E (preferably gene symbol: APOE), tumor protein p53 (preferably gene symbol: P53), sirtuin 1 protein (preferably gene symbol: SIRT1), FOXO1 transcription factor (preferably gene symbol: DAF-16), cholinergic receptor nicotin alpha 3 subunit (preferably gene symbol: CHRNA3), SH2B adapter protein 3 (preferably gene symbol: SH2B3), cyclin-dependent kinase inhibitor 2A (preferably gene symbol: CDKN2A), elongation of very-long-chain fatty acids-like protein 2 (preferably gene symbol: ELOVL2), Werner protein (preferably gene symbol: WRN), paraoxonase 1 (preferably gene symbol: PON1), superoxide dismutase 2 (preferably gene symbol: SOD2), lamin A protein (preferably gene symbol: LMNA), and cholesterol ester transfer protein.Protein (preferably gene symbol: CETP), apolipoprotein C3 (preferably gene symbol: APOC3), microsomal triglyceride transfer protein (preferably gene symbol: MTP), phosphatidylinositol 3-kinase (PI3K) (preferably gene symbol: PIK3CA), insulin-like growth factor 1 (IGF-1) receptor (preferably gene symbol: DAF-2), protein-L-isoaspartyl methyltransferase (preferably gene symbol: PIMT), growth hormone (preferably gene symbol: GH1), cAMP response element binding protein (preferably gene symbol: CREB), mitogen-activated protein kinase (preferably gene symbol: MAPK), epidermal growth factor receptor (preferably gene symbol: EGFR), nuclear factor kappa B (preferably gene symbol: NF-κB), phospholipase C beta (preferably gene symbol: PLC-β), methionine sulfoxide reductase A (preferably gene symbol: MSR-A), mediator of cell motility 1) (preferably gene symbol: MEMO1), Nei-like DNA glycosylase 1 (preferably gene symbol: NEIL1), peroxisome proliferator-activated receptor gamma 2 (preferably gene symbol: PPARγ2), eukaryotic translation initiation factor 3 subunit K (preferably gene symbol: EIF3K), ATM serine / threonine kinase (preferably gene symbol: ATM), B-cell lymphoma 2 (preferably gene symbol: BCL2), cell division cycle 42 (preferably gene symbol: CDC42), diacylglycerol O-acyltransferase 1 (preferably gene symbol: DGAT1), early growth response 11) (preferably gene symbol: EGR1), fibroblast growth factor 23 (preferably gene symbol: FGF23), fibroblast growth factor 21 (preferably gene symbol: FGF21), fructosamine 3 kinase-related protein (preferably gene symbol: FN3KRP), phosphoglycolate phosphatase (preferably gene symbol: PGP), insulin receptor substrate 1 (preferably gene symbol: IRS1), Polycomb complex protein BMI-1 (preferably gene symbol: BMI1), neuregulin 1 (preferably gene symbol: NRG-1), signal transduction and transcription activator (preferably gene symbol: STAT), E2F transcription factor 1 (preferably gene symbol: E2F1), vascular endothelial growth factor A (preferably gene symbol: VEGF-A), xenobiotic metabolizing enzymes Preferably, the proteins are enzymes (preferably gene symbol: XME), Myc proto-oncoprotein (preferably gene symbol: MYC), CXC chemokine receptor type 4 (preferably gene symbol: CXCR4), Silent information regulator 2 (preferably gene symbol: SIR-2), extracellular signal-regulated kinase (preferably gene symbol: ERK), and SLC31 (preferably gene symbol: SLC31). For each protein, the human native version is preferred. All isoforms and / or variants of these proteins are included. If multiple isoforms or variants exist, the most common isoform or variant in the human population is preferred.

[0134] In another embodiment, the present invention is a kit for administering Klotho mRNA to an individual as described in any one of the above embodiments, - Klotho mRNA according to any one of the embodiments described above, and - Device for administering Klotho mRNA Regarding the kit that includes this.

[0135] Preferably, the individual is an animal, preferably a human or a non-human animal. It is particularly preferable that the individual is a mammal, especially a human.

[0136] In preferred embodiments of the kit of the present invention, the device is for intravenous, intramuscular, subcutaneous, intradermal, transdermal, epidermal, or topical administration.

[0137] Preferably, the device for administering Klotho mRNA is a syringe, a needle, an auto-injector, and / or a needle-free injection system; preferably, the device is for intravenous and / or intramuscular injection. It is particularly preferable that the device for administering Klotho mRNA is a skin delivery device. In preferred embodiments, the skin delivery device is an intradermal delivery device, preferably selected from the group consisting of needle-based injection systems. For example, the skin delivery device may be a transdermal delivery device, preferably selected from the group consisting of transdermal patches, hollow and solid microneedle systems, microstructured transdermal systems, electrophoresis systems, and iontophoresis systems. The skin delivery device may also be an epidermal delivery device, preferably selected from the group consisting of needle-free injection systems, laser-based systems, in particular Erbium YAG laser systems, and gene gun systems.

[0138] These administration devices are, in principle, available in the art; and their adaptation for mRNA administration according to the present invention is readily possible for those skilled in the art.

[0139] A further aspect of the present invention relates to Klotho mRNA for use as a pharmaceutical.

[0140] The therapeutic use of Klotho is known in the art. The present invention provides a particularly advantageous method for bringing about these therapeutic effects in individuals who need it. The therapeutic use of Klotho is described, for example, in Prud'homme et al. “Pathobiology of the Klotho Antiaging Protein and Therapeutic Considerations.” Frontiers in Aging 3 (2022); and Sachdeva, et al. “Klotho and the treatment of human malignancies.” Cancers 12.6 (2020): 1665.

[0141] Accordingly, in one embodiment, the present invention relates to Klotho mRNA or pharmaceutical compositions of the present invention for use in the prevention or treatment of diseases selected from the group consisting of kidney diseases, cardiovascular diseases, brain diseases, lung diseases, bone diseases, and metabolic diseases.

[0142] In another aspect, the present invention relates to Klotho mRNA or pharmaceutical compositions of the present invention for use in the prevention or treatment of kidney diseases, preferably diseases selected from the group consisting of chronic kidney disease, fibrosis, hyperphosphatemia, ischemic injury, nephrectomy, toxic injury, diabetic nephropathy, and calcific proteinosis.

[0143] In another aspect, the present invention relates to the Klotho mRNA or pharmaceutical compositions of the present invention for use in the prevention or treatment of cardiovascular diseases, preferably diseases selected from the group consisting of arterial / aortic calcification, atherosclerosis, cardiomyopathy, cardiac hypertrophy, hypertension, and myocardial ischemic injury / infarction.

[0144] In another aspect, the present invention relates to Klotho mRNA or pharmaceutical compositions of the present invention for use in the prevention or treatment of brain diseases, preferably diseases selected from the group consisting of Alzheimer's disease, hippocampal neuronal loss, cognitive decline, and frailty.

[0145] In another aspect, the present invention relates to Klotho mRNA or pharmaceutical compositions of the present invention for use in the prevention or treatment of cancer, preferably cancers selected from the group consisting of colorectal cancer, esophageal cancer, gastric cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, thyroid cancer, melanoma, kidney cancer, and cervical cancer.

[0146] In another aspect, the present invention relates to Klotho mRNA or pharmaceutical compositions of the present invention for use in the prevention or treatment of lung diseases, preferably pulmonary fibrosis or chronic obstructive pulmonary disease.

[0147] In another aspect, the present invention relates to Klotho mRNA or pharmaceutical compositions of the present invention for use in the prevention or treatment of bone diseases, preferably osteoporosis or osteomalacia, and in particular osteomalacia resulting from chronic kidney disease.

[0148] In another aspect, the present invention relates to Klotho mRNA or pharmaceutical compositions of the present invention for use in the prevention or treatment of metabolic diseases, preferably diabetes, particularly type 1 diabetes and / or type 2 diabetes, pancreatic β-cell apoptosis, autoimmune disorders, and inflammation, particularly infertile inflammation.

[0149] In relation to all embodiments described herein (both therapeutic and non-therapeutic uses as described herein), it is preferable that the Klotho mRNA or compositions of the present invention be administered intravenously, intramuscularly, subcutaneously, transdermally, epidermally, or topically, particularly epidermally.

[0150] The administration of Klotho mRNA or a pharmaceutical composition containing Klotho mRNA can be carried out according to an optimized expression objective, depending on the amount of mRNA applied, the stability of the mRNA molecule, and the disease state. For example, Klotho mRNA / pharmaceutical composition may be administered at least once, at least twice, at least twice within one month, preferably weekly. For example, if Klotho mRNA / pharmaceutical composition may be administered at least twice, at least twice within one month, preferably weekly, the dose may vary.

[0151] Furthermore, the amount of mRNA delivered in a single dose may depend on the molecular stability, etc. Preferably, the Klotho mRNA / pharmaceutical composition is administered in an amount of 0.01 μg to 100 mg per dose, preferably 0.1 μg to 10 mg per dose, and particularly 1 μg to 1 mg per dose.

[0152] The present invention also relates to a method for preventing or treating a disease in an individual in need thereof, comprising administering an effective amount of the Klotho mRNA or pharmaceutical composition of the present invention to the individual. All features and preferred embodiments described herein with respect to the Klotho mRNA or pharmaceutical composition for use are also preferred embodiments of a method for treatment, for example, with respect to the disease to be prevented or treated, the administration regime, the method of administration, etc.

[0153] In another aspect, the present invention relates to the Klotho mRNA or pharmaceutical composition of the present invention for use in the prevention or treatment of aging-related disorders and / or symptoms of aging. Preferably, age-related disorders or symptoms include actinic keratosis, age-related macular degeneration (AMD), Alzheimer's disease, arthritis, atherosclerosis and cardiovascular disease, benign prostatic hyperplasia (BPH), bone atrophy, cachexia, cancer, cardiomyopathy, cataracts, chronic obstructive pulmonary disease (COPD), constipation, decreased total energy, decreased vision, delirium, dementia, depression, skin atrophy (thinning of the skin), decreased peripheral vision, increased risk of heatstroke or hypothermia, hearing loss, hypertension, increased susceptibility to infections (including influenza and pneumonia), moles (age spots), liver conditions (e.g., non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and cirrhosis), memory loss, metabolic syndrome, muscle atrophy (e.g., sarcopenia and myopenia), frailty, muscle repair or rejuvenation. One or more of the following: deficiency of cereals, muscular dystrophy, osteoarthritis, osteoporosis, periodontitis, photoaging, decreased metabolism (including increased risk of obesity), decreased reflexes and coordination (including impaired balance), respiratory diseases (including acute respiratory distress syndrome (ARDS) with or without acute lung injury (ALI) and pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis)), rheumatoid arthritis, sarcopenic obesity, sexual dysfunction, herpes zoster, type 2 diabetes, changes in the urinary tract (including incontinence), vaginal atrophy, graying or whitening of hair, delayed / impaired wound healing, wrinkles / sagging skin (including loss of skin elasticity), and xerosis (dry skin); or the disease includes asthma, hearing loss, or viral infection, and / or the symptoms of the disease include sepsis.

[0154] In another aspect, the present invention also relates to the use of Klotho mRNA or pharmaceutical compositions according to the present invention for preventing and / or alleviating symptoms of aging.

[0155] In yet another aspect, the present invention relates to the use of Klotho mRNA or pharmaceutical compositions according to the present invention for increasing the lifespan of an individual, preferably a mammal, preferably a human.

[0156] The above uses are preferably non-therapeutic, particularly for the purpose of increasing lifespan. Therefore, it is preferable that the individuals are healthy. It is preferable that the individuals are not suffering from any of the diseases described herein.

[0157] In yet another aspect, the present invention relates to a cosmetic formulation comprising Klotho mRNA according to the present invention. Preferably, the cosmetic formulation comprises a cosmetic carrier. Preferably, the cosmetic carrier is an ointment, a gel, particularly a hydrogel, a liposome, nano / microparticles, or an emulsion.

[0158] In a further embodiment, the present invention also relates to a cosmetic skincare method comprising contacting the skin of an individual, preferably a human individual, with Klotho mRNA or a cosmetic formulation according to the present invention. Preferably, the skin is aged skin. It is particularly preferable that the cosmetic skincare method reduces signs of aging such as wrinkles.

[0159] In a further embodiment, the present invention relates to a DNA molecule comprising a DNA sequence encoding the coding region of the Klotho mRNA of the present invention. This DNA molecule may be used to facilitate the synthesis of the mRNA of the present invention after transfection into mammalian cells, through both in vitro transcription (IVT) processes and intracellular transcription mechanisms.

[0160] In preferred embodiments, the DNA molecule comprises a DNA sequence encoding the 5'-UTR, coding region, and 3'-UTR of the Klotho mRNA of the present invention. It is particularly preferred that the DNA molecule comprises a DNA sequence encoding the 5'-UTR, coding region, 3'-UTR, and poly-A tail of the Klotho mRNA of the present invention. Alternatively, instead of being encoded in the DNA sequence, the poly-A tail of the Klotho mRNA of the present invention may be added post-transcriptionally by polyadenylation. Preferably, the DNA molecule comprises a DNA sequence encoding the Klotho mRNA of the present invention.

[0161] The DNA molecule preferably includes a promoter operably ligated to the DNA sequence. In a preferred embodiment, the promoter is a promoter for in vitro transcription, preferably a T7 promoter. In a further preferred embodiment, the promoter is a promoter for expression in mammals, preferably selected from the group consisting of CMV, CAG, and / or EF1a promoters.

[0162] However, in the context of the present invention, DNA molecules lacking a promoter sequence and containing only a DNA sequence encoding the coding region of the Klotho mRNA of the present invention are also useful. For example, such a DNA molecule can be used as a template for an IVT process, in which a linear DNA fragment is generated from the DNA molecule by PCR, and this linear DNA fragment is described. Features that are desirable to be present on the linear DNA fragment, such as a T7 promoter, can be added during the PCR reaction.

[0163] Further aspects of the present invention relate to cells containing the DNA molecule of the present invention. The cells of the present invention are particularly useful for the production of the Klotho mRNA of the present invention. In particularly preferred embodiments, the cells of the present invention may be used to produce extracellular vesicles (EVs), such as exosomes, containing the Klotho mRNA of the present invention, as described in detail below.

[0164] Preferably, the cells are mammalian cells, more preferably human cells. In the context of the present invention, it is particularly preferable that the cells are human embryonic kidney (HEK) 293 cells.

[0165] Particularly preferred is the case where the cells are EV-producing cells. Cell types particularly suitable for the production of EVs, especially mRNA-containing EVs, are known in the art, for example from International Publication No. 2019 / 092145 and International Publication No. 2010 / 119256. For example, particularly interesting cell lines include human umbilical cord endothelial cells (HUVEC), HEK cells, endothelial cell lines such as microvascular or lymphatic endothelial cells, erythrocytes, erythrocyte progenitor cells, chondrocytes, MSCs of different origins, amniotic cells, amniotic epithelial (AE) cells, cells obtained from amniocentesis or placenta, airway or alveolar epithelial cells, fibroblasts, and endothelial cells. Immune cells such as B cells, T cells, NK cells, macrophages, monocytes, and dendritic cells (DCs) can also be used for EV production. In general, EVs can be derived from virtually any cell.

[0166] In preferred embodiments, cells are stably transfected with the DNA molecule of the present invention. Furthermore, it is particularly preferable to expose the cells to a clonal selection protocol that enables clonal selection of single-cell clones. This can be achieved, for example, by using limiting dilution, single-cell sorting, and / or isolation of individual cells using cloning cylinders. Thus, in preferred embodiments, the cells are monoclonal cells and / or monoclonal cell lines.

[0167] In a particularly preferred embodiment, cells are encapsulated in a biocompatible scaffold. Such a biocompatible scaffold can be used for the sustained delivery of therapeutic proteins.

[0168] Similarly, in a further embodiment, the present invention relates to a therapeutic delivery device comprising the cells of the present invention, wherein the cells are encapsulated in a biocompatible scaffold.

[0169] Traditional delivery methods, such as systemic injection or infusion, often require frequent administration to maintain therapeutic levels due to the rapid clearance of proteins from the body. This not only burdens the patient but also increases the risk of side effects and reduces the overall effectiveness of the treatment. To address these challenges, cell encapsulation technology is becoming increasingly important. Cell encapsulation typically involves immobilizing cells within a biocompatible scaffold, such as a microcapsule. When these encapsulated cells are transplanted into an organism, they continuously produce and release therapeutic proteins over extended periods. This approach offers several advantages, including the possibility of sustained and localized delivery of therapeutic proteins, reduced administration frequency, and protection of encapsulated cells from the host immune system.

[0170] Methods for encapsulating cells in biocompatible scaffolds and for using such encapsulated cells for the serial delivery of therapeutic proteins are known from the art. For example, suitable methods are described in Acarregui et al. ("Therapeutic applications of encapsulated cells." Immobilization of Enzymes and Cells: Third Edition (2013): 349-364) and Wang et al. ("A nanofibrous encapsulation device for safe delivery of insulin-producing cells to treat type 1 diabetes." Science Translational Medicine 13.596 (2021): eabb4601).

[0171] The Klotho mRNA of the present invention enables particularly high levels of Klotho expression, and is therefore particularly advantageous in the context of such encapsulated cells, as it thereby increases the level of Klotho production by these cells.

[0172] In a further embodiment, the present invention relates to a therapeutic delivery device for use as a pharmaceutical product.

[0173] In the context of therapeutic delivery devices, the same therapeutic uses of the Klotho mRNA or pharmaceutical compositions of the present invention as defined above are preferred. For example, in one embodiment, the present invention relates to therapeutic delivery devices or uses in the prevention or treatment of diseases selected from the group consisting of kidney diseases, cardiovascular diseases, brain diseases, lung diseases, bone diseases, and metabolic diseases.

[0174] In a further embodiment, the present invention relates to extracellular vesicles (EVs) containing Klotho mRNA of the present invention.

[0175] Extracellular vesicles (EVs) are typically nanometer-sized vesicles produced by most cell types and function as the body's natural transport system for transporting proteins, nucleic acids, peptides, lipids, and various other molecules between cells. Due to their innate ability to encapsulate and protect nucleic acids, facilitating efficient delivery to receptor cells, EVs have emerged as a highly effective delivery system for therapeutic molecules, including mRNA. Those skilled in the art are familiar with methods for preparing such RNA-containing EVs and using them for mRNA delivery. Such methods are disclosed, for example, in International Publication No. 2010 / 119256 and International Publication No. 2019 / 092145. Furthermore, detailed discussions are provided in Lu et al. ("Exosome-based carrier for RNA delivery: progress and challenges." Pharmaceutics 15.2 (2023): 598) and Aslan et al. ("Exosomes for mRNA delivery: a novel biotherapeutic strategy with hurdles and hope." BMC biotechnology 21 (2021): 1-12).

[0176] Extracellular viable cells (EVs) containing a specific mRNA can be prepared in various ways, many of which are detailed in the references mentioned above. For example, mRNA can be loaded into EVs by simple incubation. Alternatively, EV-producing cells may be transfected with DNA encoding the desired mRNA, causing the cells to produce the target mRNA. In the context of the present invention, any suitable method may be used to load Klotho mRNA into EVs. However, in preferred embodiments, the EVs are derived from the cells of the present invention. If the cells contain a DNA molecule of the present invention that includes at least a DNA sequence encoding the coding region of the Klotho mRNA of the present invention, then the EVs derived from the cells contain the mRNA.

[0177] In the context of the present invention, EVs can be any type of vesicle obtained from any form of cell, e.g., microvesicles (e.g., any vesicles expelled from the cell membrane of a cell), exosomes (e.g., any vesicles derived from the endolysosomal pathway), apoptotic bodies (e.g., obtained from apoptotic cells), microparticles (e.g., may be derived from platelets), ectosomes (e.g., may be derived from neutrophils and monocytes in serum), prostatosomes (e.g., obtained from prostate cancer cells), or cardiosomes (e.g., derived from cardiac cells). While the size of EVs varies considerably, EVs typically have a nanoscale hydrodynamic diameter, i.e., a diameter of less than 1000 nm. EVs can be derived from any cell type in vivo, ex vivo, and in vitro. Preferred EVs include exosomes and microvesicles, but other EVs may be advantageous in various situations. Furthermore, the terms are understood to also relate to extracellular vesicle mimes, e.g., cell membrane-based vesicles obtained by membrane extrusion, sonication, or other techniques. In a particularly preferred embodiment, EV is an exosome.

[0178] In a further preferred embodiment, the EV includes a targeting moiety expressed on the surface of the EV. The targeting moiety can enable targeted delivery to cells, tissues, organs, and / or compartments of interest. The targeting moiety may be a peptide expressed as a fusion protein with a transmembrane protein typically expressed on the surface of an exosome. Suitable targeting moieties are known, for example, from International Publication No. 2010 / 119256 and International Publication No. 2019 / 092145. For example, a suitable peptide may bind to a cell surface moiety, such as a receptor or ligand, found on the cell surface of the target cell. Examples of suitable targeting moieties are short peptides, scFvs, and complete proteins, provided that the targeting moiety is expressed on the surface of an exosome and does not interfere with the insertion of membrane proteins into the exosome.

[0179] In a further embodiment, the present invention relates to a population of EVs as defined above herein. Preferably, the average number of Klotho mRNA molecules per EV in the entire population of EVs is greater than 1 per EV, preferably greater than 10 per EV, and more preferably greater than 100 per EV.

[0180] However, within the overall population, there may be EVs that do not contain Klotho mRNA molecules. In a preferred embodiment, at least 5%, at least 10%, at least 20%, at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and / or at least 95% of the total EVs contain at least one Klotho mRNA molecule.

[0181] In a further embodiment, the present invention is a method for generating an EV of the present invention or a group of EVs of the present invention, - Culturing the cells of the present invention under conditions suitable for EV formation, - Isolating EVs and Regarding methods including

[0182] Those skilled in the art are familiar with methods for generating extracellular viable cells (EVs), such as those detailed in the above-mentioned literature. Typically, EVs can be isolated from the cell culture medium in which the cells of the present invention are cultured. The EVs produced from the cells can be recovered from the culture medium by any suitable method. Typically, EVs can be isolated by centrifugation, filtration, or a combination of these methods.

[0183] In further embodiments, the present invention relates to pharmaceutical formulations comprising the EV of the present invention or a group of EVs of the present invention. The embodiments described herein relating to pharmaceutical formulations of the present invention comprising Klotho mRNA are also preferred in the context of pharmaceutical formulations of the present invention comprising the EV of the present invention or a group of EVs of the present invention.

[0184] Preferably, the pharmaceutical formulation may be formulated for parenteral administration, intramuscular administration, intracerebral administration, intravascular administration (including intravenous administration), subcutaneous administration, or transdermal administration. Formulations for parenteral administration may include sterile aqueous solutions, which may include buffers, diluents, and other suitable excipients. In addition to EV, the pharmaceutical formulation may include pharmaceutically acceptable carriers, thickeners, diluents, buffers, preservatives, and other pharmaceutically acceptable carriers or excipients.

[0185] In further embodiments, the present invention relates to the EV of the present invention, a group of EVs of the present invention, or a pharmaceutical formulation of the present invention for use as a pharmaceutical.

[0186] In this context, the same therapeutic uses of Klotho mRNA as defined above are preferred. For example, in one embodiment, the present invention relates to the EVs of the present invention, a group of EVs of the present invention, or a pharmaceutical formulation of the present invention for use in the prevention or treatment of diseases selected from the group consisting of kidney diseases, cardiovascular diseases, brain diseases, lung diseases, bone diseases, and metabolic diseases.

[0187] In a further embodiment, the present invention relates to the use of the EVs of the present invention, a group of EVs of the present invention, or a pharmaceutical formulation for preventing and / or alleviating symptoms of aging.

[0188] In yet another aspect, the present invention relates to the use of the EV of the present invention, a group of EVs of the present invention, or a pharmaceutical formulation for increasing the lifespan of an individual, preferably a mammal, preferably a human.

[0189] The above uses are preferably non-therapeutic, particularly for the purpose of increasing lifespan. Therefore, it is preferable that the individuals are healthy. It is preferable that the individuals are not suffering from any of the diseases described herein.

[0190] In yet another aspect, the present invention relates to a cosmetic formulation comprising the EV of the present invention or a group of EVs of the present invention. Preferably, the cosmetic formulation comprises a cosmetic carrier. Preferably, the cosmetic carrier is an ointment, a gel, particularly a hydrogel, a liposome, nano / microparticles, or an emulsion.

[0191] In a further embodiment, the present invention also relates to a cosmetic skincare method comprising bringing the skin of an individual, preferably a human individual, into contact with the EV of the present invention or a group of EVs of the present invention or a cosmetic formulation according to the present invention. Preferably, the skin is aged skin. It is particularly preferable that the cosmetic skincare method reduces signs of aging such as wrinkles.

[0192] To facilitate understanding of the present invention, several terms are defined below. The terms defined herein have meanings that are generally understood by those skilled in the art relating to the present invention. Terms such as "a," "an," and "the" are not intended to refer only to singular entities, but include general classifications for which specific examples may be used for illustrative purposes. The terms herein are used to describe specific embodiments of the present invention, but their use is not intended to limit the invention unless outlined in the claims.

[0193] The terms “preventing” or “prevention,” as used herein, mean preventing a disease condition or state from occurring completely, almost completely, or at least to some extent (preferably to a significant extent) in an individual, especially when the individual is predisposed to such a risk of developing the disease condition or state. However, these terms should not be interpreted as absolute success in the sense that the patient never develops the disease, reaction or state in question, but rather as a reduction in the likelihood of developing the disease, reaction or state in the event of preventive measures.

[0194] The terms “percentage of sequence identity (%)”, “X% identical” (e.g., “70% identical”) or similar terms relating to a reference nucleotide sequence are defined as the percentage of nucleotides in a candidate sequence that are identical to the nucleotides in the reference sequence after the sequences have been aligned and gaps introduced where necessary to achieve the maximum percentage of sequence identity, and no conservative substitutions are considered as part of the sequence identity. Gaps result in a lack of identity. Alignment for the purpose of determining the percentage of nucleotide sequence identity can be achieved in various ways within the scope of the art of the art using publicly available computer software, such as the “needle” pairwise sequence alignment application in the BLAST, BLAST-2, ALIGN, ALIGN-2, Megalign (DNASTAR), or EMBOSS software packages. A person skilled in the art can determine appropriate parameters for aligning sequences, including the algorithms necessary to achieve the maximum alignment over the entire length of the sequences being compared. However, for the purposes of this invention, the % value of nucleotide sequence identity is calculated using the sequence alignment of the computer program "needle" from the EMBOSS software package (publicly available from European Molecular Biology Laboratory; Rice et al., EMBOSS: the European Molecular Biology Open Software Suite, Trends Genet. 2000 Jun;16(6):276-7, PMID: 10827456).

[0195] The needle program can be accessed from the website http: / / www.ebi.ac.uk / Tools / psa / emboss_needle or downloaded locally as part of the EMBOSS package from http: / / emboss.sourceforge.net / . It runs on many widely used UNIX operating systems, including Linux.

[0196] To align two nucleotide sequences, the needle program is preferably run with the following parameters: Command line: needle -auto -stdout -asequence SEQUENCE_FILE_A -bsequence SEQUENCE_FILE_B -datafile EDNAFULL -gapopen 10.0 -gapextend 0.5 -endopen 10.0 -endextend 0.5 -aformat3 pair -snucleotide1 -snucleotide2 (Align_format:pair Report_file:stdout)

[0197] The percentage of nucleotide sequence identity of a given nucleotide sequence A to a given nucleotide sequence B, with a given nucleotide sequence B, or to a given nucleotide sequence B (which can be rephrased as a given nucleotide sequence A having, or containing, a certain percentage of nucleotide sequence identity to, with, or to a given nucleotide sequence B) is calculated as follows: 100 times the fraction X / Y In the formula, X is the number of nucleotides scored as a perfect match in the program alignment of A and B by the sequence alignment program needle, and Y is the total number of nucleotides in B. It will be understood that if the length of nucleotide sequence A is not equal to the length of nucleotide sequence B, then the percentage of nucleotide sequence identity of A to B is not equal to the percentage of nucleotide sequence identity of B to A. If "the sequence of A is at least N% identical to the entire sequence of B", then Y is the total length of B. Unless otherwise specifically stated, all nucleotide sequence identity percentage values ​​used herein are obtained using the needle computer program as described in the preceding paragraph.

[0198] The terms “percentage of sequence identity (%)”, “percentage of amino acid sequence identity (%)”, “X% identical” (e.g., “70% identical”) or similar terms relating to a reference polypeptide or protein sequence are defined in the same way as described above for nucleotide sequence identity; that is, they are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after the sequences have been aligned and gaps introduced if necessary to achieve the maximum percentage of sequence identity, and no conservative substitutions are considered as part of the sequence identity. Gaps result in a lack of identity. Alignment for determining the percentage of amino acid sequence identity is preferably performed in the same way as described above for nucleotide sequences. To align two protein sequences, the needle program is preferably run with the following parameters: Command line: needle -auto -stdout -asequence SEQUENCE_FILE_A -bsequence SEQUENCE_FILE_B -datafile EBLOSUM62 -gapopen 10.0 -gapextend 0.5 -endopen 10.0 -endextend 0.5 -aformat3 pair -sprotein1 -sprotein2 (Align_format:pair Report_file:stdout)

[0199] The percentage of amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B, or to a given amino acid sequence B (which can be rephrased as a given amino acid sequence A having, or containing, a certain percentage of amino acid sequence identity to, or to a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y In the formula, X is the number of amino acids scored as a perfect match in the alignment of programs A and B by the sequence alignment program needle, and Y is the total number of amino acids in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percentage of amino acid sequence identity of A to B is not equal to the percentage of amino acid sequence identity of B to A. If "the sequence of A is more than N% identical to the entire sequence of B", then Y is the length of the entire sequence of B (i.e., the total number of amino acid residues in B). Unless otherwise specifically stated, all percentage values ​​of amino acid sequence identity used herein are obtained using the needle computer program as described in the preceding paragraph.

[0200] In this specification, "UniProt" refers to the Universal Protein Resource. UniProt is a comprehensive resource of protein sequence and annotation data. UniProt is a collaborative project of the European Bioinformatics Institute (EMBL-EBI), the Swiss Institute of Bioinformatics (SIB), and the Protein Information Resource (PIR). These three institutes involve over 100 people involved in various tasks, including database curation, software development, and support. Website: http: / / www.uniprot.org /

[0201] An entry in the UniProt database is identified by its entrustment code (referred to herein, for example, the "UniProt entrustment code," or simply "UniProt" followed by the entrustment code), which is typically a six-character alphanumeric code (e.g., "Q1HVF7"). Unless otherwise specified, the entrustment code used herein refers to an entry in the UniProt Protein Knowledgebase (UniProtKB). Unless otherwise stated, the status of all entries in the UniProt database referred to herein is as of 2 March 2023 (UniProt / UniProtKB Release 2023_01).

[0202] In the context of this application, sequence variants (referred to as “native variants” in UniProt) are explicitly included when referring to entries in the UniProt database.

[0203] Unless otherwise specified, all parameters used herein correspond to IUPAC SATP conditions ("Standard Ambient Temperature and Pressure"), particularly the parameters at a temperature of 25°C and a pressure of 101.300 Pa.

[0204] When used herein, percentages (%) correspond to weight per volume (w / v) unless otherwise specified, such as weight per w / w. [Modes for carrying out the invention]

[0205] The present invention relates to the following preferred embodiments:

[0206] Embodiment 1. Klotho messenger RNA (mRNA) having a 5' CAP region, a 5' untranslated region (5'-UTR), a coding region encoding a Klotho polypeptide, a 3' untranslated region (3'-UTR), and a polyadenosine tail (poly-A tail), wherein the Klotho polypeptide contains the KL1 domain of human Klotho, and the Klotho mRNA.

[0207] Embodiment 2. Klotho mRNA according to any one of the embodiments, wherein the Klotho polypeptide comprises the KL1 domain and the KL2 domain of human Klotho.

[0208] Embodiment 3. Klotho mRNA according to any one of the embodiments, wherein the Klotho polypeptide comprises a signal peptide, the KL1 domain and the KL2 domain of human Klotho.

[0209] Embodiment 4. The Klotho mRNA according to any one of the above embodiments, wherein the Klotho polypeptide further comprises the transmembrane (TM) domain of human Klotho, and preferably the Klotho polypeptide further comprises the cytoplasmic tail (CT) of human Klotho.

[0210] Embodiment 5. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide has a GC content of at least 50%, preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, and more preferably at least 63%.

[0211] Embodiment 6. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide has a GC content of 50% to 74%, preferably 51% to 73%, more preferably 52% to 72%, more preferably 53% to 71%, more preferably 54% to 70%, more preferably 55% to 69%, more preferably 56% to 68%, more preferably 57% to 67%, more preferably 58% to 66%, more preferably 59% to 65%, more preferably 60% to 64%, and more preferably 61% to 63%.

[0212] Embodiment 7. The Klotho mRNA according to any one of the embodiments, wherein the Klotho mRNA has a GC content of at least 50%, preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, and more preferably at least 63%.

[0213] Embodiment 8. The Klotho mRNA according to any one of the embodiments, wherein the Klotho mRNA has a GC content of 50% to 74%, preferably 51% to 73%, more preferably 52% to 72%, more preferably 53% to 71%, more preferably 54% to 70%, more preferably 55% to 69%, more preferably 56% to 68%, more preferably 57% to 67%, more preferably 58% to 66%, more preferably 59% to 65%, more preferably 60% to 64%, and more preferably 61% to 63%.

[0214] Embodiment 9. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide includes an RNA sequence having sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% with respect to SEQ ID NO: 1 or 3, particularly SEQ ID NO: 1.

[0215] Embodiment 10. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide includes SEQ ID NO: 1, 2, 3, or 4, preferably SEQ ID NO: 1.

[0216] Embodiment 11. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide comprises an RNA sequence having sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% with respect to SEQ ID NO: 9 or 11, particularly SEQ ID NO: 9.

[0217] Embodiment 12. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide includes SEQ ID NO: 9, 10, 11, or 12, preferably SEQ ID NO: 9.

[0218] Embodiment 13. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide comprises an RNA sequence having sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% with respect to SEQ ID NO: 13, and preferably the RNA sequence is SEQ ID NO: 13 or 15, particularly SEQ ID NO: 13.

[0219] Embodiment 14. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide includes SEQ ID NOs: 13, 14, 15, or 16, preferably SEQ ID NO: 13.

[0220] Embodiment 15. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide comprises an RNA sequence having sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% with respect to SEQ ID NO: 64 or 66, particularly SEQ ID NO: 64.

[0221] Embodiment 16. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide includes SEQ ID NOs. 64, 65, 66, or 67, preferably SEQ ID NO. 64.

[0222] Embodiment 17. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide comprises an RNA sequence having sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% with respect to SEQ ID NO: 18 or 20, particularly SEQ ID NO: 18.

[0223] Embodiment 18. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide includes SEQ ID NOs: 18, 19, 20, or 21, preferably SEQ ID NO: 18.

[0224] Embodiment 19. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide comprises an RNA sequence having sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% with respect to SEQ ID NO: 68 or 70, particularly SEQ ID NO: 68.

[0225] Embodiment 20. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide includes SEQ ID NOs. 68, 69, 70, or 71, preferably SEQ ID NO. 64.

[0226] Embodiment 21. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide comprises an RNA sequence having sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% with respect to SEQ ID NO: 33 or 35, particularly SEQ ID NO: 33.

[0227] Embodiment 22. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide includes SEQ ID NOs: 33, 34, 35, or 36, preferably SEQ ID NO: 33.

[0228] Embodiment 23. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide comprises an RNA sequence having sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% with respect to SEQ ID NO: 37 or 39, particularly SEQ ID NO: 37.

[0229] Embodiment 24. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide includes SEQ ID NOs. 37, 38, 39, or 40, preferably SEQ ID NO. 37.

[0230] Embodiment 25. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide comprises an RNA sequence having sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% with respect to SEQ ID NO: 41 or 43, particularly SEQ ID NO: 41.

[0231] Embodiment 26. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide includes SEQ ID NOs: 41, 42, 43, or 44, preferably SEQ ID NO: 41.

[0232] Embodiment 27. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide has sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably 100% with respect to SEQ ID NO.

[0233] Embodiment 28. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 5.

[0234] Embodiment 29. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 6.

[0235] Embodiment 30. A Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 7.

[0236] Embodiment 31. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 8.

[0237] Embodiment 32. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide has sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably 100% with respect to SEQ ID NO: 18.

[0238] Embodiment 33. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 18.

[0239] Embodiment 34. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 19.

[0240] Embodiment 35. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 20.

[0241] Embodiment 36. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 21.

[0242] Embodiment 37. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 22.

[0243] Embodiment 38. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is Sequence ID No. 23.

[0244] Embodiment 39. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 24.

[0245] Embodiment 40. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide has sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably 100% with respect to SEQ ID NO: 68.

[0246] Embodiment 41. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 68.

[0247] Embodiment 42. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 69.

[0248] Embodiment 43. A Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 70.

[0249] Embodiment 44. A Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 71.

[0250] Embodiment 45. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide has sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably 100% with respect to SEQ ID NO: 41.

[0251] Embodiment 46. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 41.

[0252] Embodiment 47. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 42.

[0253] Embodiment 48. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 43.

[0254] Embodiment 49. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 44.

[0255] Embodiment 50. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide comprises an RNA sequence having sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% with respect to SEQ ID NO: 79 or 81, particularly SEQ ID NO: 79.

[0256] Embodiment 51. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide includes SEQ ID NO: 79, 80, 81, or 82, preferably SEQ ID NO: 79.

[0257] Embodiment 52. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide comprises an RNA sequence having sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% with respect to SEQ ID NO: 87 or 89, particularly SEQ ID NO: 87.

[0258] Embodiment 53. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide includes SEQ ID NO: 87, 88, 89, or 90, preferably SEQ ID NO: 87.

[0259] Embodiment 54. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide comprises an RNA sequence having sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% with respect to SEQ ID NO: 91 or 93, particularly SEQ ID NO: 91.

[0260] Embodiment 55. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide includes SEQ ID NOs: 91, 92, 93, or 94, preferably SEQ ID NO: 91.

[0261] Embodiment 56. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide comprises an RNA sequence having sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% with respect to SEQ ID NO: 120 or 122, particularly SEQ ID NO: 120.

[0262] Embodiment 57. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide includes SEQ ID NO: 120, 121, 122, or 123, preferably SEQ ID NO: 120.

[0263] Embodiment 58. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide comprises an RNA sequence having sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% with respect to SEQ ID NO: 96 or 98, particularly SEQ ID NO: 96.

[0264] Embodiment 59. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide includes SEQ ID NOs: 96, 97, 98, or 99, preferably SEQ ID NO: 96.

[0265] Embodiment 60. The coding region encoding the Klotho polypeptide comprises an RNA sequence having at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8% sequence identity to SEQ ID NO: 124, and preferably, the RNA sequence is SEQ ID NO: 124 or 126, particularly SEQ ID NO: 124, the Klotho mRNA according to any one of the above embodiments.

[0266] Embodiment 61. The coding region encoding the Klotho polypeptide comprises SEQ ID NO: 124, 125, 126 or 127, preferably SEQ ID NO: 124, the Klotho mRNA according to any one of the above embodiments.

[0267] Embodiment 62. The coding region encoding the Klotho polypeptide comprises an RNA sequence having at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8% sequence identity to SEQ ID NO: 104, and preferably, the RNA sequence is SEQ ID NO: 104 or 106, particularly SEQ ID NO: 104, the Klotho mRNA according to any one of the above embodiments.

[0268] Embodiment 63. The Klotho mRNA according to any one of the above embodiments, wherein the coding region encoding the Klotho polypeptide comprises SEQ ID NO: 104, 105, 106 or 107, preferably SEQ ID NO: 104.

[0269] Embodiment 64. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide comprises an RNA sequence having sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% with respect to SEQ ID NO: 108 or 110, particularly SEQ ID NO: 108.

[0270] Embodiment 65. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide includes SEQ ID NOs: 108, 109, 110, or 111, preferably SEQ ID NO: 108.

[0271] Embodiment 66. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide comprises an RNA sequence having sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.8% with respect to SEQ ID NO: 112 or 114, particularly SEQ ID NO: 112.

[0272] Embodiment 67. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide includes SEQ ID NOs: 112, 113, 114, or 115, preferably SEQ ID NO: 112.

[0273] Embodiment 68. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide has sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably 100% with respect to SEQ ID NO: 83.

[0274] Embodiment 69. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 83.

[0275] Embodiment 70. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 84.

[0276] Embodiment 71. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 85.

[0277] Embodiment 72. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 86.

[0278] Embodiment 73. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide has sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably 100% with respect to sequence number 96.

[0279] Embodiment 74. The Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 96.

[0280] Embodiment 75. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 97.

[0281] Embodiment 76. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 98.

[0282] Embodiment 77. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 99.

[0283] Embodiment 78. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide has sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably 100% with respect to SEQ ID NO: 124.

[0284] Embodiment 79. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 124.

[0285] Embodiment 80. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 125.

[0286] Embodiment 81. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 126.

[0287] Embodiment 82. Klotho mRNA according to any one of the embodiments, wherein the coding region encoding the Klotho polypeptide is sequence number 127.

[0288] Embodiment 83. The Klotho mRNA according to any one of the above embodiments, wherein the coding region encoding the Klotho polypeptide has at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably 100% sequence identity to SEQ ID NO: 112.

[0289] Embodiment 84. The Klotho mRNA according to any one of the above embodiments, wherein the coding region encoding the Klotho polypeptide is SEQ ID NO: 112.

[0290] Embodiment 85. The Klotho mRNA according to any one of the above embodiments, wherein the coding region encoding the Klotho polypeptide is SEQ ID NO: 113.

[0291] Embodiment 86. The Klotho mRNA according to any one of the above embodiments, wherein the coding region encoding the Klotho polypeptide is SEQ ID NO: 114. [[ID=1,4]]

[0292] Embodiment 87. The Klotho mRNA according to any one of the above embodiments, wherein the coding region encoding the Klotho polypeptide is SEQ ID NO: 115.

[0293] Embodiment 88. The Klotho mRNA according to any one of the embodiments, wherein the KL1 domain of the Klotho polypeptide has sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 99.5% with respect to SEQ ID NO: 52, and preferably the KL1 domain of the Klotho polypeptide is SEQ ID NO: 52 or 53, particularly SEQ ID NO: 52.

[0294] Embodiment 89. The Klotho mRNA according to any one of the embodiments, wherein the KL2 domain of the Klotho polypeptide has sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 99.5% with respect to SEQ ID NO: 54, and preferably the KL2 domain of the Klotho polypeptide is SEQ ID NO: 54.

[0295] Embodiment 90. The Klotho mRNA according to any one of the embodiments, wherein the signal peptide of the Klotho polypeptide has sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 99.5% with respect to SEQ ID NO: 51, and in particular the signal peptide of the Klotho polypeptide is SEQ ID NO: 51.

[0296] Embodiment 91. The Klotho mRNA according to any one of the embodiments, wherein the Klotho polypeptide has sequence identity of at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably 100% with respect to SEQ ID NO: 55.

[0297] Embodiment 92. The Klotho mRNA according to any one of the embodiments, wherein the Klotho polypeptide is SEQ ID NO: 55 or 56, preferably 55.

[0298] Embodiment 93. The Klotho mRNA according to any one of the embodiments, wherein the Klotho polypeptide has sequence identity of at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably 100% with respect to SEQ ID NO: 57.

[0299] Embodiment 94. The Klotho mRNA according to any one of the embodiments, wherein the Klotho polypeptide is SEQ ID NO: 57 or 58, preferably 57.

[0300] Embodiment 95. The Klotho mRNA according to any one of the embodiments, wherein the Klotho polypeptide has sequence identity of at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably 100% with respect to SEQ ID NO: 49.

[0301] Embodiment 96. Klotho mRNA according to any one of the embodiments, wherein the Klotho polypeptide is SEQ ID NO: 49 or 76, preferably 49.

[0302] Embodiment 97. The Klotho mRNA according to any one of the above embodiments, wherein the Klotho polypeptide has at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably 100% sequence identity to SEQ ID NO: 59.

[0303] Embodiment 98. The Klotho mRNA according to any one of the above embodiments, wherein the Klotho polypeptide is SEQ ID NO: 59 or SEQ ID NO: 60, preferably SEQ ID NO: 59.

[0304] Embodiment 99. The Klotho mRNA according to any one of the above embodiments, wherein the polyA tail contains at least 60 adenosine monophosphates, preferably at least 100 adenosine monophosphates, particularly at least 120 adenosine monophosphates.

[0305] Embodiment 100. The Klotho mRNA according to any one of the above embodiments, wherein the 5'-UTR or 3'-UTR or both 5'-UTR and 3'-UTR correspond to native human Klotho mRNA.

[0306] Embodiment 101. The Klotho mRNA according to any one of the above embodiments, wherein the 5'-UTR or 3'-UTR or both 5'-UTR and 3'-UTR are different from native human Klotho mRNA.

[0307] Embodiment 102. The 5'-UTR or 3'-UTR or both 5'-UTR and 3'-UTR contain at least one stabilizing sequence, preferably the general formula (C / U)CCAN x CCC(U / A)Py x UC(C / U)CC (SEQ ID NO: 63), wherein "x" is N x and Py xKlotho mRNA according to any one of the embodiments, independently of the integers 0 to 10, preferably 0 to 5, particularly 0, 1, 2, 4 and / or 5.

[0308] Embodiment 103. Klotho mRNA according to any one of the embodiments, wherein the 5'-UTR or 3'-UTR or 5'-UTR and 3'-UTR contains at least one destabilized sequence element (DSE), preferably an AU-rich element (ARE) and / or a U-rich element (URE), in particular a single copy, tandem copy, or multiple copies or duplicate copies of the nonomer UUAUUUA(U / A)(U / A).

[0309] Embodiment 104. The Klotho mRNA according to any one of the embodiments, wherein the 5'-UTR or 3'-UTR or 5'-UTR and 3'-UTR is a 5'-UTR and / or 3'-UTR of human mRNA different from native human Klotho mRNA, preferably selected from alphaglobin, betaglobin, albumin, lipoxygenase, ALOX15, alpha(1)collagen, tyrosine hydroxylase, ribosomal protein 32L, eukaryotic elongation factor 1a (EEF1A1), 5'-UTR elements present in orthopoxvirus, and mixtures thereof, in particular selected from alphaglobin, betaglobin, alpha(1)collagen, and mixtures thereof.

[0310] Embodiment 105. In Klotho mRNA, - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all cytidine residues are replaced with 5-methylcytidine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all cytidine residues are replaced with 2-amino-2-deoxycytidine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all cytidine residues are replaced with 2-fluoro-2-deoxycytidine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all cytidine residues are replaced with 2-thiocytidine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all cytidine residues are replaced with 5-iodocytidine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all uridine residues are replaced with pseudouridine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all uridine residues are replaced with 1-methyl-pseudridine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all uridine residues are replaced with 2-thiouridine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all uridine residues are replaced with 5-methyluridine residues, and / or - Klotho mRNA according to any one of the embodiments, wherein at least 5%, preferably at least 10%, more preferably at least 30%, and particularly at least 50% of all adenosine residues are replaced with N6-methyladenosine residues.

[0311] Embodiment 106. In Klotho mRNA, - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all cytidine residues are replaced with 5-methylcytidine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all uridine residues are replaced with pseudouridine residues, and / or - Klotho mRNA according to any one of the embodiments, wherein at least 5%, preferably at least 10%, more preferably at least 30%, and particularly at least 50% of all uridine residues are replaced with 2-thiouridine residues.

[0312] Embodiment 107. Klotho mRNA according to any one of the embodiments, wherein the coding region has a codon adaptation index (CAI) of at least 0.70, preferably at least 0.75, more preferably at least 0.80, more preferably at least 0.85, more preferably at least 0.86, more preferably at least 0.87, more preferably at least 0.88, more preferably at least 0.89, more preferably at least 0.90, more preferably at least 0.91, more preferably at least 0.92, more preferably at least 0.93, more preferably at least 0.94, more preferably at least 0.95, more preferably at least 0.96, more preferably at least 0.97, more preferably at least 0.98, more preferably at least 0.99, and more preferably 1.00.

[0313] Embodiment 108. Klotho mRNA according to any one of the embodiments, wherein the coding region has a codon adaptation index (CAI) of less than 1.00, preferably less than 0.99, more preferably less than 0.98, even more preferably less than 0.97, even more preferably less than 0.96, and even more preferably less than 0.95.

[0314] The Klotho mRNA according to any one of the embodiments, wherein Embodiment 109.5'-UTR has sequence identity of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably 100% with respect to SEQ ID NO: 61.

[0315] Klotho mRNA according to any one of the embodiments, wherein the 110.5'-UTR is sequence number 61.

[0316] The Klotho mRNA according to any one of the embodiments, wherein Embodiment 111.3'-UTR has sequence identity of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably 100% with respect to SEQ ID NO: 62.

[0317] Klotho mRNA according to any one of the embodiments, wherein the 112.3'-UTR is sequence number 62.

[0318] Embodiment 113. The Klotho mRNA according to any one of the embodiments, wherein the Klotho mRNA has sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably 100% with respect to SEQ ID NO: 29.

[0319] Embodiment 114. The Klotho mRNA according to any one of the embodiments, wherein the Klotho mRNA is SEQ ID NO: 29, 30, 31, or 32, preferably SEQ ID NO: 29.

[0320] Embodiment 115. The Klotho mRNA according to any one of the embodiments, wherein the Klotho mRNA has sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably 100% with respect to SEQ ID NO: 72.

[0321] Embodiment 116. The Klotho mRNA according to any one of the embodiments, wherein the Klotho mRNA is SEQ ID NO: 72, 73, 74, or 75, preferably SEQ ID NO: 72.

[0322] Embodiment 117. The Klotho mRNA according to any one of the embodiments, wherein the Klotho mRNA has sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably 100% with respect to SEQ ID NO: 45.

[0323] Embodiment 118. The Klotho mRNA according to any one of the embodiments, wherein the Klotho mRNA is SEQ ID NO: 45, 46, 47, or 48, preferably SEQ ID NO: 45.

[0324] Embodiment 119. The Klotho mRNA according to any one of the embodiments, wherein the Klotho mRNA has sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably 100% with respect to SEQ ID NO: 100.

[0325] Embodiment 120. The Klotho mRNA according to any one of the embodiments, wherein the Klotho mRNA is SEQ ID NO: 100, 101, 102, or 103, preferably SEQ ID NO: 100.

[0326] Embodiment 121. The Klotho mRNA according to any one of the embodiments, wherein the Klotho mRNA has sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably 100% with respect to SEQ ID NO: 128.

[0327] Embodiment 122. The Klotho mRNA according to any one of the embodiments, wherein the Klotho mRNA is SEQ ID NO: 128, 129, 130, or 131, preferably SEQ ID NO: 128.

[0328] Embodiment 123. The Klotho mRNA according to any one of the embodiments, wherein the Klotho mRNA has sequence identity of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably 100% with respect to SEQ ID NO: 116.

[0329] Embodiment 124. The Klotho mRNA according to any one of the embodiments, wherein the Klotho mRNA is SEQ ID NO: 116, 117, 118, or 119, preferably SEQ ID NO: 116.

[0330] Embodiment 125. A pharmaceutical formulation comprising Klotho mRNA as described in any one of the embodiments.

[0331] Embodiment 126. A pharmaceutical formulation according to any one of the above embodiments, comprising a pharmaceutically acceptable carrier, preferably a polymer-based carrier, particularly a cationic polymer including linear and branched PEIs and vilomers, lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, cationic amphiphilic lipids, e.g., SAINT-lipids, natural and synthetic exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticles, calcium phosphate nanoparticles, calcium phosphate nanoparticles, silicon dioxide nanoparticles, nanocrystalline particles, semiconductor nanoparticles, dried powders, poly(D-arginine), nanodendrimers, starch-based delivery systems, micelles, emulsions, sol-gels, niosomes, plasmids, viruses, calcium phosphate nucleotides, aptamers, peptides, peptide conjugates, vector tags, polylactic acid-coglycolic acid (PLGA) polymers; preferably a low molecular weight targeted conjugate, or a viral capsid protein, preferably a cationic polymer and liposome, particularly a cationic polymer.

[0332] Embodiment 127. A pharmaceutical formulation according to any one of the embodiments, comprising a cationic polymer containing linear and branched PEI and vilomer, lipid nanoparticles and liposomes, transfersomes, and nanoparticles, preferably calcium phosphate nanoparticles and cationic polymers, particularly cationic polymers.

[0333] Embodiment 128. A pharmaceutical formulation according to any one of the embodiments, wherein the mRNA is uncomplexed mRNA, preferably uncomplexed mRNA, contained in a suitable aqueous buffer, particularly a physiological glucose buffer aqueous solution.

[0334] Embodiment 129. A pharmaceutical formulation according to any one of the embodiments, comprising: 1 × HEPES buffer; 1 × phosphate buffer; sodium citrate buffer; sodium acetate buffer; Ringer's lactate solution; and preferably further comprising glucose, particularly 5% glucose.

[0335] Embodiment 130. A pharmaceutical formulation according to any one of the embodiments, comprising further mRNA, wherein the further mRNA has a further 5' CAP region, a further 5'-UTR, a further coding region encoding a further polypeptide, a further 3'-UTR, and a further poly(A) tail.

[0336] Embodiment 131. A pharmaceutical formulation according to any one of the embodiments, wherein the further polypeptide is a bactericidal / permeability-enhancing protein family B, member 4 (BPIFB4) protein, or an isoform or variant thereof, preferably a longevity-associated variant of BPIFB4 (LAV-BPIFB4).

[0337] Embodiment 132. The pharmaceutical formulation according to any one of the embodiments, wherein the further polypeptide is the BPIFB4 protein or its isoform or variant as described in International Publication No. 2014 / 102343 or International Publication No. 2019 / 034723.

[0338] Embodiment 133. Further polypeptides include apolipoprotein E, tumor protein p53, sirtuin 1 protein, FOXO1 transcription factor, cholinergic receptor nicotin alpha 3 subunit, SH2B adapter protein 3, cyclin-dependent kinase inhibitor 2A, extra-long chain elongation enzyme-like protein 2, Werner protein, paraoxonase 1, superoxide dismutase 2, lamin A protein, cholesteryl ester transfer protein, apolipoprotein C3, microsomal triglyceride transfer protein, phosphatidylinositol 3-kinase (PI3K), insulin-like growth factor 1 (IGF-1) receptor, protein-L-isoaspartyl methyltransferase, growth hormone, cAMP response element binding protein, mitogen-activated protein kinase, epidermal growth factor receptor, nuclear factor kappa B, phospholipase C beta, methionine sulfonate A pharmaceutical formulation according to any one of the embodiments, wherein the protein is selected from foxydoreductase A, cell motility mediator 1, Nei-like DNA glycosylase 1, peroxisome proliferator-activated receptor gamma 2, eukaryotic translation initiation factor 3 subunit K, ATM serine / threonine kinase, B-cell lymphoma 2, cell division cycle 42, diacylglycerol O-acyltransferase 1, early growth response 1, fibroblast growth factor 23, fibroblast growth factor 21, fructosamine 3 kinase-related protein, phosphoglycolate phosphatase, insulin receptor substrate 1, Polycomb complex protein BMI-1, neuregulin 1, signal transduction and transcription activator, E2F transcription factor 1, vascular endothelial growth factor A, xenobiotic metabolic enzyme, Myc proto-oncogene protein, CXC chemokine receptor type 4, silent information regulator 2, extracellular signal-regulated kinase, and SLC31.

[0339] Embodiment 134. A kit for administering Klotho mRNA to an individual according to any one of the embodiments described above, - Klotho mRNA according to any one of the embodiments described above, and - Device for administering Klotho mRNA A kit that includes this.

[0340] Embodiment 135. The kit according to any one of the embodiments, wherein the individual is a mammal, preferably a human.

[0341] Embodiment 136. The kit according to any one of the embodiments, wherein the device is for intravenous, intramuscular, subcutaneous, intradermal, transdermal, epidermal, or topical administration.

[0342] Embodiment 137. The kit according to any one of the embodiments, wherein the device for administering Klotho mRNA is a syringe, a needle, an auto-injector, and / or a needleless injection system; preferably, the device is for intravenous and / or intramuscular injection.

[0343] Embodiment 138. The kit according to any one of the embodiments, wherein the device for administering Klotho mRNA is a skin delivery device.

[0344] Embodiment 139. The kit according to any one of the embodiments, wherein the skin delivery device is an intradermal delivery device, preferably selected from the group consisting of needle-based injection systems.

[0345] Embodiment 140. The kit according to any one of the embodiments, wherein the skin delivery device is a transdermal delivery device, preferably selected from the group consisting of transdermal patches, hollow and solid microneedle systems, microstructured transdermal systems, electrophoresis systems, and iontophoresis systems.

[0346] Embodiment 141. The kit according to any one of the embodiments, wherein the skin delivery device is an epidermal delivery device, preferably selected from the group consisting of needleless injection systems, laser-based systems, in particular Erbium YAG laser systems, and gene gun systems.

[0347] Embodiment 142. A Klotho mRNA or pharmaceutical composition according to any one of the embodiments described above, for use as a pharmaceutical.

[0348] Embodiment 143. A Klotho mRNA or pharmaceutical composition according to any one of the embodiments described above, for use in the prevention or treatment of a disease selected from the group consisting of kidney disease, cardiovascular disease, brain disease, lung disease, bone disease, and metabolic disease.

[0349] Embodiment 144. Klotho mRNA or pharmaceutical composition according to any one of the above embodiments for use in the prevention or treatment of kidney disease, preferably a disease selected from the group consisting of chronic kidney disease, fibrosis, hyperphosphatemia, ischemic injury, nephrectomy, toxic injury, diabetic nephropathy, and calcific proteinosis.

[0350] Embodiment 145. Klotho mRNA or pharmaceutical composition according to any one of the above embodiments for use in the prevention or treatment of cardiovascular diseases, preferably diseases selected from the group consisting of arterial / aortic calcification, atherosclerosis, cardiomyopathy, cardiac hypertrophy, hypertension, and myocardial ischemic injury / infarction.

[0351] Embodiment 146. A Klotho mRNA or pharmaceutical composition according to any one of the embodiments described above for use in the prevention or treatment of brain diseases, preferably diseases selected from the group consisting of Alzheimer's disease, hippocampal neuronal loss, cognitive decline, and frailty.

[0352] Embodiment 147. Klotho mRNA or pharmaceutical composition according to any one of the embodiments described above for use in the prevention or treatment of cancer, preferably a cancer selected from the group consisting of colorectal cancer, esophageal cancer, gastric cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, thyroid cancer, melanoma, kidney cancer, and cervical cancer.

[0353] Embodiment 148. A Klotho mRNA or pharmaceutical composition according to any one of the embodiments described above, for use in the prevention or treatment of lung diseases, preferably pulmonary fibrosis or chronic obstructive pulmonary disease.

[0354] Embodiment 149. Klotho mRNA or pharmaceutical composition according to any one of the embodiments for use in the prevention or treatment of bone diseases, preferably osteoporosis or osteomalacia, particularly osteomalacia from chronic kidney disease.

[0355] Embodiment 150. Klotho mRNA or pharmaceutical composition according to any one of the above embodiments for use in the prevention or treatment of metabolic diseases, preferably diabetes, particularly type 1 diabetes and / or type 2 diabetes, pancreatic β-cell apoptosis, autoimmune disorders, and inflammation, particularly infertile inflammation.

[0356] Embodiment 151. Klotho mRNA for use or a pharmaceutical composition for use according to any one of the embodiments, wherein Klotho mRNA is administered intravenously, intramuscularly, subcutaneously, transdermally, epidermally, or topically, particularly epidermally.

[0357] Embodiment 152. Klotho mRNA for use or a pharmaceutical composition for use according to any one of the embodiments, wherein Klotho mRNA is administered at least twice within one month, preferably weekly.

[0358] Embodiment 153. Klotho mRNA for use or a pharmaceutical composition for use according to any one of the embodiments, wherein Klotho mRNA is administered in an amount of 0.01 μg to 100 mg per dose, preferably 0.1 μg to 10 mg per dose, and particularly 1 μg to 1 mg per dose.

[0359] Embodiment 154. A Klotho mRNA or pharmaceutical composition according to any one of the embodiments described above, for use in the prevention or treatment of age-related disorders and / or symptoms of aging.

[0360] Embodiment 155. Age-related disorders or symptoms include actinic keratosis, age-related macular degeneration (AMD), Alzheimer's disease, arthritis, atherosclerosis and cardiovascular disease, benign prostatic hyperplasia (BPH), bone atrophy, cachexia, cancer, cardiomyopathy, cataracts, chronic obstructive pulmonary disease (COPD), constipation, decreased total energy, decreased vision, delirium, dementia, depression, skin atrophy (thinning of the skin), decreased peripheral vision, increased risk of heatstroke or hypothermia, hearing loss, hypertension, increased susceptibility to infections (including influenza and pneumonia), moles (age spots), liver conditions (e.g., non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and cirrhosis), memory loss, metabolic syndrome, muscle atrophy (e.g., sarcopenia and myopenia), frailty, lack of muscle repair or rejuvenation, muscular dystrophy, and other abnormalities. Klotho mRNA for use or pharmaceutical composition for use as described in any one of the above embodiments, wherein the disease includes asthma, hearing loss, or viral infection, and / or the symptoms of the disease include sepsis.

[0361] Embodiment 156. Use of Klotho mRNA or pharmaceutical composition according to any one of the embodiments for preventing and / or alleviating symptoms of aging.

[0362] Embodiment 157. Use of Klotho mRNA or pharmaceutical composition according to any one of the embodiments for increasing the lifespan of a mammal, preferably a human.

[0363] Embodiment 158. A cosmetic formulation comprising Klotho mRNA as described in any one of the embodiments.

[0364] Embodiment 159. A cosmetic formulation according to any one of the embodiments, comprising a cosmetic carrier.

[0365] Embodiment 160. The cosmetic formulation according to any one of the embodiments, wherein the cosmetic carrier is an ointment, a gel, in particular a hydrogel, a liposome, nano / microparticles, or an emulsion.

[0366] Embodiment 161. A cosmetic skincare method comprising contacting the skin of an individual with Klotho mRNA as described in any one of the embodiments.

[0367] Embodiment 162. The cosmetic skincare method according to any one of the embodiments, wherein the individual is a human individual.

[0368] Embodiment 163. A cosmetic skincare method according to any one of the embodiments, wherein the skin is aged skin.

[0369] Embodiment 164. A DNA molecule comprising a DNA sequence encoding the coding region of a Klotho mRNA as defined in any one of the embodiments described above.

[0370] Embodiment 165. The DNA molecule according to the embodiment, wherein the DNA molecule comprises a DNA sequence encoding the 5'-UTR, coding region, and 3'-UTR of the Klotho mRNA as defined in any one of the embodiments.

[0371] Embodiment 166. A DNA molecule according to any one of the embodiments, comprising a DNA sequence encoding the 5'-UTR, coding region, 3'-UTR, and poly-A tail of Klotho mRNA as defined in any one of the embodiments.

[0372] Embodiment 167. A DNA molecule according to any one of the embodiments, comprising a DNA sequence encoding Klotho mRNA as defined in any one of the embodiments.

[0373] Embodiment 168. A DNA molecule according to any one of the embodiments, comprising a promoter operably ligated to the DNA sequence.

[0374] Embodiment 169. The DNA molecule according to the embodiment, wherein the promoter is a promoter for in vitro transcription, preferably a T7 promoter.

[0375] Embodiment 170. The DNA molecule according to the embodiment, wherein the promoter is a promoter for mammalian expression, preferably selected from the group consisting of CMV, CAG, and / or EF1a promoters.

[0376] Embodiment 171. A cell containing a DNA molecule as defined in any one of the embodiments described above.

[0377] Embodiment 172. The cells according to the embodiment, which are mammalian cells, preferably human cells, and more particularly human embryonic kidney (HEK) 293 cells.

[0378] Embodiment 173. A cell according to any one of the embodiments, which is a monoclonal cell and / or monoclonal cell line.

[0379] Embodiment 174. Cells according to any one of the embodiments, encapsulated in a biocompatible scaffold.

[0380] Embodiment 175. A therapeutic delivery device comprising cells as defined in any one of the embodiments, wherein the cells are encapsulated in a biocompatible scaffold.

[0381] Embodiment 176. A therapeutic delivery device according to the embodiment for use as a pharmaceutical, preferably, the use being as defined in any one of Embodiments 144 to 155.

[0382] Embodiment 177. An extracellular vesicle (EV) containing Klotho mRNA as defined in any one of the embodiments described above.

[0383] Embodiment 178. An EV according to the embodiment, which is derived from a cell as defined in any one of the embodiments.

[0384] Embodiment 179. The EV according to any one of the embodiments, wherein the EV includes a targeted portion expressed on the surface of the EV.

[0385] Embodiment 180. The EV according to any one of the embodiments, wherein the EV is an exosome.

[0386] Embodiment 181. A group of EVs as defined in any one of the above embodiments.

[0387] Embodiment 182. The population of EVs according to the embodiment, wherein the average number of Klotho mRNA molecules per EV is greater than 1 per EV throughout the entire population of EVs.

[0388] Embodiment 183. A population of EVs according to any one of the embodiments, wherein at least 5%, at least 10%, at least 20%, at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and / or at least 95% of the total EVs contain at least one Klotho mRNA molecule.

[0389] Embodiment 184. A method for generating an EV or a group of EVs according to any one of the embodiments described above, - Culturing the cells defined in any one of the above embodiments under conditions suitable for EV formation, - Isolating EVs and A method that includes this.

[0390] Embodiment 185. A pharmaceutical formulation comprising an EV or a group of EVs as defined in any one of the embodiments, preferably comprising a pharmaceutically acceptable carrier, preferably as defined in any one of embodiments 126 to 133.

[0391] Embodiment 186. An EV, a group of EVs, or a pharmaceutical formulation according to any one of the embodiments, for use as a pharmaceutical, wherein the use is defined in any one of embodiments 144 to 155.

[0392] Embodiment 187. Use of an EV, a group of EVs, or a pharmaceutical formulation as defined in any one of the embodiments, to prevent and / or alleviate the symptoms of aging.

[0393] Embodiment 188. Use of an EV, a population of EVs, or a pharmaceutical formulation as defined in any one of the embodiments, to increase the lifespan of a mammal, preferably a human.

[0394] Embodiment 189. A cosmetic formulation comprising an EV or a group of EVs as defined in any one of the embodiments, preferably as defined in any one of embodiments 159 to 160.

[0395] Embodiment 190. A cosmetic skincare method comprising bringing the skin of an individual into contact with an EV or a group of EVs as defined in any one of the embodiments, preferably as defined in any one of embodiments 162 to 163.

[0396] The present invention is not limited thereto, but is further illustrated by the following drawings and embodiments. [Brief explanation of the drawing]

[0397] [Figure 1A] Quantification of protein levels obtained after transfection of mammalian cell lines with different Klotho mRNA mutants. Three different cell lines were used: (A) Hs27; (B) HEK293; (C) 3T3. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 2A] Results of biologically independent replication of the experiment shown in Figure 1. (A) Hs27; (B) HEK293; (C) 3T3. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 3A] Quantification of Klotho protein levels obtained over long periods after transfection of HEK293 cells with different Klotho mRNA mutants. (A) Time-course experiments up to 144 hours. (B) Cumulative Klotho protein levels over 7 days. [Figure 3B] Same as above. [Figure 4A] A comparative study to quantify Klotho protein levels after transfection of HEK293 cells with different Klotho mRNA variants. Sequences designated as WO666_SEQ_xxxx correspond to mRNA sequences disclosed in International Publication No. 2013 / 151666. These were tested against the GC62, GC60, GC57, GC55, GC53 variants and native Klotho mRNA sequences disclosed herein. (A) Klotho protein expression levels. (B) Correlation between Klotho protein expression levels (relative to expression levels obtained with native Klotho mRNA sequences) and sequence identity for mutant GC62. [Figure 4B] Same as above. [Figure 5]Klotho protein expression from monoclonal HEK293 cells stably transfected with a DNA vector encoding the Klotho mRNA mutant GC62 ("clone_10") compared to transient transfection with GC62 mRNA and untreated cells ("untr."). [Figure 6A] Serum Klotho protein levels from samples of mice injected with the Klotho mRNA mutant GC62, compared to the native sequence. Serum protein levels were determined at various time points after injection. [Figure 6B] Same as above. [Examples]

[0398] [Example 1] Klotho expression levels obtained from different mRNA sequence variants. To quantify the effects of different mRNA sequences on expression levels in different cell lines, Klotho protein expression levels obtained from different sequence variants were tested.

[0399] Sequence variants We used the mRNA sequence of native human Klotho isoform 1, NM_004795.4, according to the NCBI reference sequence, as a starting point. From this, we extracted the sequence encoding soluble Klotho, i.e., amino acids M1 to S981 (SEQ ID NO: 17). This sequence is 2946 nt long and can be divided into a signal peptide (amino acids 1-33; nt1-99) and KL1 and KL2 domains (amino acids E34-S981; nt100-2946) containing adjacent linkage regions. The portion encoding the signal peptide was kept constant in the following comparative experiments. The latter part of the sequence corresponding to nt100-2946 was varied. Four different publicly available codon optimization algorithms were used, as described above in this specification. Furthermore, custom mutants with increased GC content were created. The signal peptide sequence was appended to all of these mutants. This resulted in the following mutants: - Mutant "GC62" (SEQ ID NO: 18); GC content: 61.6% - Mutant "GC60" (SEQ ID NO: 19); GC content: 60.1% - Mutant "GC57" (SEQ ID NO: 22); GC content: 57.4% - Mutant "GC55" (SEQ ID NO: 23); GC content: 54.9% - Mutant "GC53" (SEQ ID NO: 24); GC content: 52.8% - "GENEius algorithm" (SEQ ID NO: 25); GC content: 52.6% - "ExpOptimizer" (SEQ ID NO: 27); GC content: 51.1% - "IDT Codon Optimization Tool" (SEQ ID NO: 26); GC content: 49.9% - "Twist Codon Optimization Tool" (SEQ ID NO: 28); GC content: 48.7%

[0400] To finalize the construct for synthesis, the 5'-UTR sequence (SEQ ID NO: 61), the 3'-UTR sequence (SEQ ID NO: 62), and the T7 promoter sequence were added. The complete mRNA sequences of the "GC62" and "GC60" mutants are given in SEQ ID NOs: 29 and 30, respectively.

[0401] Cloning and in vitro transcription (IVT) Using standard gene synthesis services and cloning techniques, vectors containing the coding sequences of the different Klotho variants described above were obtained. The poly(A) tail required for in vitro transcription was added by polymerase chain reaction (PCR). Vectors containing the different coding sequences were used as templates. Amplification was performed using Phusion according to the manufacturer's instructions. (商標) I used the Plus DNA Polymerase Kit (Thermo Fisher).

[0402] For PCR, 10 ng of plasmid DNA, a 0.7 μM forward primer, 5'-TGCTGCCGTAATACGACTCACTATAAGG-3' (SEQ ID NO: 77), and a 0.7 μM reverse primer, 5'-T120-TGCCGCCCACTCAGACTTTATTC-3' (SEQ ID NO: 78) (IDT Technologies) were used. PCR was performed using the following cycling protocol: initial activation step at 98°C for 30 seconds, followed by denaturation at 98°C for 5 seconds, annealing at 60°C for 10 seconds, extension at 72°C for 1:45 minutes, and final extension at 72°C for 5 minutes, for a total of 35 cycles. After DNA amplification, the PCR product was purified using the QIAquick PCR purification kit (Qiagen) and eluted in 2 × 20 μl of nuclease-free water (Qiagen). DNA quality and purity were evaluated by 1% agarose gel electrophoresis.

[0403] DNA to mRNA in vitro transpiration (IVT) was performed using the MEGAscript® T7 Kit (Life Technologies, Darmstadt, Germany) according to the manufacturer's instructions. Briefly, a 20 μl IVT reaction mixture was prepared containing 7.5 mM ATP, GTP, CTP, UDP, 6 mM CleanCAP (TriLink), 40 U RiboLock RNase inhibitor (Thermo Fisher Scientific, Waltham, USA), 100 ng of PCR product, 1 × reaction buffer, and 1 × T7 RNA polymerase enzyme mix. After incubation at 37°C for 4 hours, 1 μl of TURBO DNase (from the MEGAscript® T7 Kit) was added to the IVT reaction mixture, and the mixture was incubated at 37°C for 15 minutes to remove the template DNA. Following incubation, the mRNA was purified using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions and eluted in 2 × 35 μl of nuclease-free water. Subsequently, dephosphorylation was performed using 10 U of Antarctic phosphatase (New England Biolabs) at 37°C for 30 minutes. The mRNA was purified and eluted in 2 × 35 μL of nuclease-free water using the RNeasy Mini Kit. The quality and purity of the synthesized and modified mRNA were confirmed on a 1% agarose gel. The synthesized mRNA was stored at -80°C and used for transfection.

[0404] In vitro transfection In preparing mRNA transfections, cells were plated to approximately 80% density (human: HEK, Hs27; mouse: 3T3). After 4 hours, mRNA was complexed with JetMessenger (Polyplus) in a 1:2 ratio (μg mRNA:μL JetMessenger) according to the manufacturer's instructions. After 15 minutes of incubation, the mRNA complex was added dropwise to the cells.

[0405] 0.75 μg of mRNA was added to cells contained in a 24-well plate. The number of cells per well was as follows: 70,000 (for Hs27), 200,000 (for HEK), and 100,000 (for 3T3).

[0406] Depending on the type of experiment, the cell pellet and / or supernatant were collected and frozen at -80°C until further analysis was performed.

[0407] ELISA Klotho levels in cell culture supernatant were measured by ELISA (R&D Systems, DY5334-05) according to the manufacturer's instructions.

[0408] cell line All cell lines were cultured at 37°C in a 5% CO2 atmosphere. The culture medium and supplements were used as recommended by ATCC.

[0409] NIH / 3T3 fibroblasts (ATCC, Manassas, Virginia, USA) were cultured in high-glucose DMEM containing 10% fetal bovine serum (FCS) and 1% penicillin / streptomycin. Cell culture media and supplements were obtained from Thermo Fisher Scientific (Waltham, USA). Cells were maintained at 37°C and 5% CO2, and the medium was changed every 3 days. TrypLE (商標) Cells were passaged using Express Enzyme (Thermo Fisher Scientific, 1260401).

[0410] Hs27 human fibroblasts (ATCC, Manassas, Virginia, USA) were cultured in high-glucose DMEM containing 20% ​​fetal bovine serum (FCS) and 1% penicillin / streptomycin. Cell culture media and supplements were obtained from Thermo Fisher Scientific (Waltham, USA). Cells were maintained at 37°C and 5% CO2, and the medium was changed every 3 days. TrypLE (商標)Cells were passaged using Express Enzyme (Thermo Fisher Scientific, 1260401).

[0411] HEK293 cells (ATCC, Manassas, Virginia, USA) were cultured in high-glucose DMEM containing 10% fetal bovine serum (FCS) and 1% penicillin / streptomycin. Cell culture media and supplements were obtained from Thermo Fisher Scientific (Waltham, USA). Cells were kept at 37°C and 5% CO2, and the medium was changed every 3 days. TrypLE (商標) Cells were passaged using Express Enzyme (Thermo Fisher Scientific, 1260401).

[0412] result Figures 1 and 2 show two biologically independent in vitro experiments in three different cell lines (human: Hs27 and HEK, mouse: 3T3). Cell culture supernatants were collected 24 hours after mRNA transfection and analyzed by ELISA.

[0413] In all experiments and cell lines, the "GC62" mutant outperformed all other mutants, as well as the native Klotho sequence. The increase in expression levels compared to the native sequence ranged from 3 to 6 times in HEK and 3T3 cells, and more than 10 times in Hs27 cells. Furthermore, surprisingly, a direct relationship was found between GC content and expression levels.

[0414] [Example 2] Time-course experiments using different mRNA sequence variants To investigate how mRNA sequences affect Klotho expression levels over time, experiments were conducted on longer timescales. These experiments were basically performed in the same manner as in Example 1 above. HEK293 cells were transfected as described above. After transfection, samples were taken from the cell culture supernatant at various time points, and Klotho protein levels were analyzed as described above. The supernatant was changed every 24 hours during the experiment.

[0415] The following Klotho mRNA variants were tested: native human Klotho (SEQ ID NO: 17), variant "GC62" (SEQ ID NO: 18), and variant "GC60" (SEQ ID NO: 19). Based on these sequences, mRNA was synthesized as described in Example 1.

[0416] The results of this experiment are shown in Figures 3A and 3B. As can be seen from the time course in Figure 3A, the Klotho expression levels after transfection with the mutants GC62 and GC60 of the present invention were already significantly higher 24 hours after transfection. Surprisingly, however, this difference became even larger after 48 hours. At this point, the expression level obtained from the native sequence had already decreased, while the expression level obtained from the mutants of the present invention continued to increase. After 72 hours, the expression levels obtained from the GC62 and GC60 mutants were at least the same as the peak expression level obtained from the native sequence, while the expression level obtained from the native sequence had already decreased to near zero. This indicates that not only is the total expression level more strongly increased in the mutants of the present invention compared to the native sequence (see Figure 3B for cumulative expression over 7 days), but the time window in which expression remains high is also much longer (Figure 3A).

[0417] [Example 3] Comparative study with Klotho mRNA sequences described in International Publication No. 2013 / 151666 The inventors learned after the invention was made that other Klotho mRNA sequences were described in the prior art. International Publication No. 2013 / 151666 ("WO'666") relates broadly to mRNA for therapeutic use. In Table 6, WO'666 refers to 655 different "targets," among which klotho is mentioned as target number 364. For each target, numerous mRNA sequences are mentioned. This is also true for klotho as target number 364, and the following sequence identification numbers are listed in Table 6: "1493, 2019, 2639, 3259, 3879, 4499, 14948~15347." Although these sequences are merely arbitrarily mentioned in WO'666 without any clear evidence, let alone experimental data, the inventors undertook to investigate how these sequences behave compared to the Klotho mRNA sequence according to the present invention.

[0418] The sequence disclosed in WO'666 encodes the full-length human Klotho isoform 1 (amino acids M1 to K1012). Therefore, as described in Example 1, the sequence encoding soluble Klotho, i.e., amino acids M1 to S981, was extracted. These sequences are referred to herein as follows: - "WO666_SEQ_1493": Sequence ID 132 - "WO666_SEQ_14948": Sequence ID 133 - "WO666_SEQ_4499": Sequence ID 134 - "WO666_SEQ_2639": Sequence ID 135 - "WO666_SEQ_3879": Sequence ID 136 - "WO666_SEQ_3259": Sequence ID 137 - "WO666_SEQ_2019": Sequence ID 138

[0419] These sequences were tested against the GC62, GC60, GC57, GC55, GC53 variants and native Klotho mRNA sequences disclosed herein. mRNA based on all of these sequences was synthesized, transfected into HEK293 cells, and analyzed as described in Example 1.

[0420] The results are shown in Figure 4A. The highest expression levels of the Klotho protein were observed in the mutants GC62 and GC60 of the present invention. Among the sequences mentioned in WO'666, the highest expression levels were observed in sequences 2019 (SEQ ID NO: 138) and 3259 (SEQ ID NO: 137), but these were significantly lower than those of the mutants GC62 and GC60 of the present invention.

[0421] The results obtained, along with the GC content of each sequence and its sequence identity to the GC62 variant (SEQ ID NO: 18), are shown in the table below:

[0422] [Table 4]

[0423] As can be seen from the table above, there is a significant correlation between expression levels and sequence identity to the GC62 variant of the present invention. The higher the sequence identity to GC62, the higher the expression level. The only constructs in WO'666 that do not follow this trend are two sequences with extremely high GC content (sequence 1493, 67.0%) and extremely low GC content (sequence number 132). After removing these outliers with extreme GC content, the remaining 11 data points (including the native sequence and GC53-GC62 variants) show a significant linear correlation, and R 2 The value was 0.88 (see Figure 4B).

[0424] These results demonstrate that sequence identity with the GC62 variant highly predicts the resulting expression levels.

[0425] [Example 4] Monoclonal cells for Klotho expression To create a monoclonal cell line expressing Klotho, HEK293 cells were stably transfected with a DNA vector encoding the Klotho mRNA mutant GC62.

[0426] Plasmid design: The plasmid was designed in silico. The plasmid backbone used was pcDNA3.1 / Hygro(+), which contained a hygromycin selection cassette and a CMV promoter. The DNA sequence encoding the GC62 mRNA variant of the present invention (SEQ ID NO: 18) was inserted into the multicloning site immediately following the CMV promoter to ensure potent and stable expression of the target gene.

[0427] Transfection and selection: HEK293 cells were transfected with Lipofectamine 3000 according to the manufacturer's protocol. 72 hours after transfection, growth medium was supplemented with 100 ug / mL of hygromycin, and transfected cells were selected. After 7 days, all untransfected cells had died.

[0428] Single-cell cloning: Transfected and selected cells were trypsin-treated and counted. The cell suspension was diluted to 8 cells / mL and plated into several 96-well plates (100 μL / well). After 10 days, wells with single colonies were expanded to 24-well plates, and then to 6-well plates.

[0429] Characterization of single-cell clones: To characterize single-cell clones, 240,000 cells from each clone were seeded in 24-well plates, and the supernatant was collected and replaced at 24, 48, and 72 hours. The Klotho concentration in the supernatant was determined by ELISA as described in Example 1. The best clone was selected and further experiments were conducted.

[0430] result: Figure 5 shows the Klotho protein expression levels from cells of a single high-expression clone compared to transient transfection using GC62 mRNA. As can be seen from the figure, the expression levels obtained from stably transfected cells continued to increase even after 72 hours, while the expression levels obtained from transiently transfected cells began to decrease.

[0431] [Example 5] In vivo experiment To test the effects of the mRNA of the present invention in vivo, experiments were conducted using mice. In these experiments, serum levels of Klotho protein obtained from treatment with the Klotho mRNA mutant GC62 of the present invention were compared to those obtained with the native Klotho mRNA sequence. In these experiments, modified versions of the mRNA sequence, in which certain restriction enzyme recognition sites were removed, were used to facilitate molecular cloning. Therefore, SEQ ID NO: 96 of the Klotho mRNA mutant GC62 was used instead of SEQ ID NO: 18, and SEQ ID NO: 95 of the native Klotho mRNA sequence was used instead of SEQ ID NO: 17. In both cases, the mRNA sequence encoded soluble Klotho (amino acids M1 to S981). Aside from the modification to remove the restriction enzyme recognition site, the mRNA construct was the same as that described in Example 1.

[0432] These mRNA constructs were formulated using LNPs. 100 μg of the LNP-formulated mRNA was injected into each mouse. Five mice were used at each time point for each native sequence and GC62 variant. Terminal blood samples were collected at 6, 24, 48, and 72 hours. Serum Klotho protein levels were determined by ELISA.

[0433] The results are shown in Figure 6. Serum Klotho levels were significantly higher when using the Klotho mRNA mutant GC62 of the present invention compared to when using the native sequence, even when the mRNA injection volume and amino acid sequence were the same. Therefore, the strong increase in Klotho serum levels was directly caused by the improvement of the mRNA sequence.

[0434] The difference in Klotho serum levels was particularly pronounced at later time points. As shown in Figure 6B, serum Klotho levels 72 hours after injection were more than twice the values ​​obtained from native Klotho mRNA sequences. Thus, the mRNA of the present invention not only enhanced total expression but also significantly expanded the expression time window.

[0435] array The following table provides an overview of the RNA sequences disclosed herein.

[0436] [Table 5] TIFF2026511117000009.tif36162

[0437] [Table 6] TIFF2026511117000011.tif55169

[0438] The following table provides an overview of the protein sequences disclosed herein.

[0439] [Table 7]

[0440] Sequence ID 1: Sequence ID 2: Sequence ID 3: Sequence ID 4: Sequence ID 5: Sequence ID 6: Sequence ID 7: Sequence ID 8: Sequence ID 9: Sequence ID 10: Sequence ID 11: Sequence ID 12: Sequence ID 13: Sequence ID 14: Sequence ID 15: Sequence ID 16: Sequence ID 17: Sequence ID 18: Sequence ID 19: Sequence ID 20: Sequence ID 21: Sequence ID 22: Sequence ID 23: Sequence ID 24: Sequence ID 25: Sequence ID 26: Sequence ID 27: Sequence ID 28: Sequence ID 29: Sequence ID 30: Sequence ID 31: Sequence ID 32: Sequence ID 33: Sequence ID 34: Sequence ID 35: Sequence ID 36: Sequence ID 37: Sequence ID 38: Sequence ID 39: Sequence ID 40: Sequence ID 41: Sequence ID 42: Sequence ID 43: Sequence ID 44: Sequence ID 45: Sequence ID 46: Sequence ID 47: Sequence ID 48: Sequence ID 64: Sequence ID 65: Sequence ID 66: Sequence ID 67: Sequence ID 68: Sequence ID 69: Sequence ID 70: Sequence ID 71: Sequence ID 72: Sequence ID 73: Sequence ID 74: Sequence ID 75: Sequence ID 79: Sequence ID 80: Sequence ID 81: Sequence ID 82: Sequence ID 83: Sequence ID 84: Sequence ID 85: Sequence ID 86: Sequence ID 87: Sequence ID 88: Sequence ID 89: Sequence ID 90: Sequence ID 91: Sequence ID 92: Sequence ID 93: Sequence ID 94: Sequence ID 95: Sequence ID 96: Sequence ID 97: Sequence ID 98: Sequence ID 99: Sequence ID 100: Sequence ID 101: Sequence ID 102: Sequence ID 103: Sequence ID 104: Sequence ID 105: Sequence ID 106: Sequence ID 107: Sequence ID 108: Sequence ID 109: Sequence ID 110: Sequence ID 111: Sequence ID 112: Sequence ID 113: Sequence ID 114: Sequence ID 115: Sequence ID 116: Sequence ID 117: Sequence ID 118: Sequence ID 119: Sequence ID 120: Sequence ID 121: Sequence ID 122: Sequence ID 123: Sequence ID 124: Sequence ID 125: Sequence ID 126: Sequence ID 127: Sequence ID 128: Sequence ID 129: Sequence ID 130: Sequence ID 131: Sequence ID 132: Sequence ID 133: Sequence ID 134: Sequence ID 135: Sequence ID 136: Sequence ID 137: Sequence ID 138: Sequence ID 49: Sequence ID 50: MPASAPPRRPRPPPPSLSLLLVLLGLGGRRLRAEPGDGAQTWARFSRPPAPEAAGLFQGTFPDGFLWAVGSAAYQTEGGWQQHGKGASIWDTFTHHPLAPPGDSRNASLPLGAPSPLQPATGDVASDSYNNVFRDTE ALRELGVTHYRFSISWARVLPNGSAGVPNREGLRYYRRLLERLRELGVQPVVTLYHWDLPQRLQDAYGGWANRALADHFRDYAELCFRHFGGQVKYWITIDNPYVVAWHGYATGRLAPGIRGSPRLGYLVAHNLLLA HAKVWHLYNTSFRPTQGGQVSIALSSHWINPRRMTDHSIKECQKSLDFVLGWFAKPVFIDGDYPESMKNNLSSILPDFTESEKKFIKGTADFFALCFGPTLSFQLLDPHMKFRQLESPNLRQLLSWIDLEFNHPQIF IVENGWFVSGTTKRDDAKYMYYLKKFIMETLKAIKLDGVDVIGYTAWSLMDGFEWHRGYSIRRGLFYVDFLSQDKMLLPKSSALFYQKLIEKNGFPPLPENQPLEGTFPCDFAWGVVDNYIQVSQLTKPISSLTKPYH Sequence ID 51: MPASAPPRRPRPPPPSLSLLLVLLGLGGRRLRA Sequence ID 52: LFQGTFPDGFLWAVGSAAYQTEGGWQQHGKGASIWDTFTHHPLAPPGDSRNASLPLGAPSPLQPATGDVASDSYNNVFRDTEALRELGVTHYRFSISWARVLPNGSAGVPNR EGLRYYRRLLERLRELGVQPVVTLYHWDLPQRLQDAYGGWANRALADHFRDYAELCFRHFGGQVKYWITIDNPYVVAWHGYATGRLAPGIRGSPRLGYLVAHNLLLAHAKVWH LYNTSFRPTQGGQVSIALSSHWINPRRMTDHSIKECQKSLDFVLGWFAKPVFIDGDYPESMKNNLSSILPDFTESEKKFIKGTADFFALCFGPTLSFQLLDPHMKFRQLESPN LRQLLSWIDLEFNHPQIFIVENGWFVSGTTKRDDAKYMYYLKKFIMETLKAIKLDGVDVIGYTAWSLMDGFEWHRGYSIRRGLFYVDFLSQDKMLLPKSSALFYQKLIEKNGF Sequence ID 53: LFQGTFPDGFLWAVGSAAYQTEGGWQQHGKGASIWDTFTHHPLAPPGDSRNASLPLGAPSPLQPATGDVASDSYNNVFRDTEALRELGVTHYRFSISWARVLPNGSAGVPNR EGLRYYRRLLERLRELGVQPVVTLYHWDLPQRLQDAYGGWANRALADHFRDYAELCFRHFGGQVKYWITIDNPYVVAWHGYATGRLAPGIRGSPRLGYLVAHNLLLAHAKVWH LYNTSFRPTQGGQVSIALSSHWINPRRMTDHSIKECQKSLDFVLGWFAKPVFIDGDYPESMKNNLSSILPDVTESEKKFIKGTADFFALSFGPTLSFQLLDPHMKFRQLESPN LRQLLSWIDLEFNHPQIFIVENGWFVSGTTKRDDAKYMYYLKKFIMETLKAIKLDGVDVIGYTAWSLMDGFEWHRGYSIRRGLFYVDFLSQDKMLLPKSSALFYQKLIEKNGF Sequence ID 54: LEGTFPCDFAWGVVDNYIQVDTTLSQFTDLNVYLWDVHHSKRLIKVDGVVTKKRKSYCVDFAAIQPQIALLQEMHVTHFRFSLDWALILPLGNQSQVNHTILQYYRCMA SELVRVNITPVVALWQPMAPNQGLPRLLARQGAWENPYTALAFAEYARLCFQELGHHVKLWITMNEPYTRNMTYSAGHNLLKAHALAWHVYNEKFRHAQNGKISIALQAD WIEPACPFSQKDKEVAERVLEFDIGWLAEPIFGSGDYPWVMRDWLNQRNNFLLPYFTEDEKKLIQGTFDFLALSHYTTILVDSEKEDPIKYNDYLEVQEMTDITWLNSP SQVAVVPWGLRKVLNWLKFKYGDLPMYIISNGIDDGLHAEDDQLRVYYMQNYINEALKAHILDGINLCGYFAYSFNDRTAPRFGLYRYAADQFEPKASMKHYRKIIDSNG Sequence ID 55: MPASAPPRRPRPPPPSLSLLLVLLGLGGRRLRAEPGDGAQTWARFSRPPAPEAGLFQGTFPDGFLWAVGSAAYQTEGGWQQHGKGASIWDTFTHHPLAPPGDSRNASLPLGAPSPLQPATGDVAS DSYNNVFRDTEALRELGVTHYRFSISWARVLPNGSAGVPNREGLRYYRRLLERLRELGVQPVVTLYHWDLPQRLQDAYGGWANRALADHFRDYAELCFRHFGGQVKYWITIDNPYVVAWHGYATGRL APGIRGSPRLGYLVAHNLLLAHAKVWHLYNTSFRPTQGGQVSIALSSHWINPRRMTDHSIKECQKSLDFVLGWFAKPVFIDGDYPESMKNNLSSILPDFTESEKKFIKGTADFFALCFGPTLSFQL LDPHMKFRQLESPNLRQLLSWIDLEFNHPQIFIVENGWFVSGTTKRDDAKYMYYLKKFIMETLKAIKLDGVDVIGYTAWSLMDGFEWHRGYSIRRGLFYVDFLSQDKMLLPKSSALFYQKLIEKNGF Sequence ID 56: MPASAPPRRPRPPPPSLSLLLVLLGLGGRRLRAEPGDGAQTWARFSRPPAPEAGLFQGTFPDGFLWAVGSAAYQTEGGWQQHGKGASIWDTFTHHPLAPPGDSRNASLPLGAPSPLQPATGDVAS DSYNNVFRDTEALRELGVTHYRFSISWARVLPNGSAGVPNREGLRYYRRLLERLRELGVQPVVTLYHWDLPQRLQDAYGGWANRALADHFRDYAELCFRHFGGQVKYWITIDNPYVVAWHGYATGRL APGIRGSPRLGYLVAHNLLLAHAKVWHLYNTSFRPTQGGQVSIALSSHWINPRRMTDHSIKECQKSLDFVLGWFAKPVFIDGDYPESMKNNLSSILPDVTESEKKFIKGTADFFALSFGPTLSFQL LDPHMKFRQLESPNLRQLLSWIDLEFNHPQIFIVENGWFVSGTTKRDDAKYMYYLKKFIMETLKAIKLDGVDVIGYTAWSLMDGFEWHRGYSIRRGLFYVDFLSQDKMLLPKSSALFYQKLIEKNGF Sequence ID 57: MPASAPPRRPRPPPPSLSLLLVLLGLGGRRLRAEPGDGAQTWARFSRPPAPEAAGLFQGTFPDGFLWAVGSAAYQTEGGWQQHGKGASIWDTFTHHPLAPPGDSRNASLPLGAPSPLQPATG DVASDSYNNVFRDTEALRELGVTHYRFSISWARVLPNGSAGVPNREGLRYYRRLLERLRELGVQPVVTLYHWDLPQRLQDAYGGWANRALADHFRDYAELCFRHFGGQVKYWITIDNPYVVAW HGYATGRLAPGIRGSPRLGYLVAHNLLLAHAKVWHLYNTSFRPTQGGQVSIALSSHWINPRRMTDHSIKECQKSLDFVLGWFAKPVFIDGDYPESMKNNLSSILPDFTESEKKFIKGTADFF ALCFGPTLSFQLLDPHMKFRQLESPNLRQLLSWIDLEFNHPQIFIVENGWFVSGTTKRDDAKYMYYLKKFIMETLKAIKLDGVDVIGYTAWSLMDGFEWHRGYSIRRGLFYVDFLSQDKMLLP KSSALFYQKLIEKNGFPPLPENQPLEGTFPCDFAWGVVDNYIQVDTTLSQFTDLNVYLWDVHHSKRLIKVDGVVTKKRKSYCVDFAAIQPQIALLQEMHVTHFRFSLDWALILPLGNQSQVN HTILQYYRCMASELVRVNITPVVALWQPMAPNQGLPRLLARQGAWENPYTALAFAEYARLCFQELGHHVKLWITMNEPYTRNMTYSAGHNLLKAHALAWHVYNEKFRHAQNGKISIALQADWI EPACPFSQKDKEVAERVLEFDIGWLAEPIFGSGDYPWVMRDWLNQRNNFLLPYFTEDEKKLIQGTFDFLALSHYTTILVDSEKEDPIKYNDYLEVQEMTDITWLNSPSQVAVVPWGLRKVLNW LKFKYGDLPMYIISNGIDDGLHAEDDQLRVYYMQNYINEALKAHILDGINLCGYFAYSFNDRTAPRFGLYRYAADQFEPKASMKHYRKIIDSNGFPGPETLERFCPEEFTVCTECSFFHTRKS Sequence ID 58: MPASAPPRRPRPPPPSLSLLLVLLGLGGRRLRAEPGDGAQTWARFSRPPAPEAAGLFQGTFPDGFLWAVGSAAYQTEGGWQQHGKGASIWDTFTHHPLAPPGDSRNASLPLGAPSPLQPATGDVASDSYNNVFRDTEALRELGVTHYRFSISWARVLPNGSAGVPNREGLRYYRRLLERLRELGVQPVVTLYHWDLPQRLQDAYGGWANRALADHFRDYAELCFRHFGGQVKYWITIDNPYVVAWHGYATGRLAPGIRGSPRLGYLVAHNLLLAHAKVWHLYNTSFRPTQGGQVSIALSSHWINPRRMTDHSIKECQKSLDFVLGWFAKPVFIDGDYPESMKNNLSSILPDVTESEKKFIKGTADFFALSFGPTLSFQLLDPHMKFRQLESPNLRQLLSWIDLEFNHPQIFIVENGWFVSGTTKRDDAKYMYYLKKFIMETLKAIKLDGVDVIGYTAWSLMDGFEWHRGYSIRRGLFYVDFLSQDKMLLPKSSALFYQKLIEKNGFPPLPENQPLEGTFPCDFAWGVVDNYIQVDTTLSQFTDLNVYLWDVHHSKRLIKVDGVVTKKRKSYCVDFAAIQPQIALLQEMHVTHFRFSLDWALILPLGNQSQVNHTILQYYRCMASELVRVNITPVVALWQPMAPNQGLPRLLARQGAWENPYTALAFAEYARLCFQELGHHVKLWITMNEPYTRNMTYSAGHNLLKAHALAWHVYNEKFRHAQNGKISIALQADWIEPACPFSQKDKEVAERVLEFDIGWLAEPIFGSGDYPWVMRDWLNQRNNFLLPYFTEDEKKLIQGTFDFLALSHYTTILVDSEKEDPIKYNDYLEVQEMTDITWLNSPSQVAVVPWGLRKVLNWLKFKYGDLPMYIISNGIDDGLHAEDDQLRVYYMQNYINEALKAHILDGINLCGYFAYSFNDRTAPRFGLYRYAADQFEPKASMKHYRKIIDSNGFPGPETLERFCPEEFTVCTECSFFHTRKS Sequence ID 59: MPASAPPRRPRPPPPSLSLLLVLLGLGGRRLRAEPGDGAQTWARFSRPPAPEAAGLFQGTFPDGFLWAVGSAAYQTEGGWQQHGKGASIWDTFTHHPLAPPGDSRNASLPLGAPSPLQPATGDVASDSYNNVFRDTE ALRELGVTHYRFSISWARVLPNGSAGVPNREGLRYYRRLLERLRELGVQPVVTLYHWDLPQRLQDAYGGWANRALADHFRDYAELCFRHFGGQVKYWITIDNPYVVAWHGYATGRLAPGIRGSPRLGYLVAHNLLLA HAKVWHLYNTSFRPTQGGQVSIALSSHWINPRRMTDHSIKECQKSLDFVLGWFAKPVFIDGDYPESMKNNLSSILPDFTESEKKFIKGTADFFALCFGPTLSFQLLDPHMKFRQLESPNLRQLLSWIDLEFNHPQIF IVENGWFVSGTTKRDDAKYMYYLKKFIMETLKAIKLDGVDVIGYTAWSLMDGFEWHRGYSIRRGLFYVDFLSQDKMLLPKSSALFYQKLIEKNGFPPLPENQPLEGTFPCDFAWGVVDNYIQVSQLTKPISSLTKPYH Sequence ID 60: MPASAPPRRPRPPPPSLSLLLVLLGLGGRRLRAEPGDGAQTWARFSRPPAPEAAGLFQGTFPDGFLWAVGSAAYQTEGGWQQHGKGASIWDTFTHHPLAPPGDSRNASLPLGAPSPLQPATGDVASDSYNNVFRDTE ALRELGVTHYRFSISWARVLPNGSAGVPNREGLRYYRRLLERLRELGVQPVVTLYHWDLPQRLQDAYGGWANRALADHFRDYAELCFRHFGGQVKYWITIDNPYVVAWHGYATGRLAPGIRGSPRLGYLVAHNLLLA HAKVWHLYNTSFRPTQGGQVSIALSSHWINPRRMTDHSIKECQKSLDFVLGWFAKPVFIDGDYPESMKNNLSSILPDVTESEKKFIKGTADFFALSFGPTLSFQLLDPHMKFRQLESPNLRQLLSWIDLEFNHPQIF IVENGWFVSGTTKRDDAKYMYYLKKFIMETLKAIKLDGVDVIGYTAWSLMDGFEWHRGYSIRRGLFYVDFLSQDKMLLPKSSALFYQKLIEKNGFPPLPENQPLEGTFPCDFAWGVVDNYIQVSQLTKPISSLTKPYH Sequence ID 76: sequence number 61: GGGAGACAUAAACCCUGGCGCGCUCGCGGCCCGCACUCUUCUGGUCCCCACAGACUCAGAGAGAACCCACC sequence number 62: GCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCA sequence number 63: YCCANCCCWNUCYCC sequence number 77: TGCTGCCGTAATACGACTCACTATAAGG sequence number 78: TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT GCCGCCCACTCAGACTTTATTC

Claims

1. Klotho messenger RNA (mRNA) having a 5' CAP region, a 5' untranslated region (5'-UTR), a coding region encoding a Klotho polypeptide, a 3' untranslated region (3'-UTR), and a polyadenosine tail (poly-A tail), The Klotho polypeptide contains the KL1 domain of human Klotho. The coding region encoding the Klotho polypeptide includes an RNA sequence having at least 80% sequence identity with SEQ ID NO: 1 or 79. Klotho mRNA in which the coding region encoding the Klotho polypeptide has a GC content of at least 54%.

2. The Klotho polypeptide contains the KL1 and KL2 domains of human Klotho. The Klotho mRNA according to claim 1, wherein the coding region encoding the Klotho polypeptide includes an RNA sequence having at least 80% sequence identity with SEQ ID NO: 13 or 91.

3. The Klotho mRNA according to any one of the claims, wherein the coding region encoding the Klotho polypeptide has at least 80% sequence identity with SEQ ID NOs. 18, 96, 41, 112, 68, or 124.

4. Klotho mRNA according to any one of the claims, wherein the coding region encoding the Klotho polypeptide has at least 93% sequence identity with at least one of sequence numbers 1, 79, 13, 91, 18, 96, 41, 112, 68, or 124, and the coding region encoding the Klotho polypeptide has a GC content of 54% to 66%.

5. The Klotho mRNA according to any one of the claims, wherein the coding region encoding the Klotho polypeptide has at least 93% sequence identity with SEQ ID NO: 18 or 96, the coding region encoding the Klotho polypeptide has a GC content of 54% to 66%, and the Klotho polypeptide is SEQ ID NO:

57.

6. The code regions that encode the Klotho polypeptide are sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, 24, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 64, 65, 66, 67, 68, 69, 70, 71, 79, 80, 81, 82, 83 Klotho mRNA according to any one of the claims, comprising a sequence selected from 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 120, 121, 122, 123, 124, 125, 126, 127.

7. Klotho mRNA according to any one of the claims, wherein the coding region encoding the Klotho polypeptide is selected from SEQ ID NOs: 18, 19, 20, 21, 96, 97, 98, 99, 41, 42, 43, 44, 112, 113, 114, 115, 68, 69, 70, 71, 124, 125, 126, 127.

8. The Klotho mRNA according to any one of the claims, wherein the Klotho polypeptide has at least 90% sequence identity with SEQ ID NOs: 49, 55, 57, or 59, and preferably the Klotho polypeptide is selected from SEQ ID NOs: 49, 55, 56, 57, 58, 59, 60, or 76, preferably 49, 57, or 59.

9. Klotho mRNA according to any one of the claims, wherein the Klotho mRNA is sequence numbers 29, 30, 31, 32, 100, 101, 102, 103, 45, 46, 47, 48, 116, 117, 118, 119, 72, 73, 74, 75, 128, 129, 130, 131; preferably sequence numbers 29, 100, 45, 116, 72, 128.

10. In Klotho mRNA, - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of the total cytidine residues are replaced with 5-methylcytidine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all cytidine residues are replaced with 2-amino-2-deoxycytidine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all cytidine residues are replaced with 2-fluoro-2-deoxycytidine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all cytidine residues are replaced with 2-thiocytidine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all cytidine residues are replaced with 5-iodocytidine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all uridine residues are replaced with pseudouridine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all uridine residues are replaced with 1-methylpseudridine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all uridine residues are replaced with 2-thiouridine residues, and / or - At least 5%, preferably at least 10%, more preferably at least 30%, and especially at least 50% of all uridine residues are replaced with 5-methyluridine residues, and / or - Klotho mRNA according to any one of the claims, wherein at least 5%, preferably at least 10%, more preferably at least 30%, and particularly at least 50% of the total adenosine residues are replaced with N6-methyladenosine residues.

11. A pharmaceutical formulation comprising Klotho mRNA and a pharmaceutically acceptable carrier as described in any one of the preceding claims.

12. A pharmaceutical formulation according to any one of the claims, comprising further mRNA, wherein the further mRNA has a further 5'-CAP region, a further 5'-UTR, a further coding region encoding a further polypeptide, a further 3'-UTR, and a further poly-A tail, and preferably the further polypeptide is a bactericidal / permeability-enhancing protein family B, member 4 (BPIFB4) protein, or an isoform or variant thereof, preferably a longevity-associated variant of BPIFB4 (LAV-BPIFB4).

13. Further polypeptides include apolipoprotein E, oncoprotein p53, sirtuin 1 protein, FOXO1 transcription factor, cholinergic receptor nicotin alpha 3 subunit, SH2B adapter protein 3, cyclin-dependent kinase inhibitor 2A, and very-long-chain elongation enzyme-like protein 2 (Elongation of very-long-chain fatty acid-like protein 2). 2) Werner protein, paraoxonase 1, superoxide dismutase 2, lamin A protein, cholesteryl ester transfer protein, apolipoprotein C3, microsomal triglyceride transfer protein, phosphatidylinositol 3-kinase (PI3K), insulin-like growth factor 1 (IGF-1) receptor, protein-L-isoaspartyl methyltransferase, growth hormone, cAMP response element binding protein, mitogen-activated protein kinase, epidermal growth factor receptor, nuclear factor kappa B, phospholipase C beta, methionine sulfoxide reductase A, cell motility mediator 1, Nei-like DNA glycosylase 1, peroxisome proliferator-activated receptor gamma 2, eukaryotic translation initiation factor 3 subunit K, ATM serine / threonine kinase, B-cell lymphoma 2, cell division cycle 42 42) A pharmaceutical formulation according to any one of the claims, wherein the protein is selected from diacylglycerol O-acyltransferase 1, Early growth response 1, fibroblast growth factor 23, fibroblast growth factor 21, fructosamine 3 kinase-related protein, phosphoglycolate phosphatase, insulin receptor substrate 1, Polycomb complex protein BMI-1, Neureglin 1, signal transduction and transcription activator, E2F transcription factor 1, vascular endothelial growth factor A, xenobiotic metabolizing enzymes, Myc proto-oncogene protein, C-X-C chemokine receptor type 4, Silent information regulator 2, extracellular signal-regulated kinase, and SLC31.

14. A kit for administering Klotho mRNA according to any one of the above claims to an individual, preferably a human, - Klotho mRNA according to any one of the above claims, and - A device for administering Klotho mRNA. A kit that includes this.

15. A DNA molecule comprising a DNA sequence encoding the coding region of Klotho mRNA as defined in any one of the above claims.

16. A cell comprising the DNA molecule defined in the above claim, preferably a mammalian cell, preferably a human embryonic kidney (HEK) 293 cell.

17. A therapeutic delivery device comprising cells as defined in the above claims, wherein the cells are encapsulated in a biocompatible scaffold.

18. An extracellular vesicle (EV) comprising Klotho mRNA as defined in any one of the above claims, preferably an EV derived from a cell as defined in any one of the above claims.

19. A population of EVs as defined in the claim, wherein the average number of Klotho mRNA molecules per EV is greater than 1 per EV throughout the entire population of EVs.

20. A method for generating an EV or a group of EVs according to any one of the above claims, - Culturing cells as defined in any one of the above claims under conditions suitable for EV formation, - Isolating EVs and A method that includes this.

21. A pharmaceutical formulation comprising an EV or a group of EVs as defined in any one of the preceding claims and a pharmaceutically acceptable carrier.

22. A Klotho mRNA, a pharmaceutical formulation, a therapeutic delivery device, an EV, or a group of EVs as described in any one of the claims, for use as a pharmaceutical.

23. Klotho mRNA, a pharmaceutical formulation, a therapeutic delivery device, an EV, or a group of EVs according to any one of the claims, for use in the prevention or treatment of age-related disorders and / or symptoms of aging.

24. Use of Klotho mRNA, EV, or population of EV according to any one of the claims for increasing the lifespan of a mammal, preferably a human.

Images