Secreted splicing variants of Klotho for lifespan extension

Abstract

The present invention provides a polypeptide consisting of the sequence of SEQ ID NO: 1, or a variant thereof consisting of a sequence at least 85% identical to SEQ ID NO: 1, for use in extending the lifespan of a subject. The present invention also provides a nucleic acid sequence encoding the polypeptide, a genetic construct comprising the nucleic acid sequence, or an expression vector comprising the genetic construct, for said use. The polypeptide, nucleic acid sequence, genetic construct, or expression vector of the present invention may be administered in the form of a pharmaceutical composition, together with at least one pharmaceutically acceptable excipient, diluent, or carrier.

Description

[Technical Field]

[0001] This application claims the benefit of European Patent Application No. 22383171.0, filed December 2, 2022.

[0002] [Technical field] The present invention relates to methods for extending the lifespan of organisms.

[0003] [Background technology] Calendar age is well understood to be the single greatest risk factor for nearly all major causes of mortality and morbidity in living organisms, including humans. Even before the onset of observable disease, the physiology of organ systems and tissues progressively declines throughout life. Therefore, interventions to extend lifespan, i.e., slow the aging process itself, may be able to delay the onset of age-related diseases and death overall, which would be a much more efficient therapeutic approach than treating age-related diseases individually.

[0004] Although our understanding of the biology of aging and longevity has increased greatly over the past several decades, identifying lifespan-extending treatments and elucidating the molecular mechanisms underlying longevity have proven difficult. Accordingly, products and methods for extending lifespan have generally proven ineffective and / or unsafe.

[0005] Most studies reporting lifespan extension in animals have been conducted in animal models with a specific disease or condition. As a result, in most cases, interventions are considered to be the result of preventing or treating a specific condition, rather than extending lifespan itself. Therefore, identifying lifespan-extending effects per se, as opposed to disease treatments that indirectly extend lifespan in diseased animals, has proven difficult.

[0006] The Klotho gene was discovered in 1997 as a gene whose absence in mice conferred an accelerated or premature aging phenotype and a dramatically shortened lifespan. Yet, despite more than 25 years of intensive research since the discovery of Klotho, there are currently no available Klotho-based treatments or clinical trials for lifespan extension.

[0007] Thus, despite the efforts made to date, there remains a need in the art for safe compounds that have the ability to extend the lifespan of a subject.

[0008] [Summary of the Invention] The present inventors have developed a novel treatment for lifespan extension based on the administration of the secreted splicing isoform of Klotho, s-KL.

[0009] The Klotho gene expresses two major transcripts: full-length Klotho mRNA and alternatively spliced ​​Klotho mRNA. Full-length Klotho mRNA transcribes a 135-kDa, single-pass transmembrane protein called m-KL. The extracellular domain of m-KL can be released from the membrane by protease-mediated shedding to generate soluble, circulating processed Klotho (p-KL, 130 kDa), sometimes simply called soluble Klotho, which has two active domains. Alternatively spliced ​​Klotho mRNA displays a premature stop codon and generates a secreted protein, s-KL (70 kDa), which contains only one of the active domains and an extra 15 amino acids at the C-terminus of s-KL. Although the same abbreviation (s-KL) is sometimes used in the prior art to refer to soluble Klotho, a processed version of the full-length transmembrane Klotho, and secreted Klotho, a splicing isoform, these two isoforms exhibit completely different structures, sizes, and biological activities. Indeed, full-length Klotho has been described to be involved in FGF23 receptor binding, PTH synthesis, regulation of parathyroid growth, and alteration of vitamin D metabolism and calcium ion blood levels, whereas the secreted splicing isoform (s-KL) of Klotho has not.

[0010] The present invention is based exclusively on the use of secreted Klotho, a splicing isoform whose biological function remains unknown, and the abbreviation s-KL is used herein to refer exclusively to secreted Klotho.

[0011] Surprisingly, the present inventors found that s-KL administration increased both median and total lifespan in mice without causing any adverse effects, whether administered at the juvenile (i.e., 6 months) or adult (i.e., 12 months) developmental stage.

[0012] It should be noted that the results provided herein were obtained in a naive (i.e., non-diseased) animal model, thus allowing conclusions to be drawn regarding the positive direct effect of s-KL on the lifespan of animals and excluding the results as an indirect consequence of disease treatment. Furthermore, the results were obtained by administering exogenous s-KL rather than by genetic manipulation of animals, clearly demonstrating that the anti-aging effects of s-KL proposed herein can be readily translated into clinical applications for promoting longevity.

[0013] In view of the above, the present invention constitutes a significant advance in the field of life extension strategies and may help indirectly prevent all kinds of age-related diseases.

[0014] Thus, in a first aspect, the present invention provides a polypeptide consisting of the sequence of SEQ ID NO: 1, or a variant thereof consisting of a sequence which is at least 85% identical to SEQ ID NO: 1, for use in extending the lifespan of a subject.

[0015] In a second aspect, the present invention provides a nucleic acid sequence encoding a polypeptide as defined in the first aspect or a variant thereof for use in extending the lifespan of a subject.

[0016] In a third aspect, the present invention provides a genetic construct comprising a nucleic acid sequence as defined in the second aspect operably linked to an expression promoter, for use in extending the lifespan of a subject.

[0017] In a fourth aspect, the present invention provides an expression vector comprising a genetic construct as defined in the third aspect for use in extending the lifespan of a subject.

[0018] In a fifth aspect, the present invention provides a host cell transformed or transfected with a nucleic acid sequence as defined in the second aspect, a genetic construct as defined in the third aspect, or an expression vector as defined in the fourth aspect, for use in extending the lifespan of a subject.

[0019] In a sixth aspect, the present invention provides a method for extending the lifespan of a subject, comprising administering to the subject a polypeptide consisting of the sequence of SEQ ID NO: 1 or a variant thereof consisting of a sequence that is at least 85% identical to SEQ ID NO: 1, or a nucleic acid sequence encoding the polypeptide or variant thereof.

[0020] In a seventh aspect, the present invention provides the use of a polypeptide consisting of the sequence of SEQ ID NO: 1, or a variant thereof consisting of a sequence which is at least 85% identical to SEQ ID NO: 1, or a nucleic acid sequence encoding said polypeptide or said variant, for extending the lifespan of a subject. [Brief explanation of the drawings]

[0021] [Figure 1] Figure 1 shows that s-KL treatment efficiently increased s-KL protein concentrations. a) Schematic of experimental design. b) Analysis of s-KL gene expression in the liver of males (left panel) and females (right panel). c) Quantification of total s-KL protein concentrations in the serum of males (left panel) and females (right panel). Analysis was performed using samples from a subset of animals euthanized at 24 months of age. In (b), data are expressed as fold changes compared to null-treated animals. Mean ± standard error (SEM), n = 4, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001. [Figure 2] Figure 1: s-KL treatment increases median and total lifespan in wild-type mice. a) Body weight follow-up. b) Violin plot of median survival times for the different treatments studied. Mean ± standard error of mean (SEM), n = 11-12 mice, *p < 0.05. c) Kaplan-Meier longevity curves are used to depict the longevity evolution of the different groups.

[0022] [Detailed Description of the Invention] In this application, all terms used herein should be understood in their ordinary meaning as known in the art unless otherwise stated. Other, more specific definitions for certain terms used in this application are set forth below and are intended to be applied uniformly throughout the specification and claims, unless a definition expressly set forth otherwise provides a broader definition.

[0023] As used herein, the indefinite articles "a" and "an" are synonymous with "at least one" or "one or more." Unless otherwise indicated, definite articles used herein, such as "the," also include the plural of the noun in question.

[0024] As noted above, in a first aspect, the present invention provides a polypeptide consisting of the sequence of SEQ ID NO: 1, or a variant thereof consisting of a sequence which is at least 85% identical to SEQ ID NO: 1, for use in extending the lifespan of a subject.

[0025] This aspect can also be formulated as the use of a polypeptide as defined above for the manufacture of a medicament for extending the lifespan of a subject. The present invention also relates to a method for extending the lifespan of a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide as defined above, together with a pharmaceutically acceptable excipient or carrier.

[0026] In more particular embodiments of the first aspect of the invention, the polypeptide consists of the sequence of SEQ ID NO: 1 or a variant thereof consisting of a sequence that is at least 85%, 86%, 87%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to SEQ ID NO: 1. In even more particular embodiments, the polypeptide consists of the sequence of SEQ ID NO: 1 or a variant thereof consisting of a sequence that is at least 88% or 98% identical to SEQ ID NO: 1.

[0027] In more particular embodiments of the first aspect of the invention, the polypeptide consists of the sequence of SEQ ID NO:1 or a variant thereof consisting of a sequence that is at least 85%, 86%, 87%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to SEQ ID NO:1, which variant substantially maintains or improves the lifespan extending effect of SEQ ID NO:1.

[0028] In another embodiment of the first aspect of the invention, the polypeptide consists of the sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0029] Protein variants are well understood to those of skill in the art and can involve amino acid sequence modifications, which typically fall into one or more of three classes: substitutional, insertional, or deletional variants.

[0030] In the present invention, the term "identity" refers to the percentage of residues that are identical in two sequences when the sequences are optimally aligned. If a position in a first sequence is occupied by the same amino acid residue as the corresponding position in a second sequence during optimal alignment, the sequences are said to be identical at that position. The percentage of identity determines the number of identical residues over a defined length in a given alignment. Thus, the level of identity between two sequences ("percent sequence identity") is measured by the ratio of the number of identical positions shared by the sequences to the number of compared positions (i.e., percent sequence identity = (number of identical positions / total number of compared positions) x 100). Gaps, i.e., positions in the alignment where a residue is present in one sequence but not in the other, are considered as positions with non-identical residues and are counted as compared positions.

[0031] By way of example, a polypeptide having an amino acid sequence that is at least, for example, 95% identical to the reference amino acid sequence of SEQ ID NO: 1 intends that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may contain up to 5 amino acid changes for each 100 amino acids of the reference amino acid sequence of SEQ ID NO: 1. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These changes in the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence, or anywhere between these terminal positions, either individually among residues in the reference sequence, or scattered in one or more contiguous groups within the reference sequence.

[0032] Several mathematical algorithms for rapidly obtaining optimal alignments and calculating identity between two or more sequences are known and are incorporated into several available software programs. For purposes of the present invention, sequence identity between two amino acid sequences is preferably determined using default settings using an algorithm based on global alignment, such as the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol., 48:443-453), preferably as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite; Rice et al., 2000, Trends Genet., 16:276-277) or the BLAST global alignment tool (Altschul et al., "Basic local alignment search tool," 1990, J. Mol. Biol., 215, 403-410). Local alignments can also be used if the sequences being compared are of substantially the same length.

[0033] Polypeptides having at least 88% percent identity with either SEQ ID NO: 1 or SEQ ID NO: 2 include s-KL of mammals other than mouse and human.

[0034] SEQ ID NO: 1 is the amino acid sequence of a transcript from alternative splicing of the α-Klotho human gene, which contains the KL1 domain sequence, has an estimated weight of 70 kDa, and has a specific secretion signal consisting of a 15-amino acid tail not found in the m-KL transcript. The α-Klotho human gene is located in chromosome 13NC_000013.11 (33016063..33066145) of the human genome assembly GRCh38 (24.12.2013) maintained by the Genome Reference Consortium. SEQ ID NO: 1 is derived from the cDNA corresponding to SEQ ID NO: 3, which is derived from an alternatively spliced ​​transcript of a 5012-base pair mRNA sequence with GenBank database accession number NM_004795, version 3, as of May 3, 2014.

[0035] SEQ ID NO:2 is the amino acid sequence of a transcript from alternative splicing of the α-Klotho mouse gene, which contains the KL1 domain sequence, an estimated weight of 70 kDa, and a specific secretory signal consisting of a 15-amino acid tail not found in the m-KL transcript. The α-Klotho mouse gene is located in chromosome 5 (150,952,607-150,993,809) of the UCSC Genome Browser of the Mouse July 2007 (NCBI37 / mm9) assembly of the mouse genome. SEQ ID NO:2 is derived from the corresponding cDNA of SEQ ID NO:4, which in turn is derived from the alternatively spliced ​​transcript of the 5124 base pair mRNA sequence with GenBank database accession number NM_013823, version 2, dated February 15, 2015.

[0036] In another embodiment of the first aspect of the invention, the polypeptide variant consists of the sequence of SEQ ID NO:5 or SEQ ID NO:6.

[0037] In further embodiments of the first aspect of the invention, the polypeptide has a length of 645 amino acids or less, 600 amino acids or less, or 550 amino acids or less. In even more particular embodiments, the polypeptide consists of the sequence of SEQ ID NO: 1 or a variant thereof that consists of a sequence at least 85% identical to SEQ ID NO: 1, and has a length of 645 amino acids or less, 600 amino acids or less, or 550 amino acids or less. In particular embodiments, the polypeptide consists of the sequence of SEQ ID NO: 1 or a variant thereof that consists of a sequence at least 85% identical to SEQ ID NO: 1, said variant having one of the following sequences: 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, and 600 amino acids, or a length of 545 to 600 amino acids.

[0038] In one embodiment of the first aspect of the present invention, the polypeptide is a secreted splicing isoform (s-KL) of a mammalian Klotho protein. In even more particular embodiments, the polypeptide is human s-KL. Secreted splicing isoforms (s-KL) of mammalian Klotho proteins have been disclosed in the prior art (see, for example, WO 2017085317A1). Thus, the present invention can be formulated as a secreted splicing isoform (s-KL) of a mammalian Klotho protein, particularly human s-KL, or a nucleic acid sequence encoding the same, for use in extending the lifespan of a subject.

[0039] The term "secreted spliced ​​isoform of mammalian Klotho," abbreviated as "s-KL," refers to a protein resulting from a transcript from alternative splicing, which generates a truncated form of the protein (s-KL) formed by the KL1 domain, with an estimated weight of 70 kDa, along with a specific secretion signal consisting of a 15-amino acid tail not found in the m-KL transcript. Therefore, it is also referred to as the secreted isoform of Klotho, s-KL, or secreted spliced ​​isoform of the Klotho protein. s-KL differs from other forms of soluble Klotho, namely, p-KL, p-KL1, and p-KL2. In this description, m-KL refers to the full-length transmembrane form, p-KL refers to the soluble proteolyzed Klotho resulting from cleavage of m-KL, and p-KL1 and p-KL2 refer to soluble Klotho forms consisting of the KL1 and KL2 domains of p-KL, respectively. m-KL is derived from a full-length transcript encoding a single-pass transmembrane protein (m-KL) with a molecular weight of approximately 130 kDa. The protein contains three domains: a short C-terminal transmembrane domain, an extracellular domain consisting of two internal repeats of approximately 550 amino acids, designated KL1 and KL2, respectively, and a very short intracellular domain of 10 amino acids. The transmembrane extracellular domain can be cleaved by the metalloproteinases ADAM10 and ADAM17 to yield another form of soluble Klotho of approximately 130 kDa (abbreviated as p-KL for proteolyzed membrane isoform). In addition, there is a second recognition site for the proteases ADAM10 and 17 located between the KL1 and KL2 domains, which generates two new 70 kDa isoforms, one contained only in the KL1 domain (e.g., resulting from alternative splicing but without the specific amino acid tail) and the other contained in the KL2 domain. However, proteolysis of p-KL into p-KL1 and p-KL2 has not been demonstrated in vivo.

[0040] As used herein, "extending lifespan" refers to increasing the average or maximum length of time that an organism can be expected to survive or persist. In particular, it refers to increasing maximum lifespan. "Maximum lifespan" refers to the age at which the oldest member of the species dies. The present invention is particularly directed to increasing maximum lifespan. The terms "extending lifespan," "extending lifespan," and "promoting longevity" are used interchangeably in the present invention.

[0041] In certain embodiments of the first aspect, optionally in combination with any of the embodiments provided above and below, the polypeptide is linked to a heterologous moiety.

[0042] As used herein, a "heterologous moiety" refers to any molecule that is coupled to a polypeptide via either a covalent or non-covalent bond. In certain embodiments, the heterologous moiety is located at either the N-terminus or the C-terminus of the polypeptide. In certain embodiments, the heterologous moiety is located at both the N-terminus and the C-terminus of the polypeptide.

[0043] The heterologous moiety can be, for example, a molecule that facilitates purification of the polypeptide. In particular embodiments, the heterologous moiety is a peptide. In even more particular embodiments, the heterologous moiety is a polyhistidine track. As will be appreciated by those skilled in the art, small peptides that aid in the purification of a protein can be maintained in the final compound without affecting its functionality.

[0044] The heterologous moiety can also be any vehicle to facilitate absorption, transport, and delivery of the polypeptide.

[0045] These polypeptides, particularly those derived from KL proteins such as s-KL, may be used directly in protein form or may be expressed in target cells of tissues of interest by gene therapy. To this end, the present invention also provides in a second aspect a nucleic acid sequence encoding a polypeptide as defined in the first aspect or a variant thereof for use in extending the lifespan of a subject.

[0046] The term "nucleic acid sequence encoding a polypeptide" is to be understood in particular as meaning an mRNA encoding said polypeptide or a cDNA sequence resulting from reverse transcription (RT-PCR) of an mRNA encoding said polypeptide.

[0047] This aspect can also be formulated as the use of a nucleic acid sequence as defined above for the manufacture of a medicament for extending the lifespan of a subject. The present invention also relates to a method for extending the lifespan of a subject, comprising administering to the subject a therapeutically effective amount of a nucleic acid sequence as defined above, together with a pharmaceutically acceptable excipient or carrier.

[0048] In particular embodiments of the second aspect, the nucleic acid sequence comprises SEQ ID NO: 3 or SEQ ID NO: 4. In even more particular embodiments, the nucleic acid sequence consists of SEQ ID NO: 3 or SEQ ID NO: 4.

[0049] In more particular embodiments of the second aspect of the invention, the nucleic acid sequence consists of the sequence of SEQ ID NO:3 or SEQ ID NO:4, or a variant thereof consisting of a sequence that is at least 85%, 86%, 87%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to SEQ ID NO:3 or SEQ ID NO:4.

[0050] In more particular embodiments of the second aspect of the invention, the nucleic acid sequence consists of the sequence of SEQ ID NO:3 or SEQ ID NO:4, or a variant thereof consisting of a sequence that is at least 85%, 86%, 87%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to SEQ ID NO:3 or SEQ ID NO:4, which variant substantially maintains or improves the lifespan extending effect of SEQ ID NO:3 or SEQ ID NO:4.

[0051] In a third aspect, the present invention provides a genetic construct comprising a nucleic acid sequence as defined in the second aspect operably linked to an expression promoter, for use in extending the lifespan of a subject.

[0052] This aspect can also be formulated as the use of a genetic construct as defined above for the manufacture of a medicament for extending the lifespan of a subject. The present invention also relates to a method for extending the lifespan of a subject, comprising administering to the subject a therapeutically effective amount of a genetic construct as defined above, together with a pharmaceutically acceptable excipient or carrier.

[0053] In a specific embodiment of the third aspect, the operably linked expression promoter is selected from the group consisting of a constitutive expression promoter, an inducible promoter, a muscle-specific expression promoter, and a neuron-specific expression promoter. In a more specific embodiment, the genetic construct according to the present invention comprises a cytomegalovirus intermediate-early (CMV IE) promoter, a sequence encoding s-KL (mouse or human s-KL cDNA), and a polyadenylation sequence (polyA). In another specific embodiment, the genetic construct according to the present invention comprises a CAG promoter, a sequence encoding s-KL (mouse or human s-KL cDNA), and a polyadenylation sequence (polyA).

[0054] In particular embodiments of the third aspect, optionally in combination with any of the embodiments provided above and below, the genetic construct comprises or consists of SEQ ID NO:7 or SEQ ID NO:8.

[0055] All these genetic constructs are capable of expressing a protein of interest once in a cell. To facilitate administration of the construct, the present invention also provides in a fourth aspect an expression vector comprising a genetic construct as defined in the third aspect for use in extending the lifespan of a subject, and thus comprising a nucleic acid sequence of the second aspect encoding a polypeptide of the first aspect operably linked to an expression promoter, in particular a constitutive expression promoter.

[0056] This aspect can also be formulated as the use of an expression vector as defined above for the manufacture of a medicament for extending the lifespan of a subject. The present invention also relates to a method for extending the lifespan of a subject, comprising administering to the subject a therapeutically effective amount of an expression vector as defined above, together with a pharmaceutically acceptable excipient or carrier.

[0057] In certain embodiments of the fourth aspect, optionally in combination with any of the embodiments provided above and below, the expression vector is a viral vector.

[0058] In a particular embodiment of the third aspect, optionally in combination with any of the embodiments provided above and below, the expression vector consists of the sequence of SEQ ID NO:9.

[0059] In certain embodiments of the fourth aspect, optionally in combination with any of the embodiments provided above and below, the viral vector is an adeno-associated virus. In certain embodiments, it is an adeno-associated virus of a serotype selected from the group consisting of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, PHPeB, and 9P31, which has the ability to cross the BBB. In more specific embodiments, it is an adeno-associated virus of the serotype AAV9.

[0060] In another embodiment of the first, second, third and fourth aspect, optionally in combination with any of the embodiments provided above and below, the polypeptide for use according to the first aspect, the nucleic acid sequence for use according to the second aspect, the genetic construct for use according to the third aspect or the expression vector for use according to the fourth aspect is administered together with at least one pharmaceutically acceptable excipient, diluent or carrier in the form of a pharmaceutical composition.

[0061] The expression "pharmaceutical composition" encompasses compositions intended for both humans and non-human animals. Those skilled in the art will understand that a pharmaceutical composition must contain a therapeutically effective amount of a compound. As used herein, the expression "therapeutically effective amount" refers to an amount of a polypeptide, nucleic acid sequence, gene construct, or expression vector that, when administered, is sufficient to prevent the occurrence of, or alleviate to some extent, one or more symptoms of the disease being addressed. The specific dosage of a compound to be administered in accordance with the present invention will, of course, be determined by the specific circumstances surrounding the case, including the compound to be administered, the route of administration, the specific condition being treated, and similar considerations.

[0062] The expression "pharmaceutically acceptable excipient, diluent, or carrier" refers to a pharmaceutically acceptable material, composition, or vehicle. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with the tissues or organs of humans and non-human animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problem or complication commensurate with a reasonable benefit-to-risk ratio.

[0063] Examples of suitable pharmaceutically acceptable excipients are solvents, dispersion media, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, tonicity adjusting agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, etc. Use of any conventional excipient medium is contemplated within the scope of the present invention so long as it is not incompatible with the substance or its derivatives, such as by producing any undesirable biological effects or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition.

[0064] The relative amounts of the active ingredient, pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition of the present invention will vary depending on the identity, size, and / or condition of the subject being treated, and further depending on the route by which the composition is intended to be administered.

[0065] Pharmaceutically acceptable excipients for use in preparing pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surfactants and / or emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants, and / or oils. Excipients such as coloring agents, coating agents, sweetening agents, and flavoring agents can be present in the composition, according to the judgment of the formulator.

[0066] Pharmaceutical compositions containing the proteins or nucleic acids of the invention can be in any dosage form, e.g., solid or liquid, and can be administered by any suitable route, e.g., oral, parenteral, rectal, topical, intranasal, intraocular, intraperitoneal, or sublingual, and therefore, they contain pharmaceutically acceptable excipients necessary for formulation into the desired dosage form, e.g., topical formulations (ointments, creams, lipogels, hydrogels, etc.), eye drops, aerosol sprays, injectable hydrogels, injectable solutions, osmotic pumps, etc.

[0067] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and combinations thereof.

[0068] Exemplary granulating and / or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinylpyrrolidone (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and combinations thereof.

[0069] Exemplary binders include, but are not limited to, starches (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol), natural and synthetic gums (e.g., acacia, sodium alginate, Irish moss extract, panwar gum, ghatti gum, isapol husk mucilage, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, polyvinylpyrrolidone), magnesium aluminum silicate (veegum), and larch arabinogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and combinations thereof.

[0070] Exemplary preservatives may include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. Exemplary antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, ascorbyl palmitate, ascorbyl stearate, ascorbyl oleate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, edetate disodium, edetate dipotassium, edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and edetate trisodium.

[0071] Exemplary buffering agents include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and combinations thereof.

[0072] Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0073] In another embodiment of the first, second, third, and fourth aspects, optionally in combination with any of the embodiments provided above and below, the pharmaceutical composition is for administration to a patient mucosally (e.g., nasally, sublingually, vaginally, buccally, or rectally), parenterally (e.g., subcutaneous, intravenous, intramuscular, or intraarterial injection, either bolus or infusion), orally, transdermally, or via inhalation, e.g., by aerosol. Formulations suitable for parenteral administration, e.g., by intraarticular, intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Injection solutions and suspensions may also be prepared from sterile powders, granules, and tablets. In some embodiments, the compositions are administered, for example, by subcutaneous, intraperitoneal, intravesical, intravenous, or intracerebroventricular injection; by infusion, e.g., using a reservoir or osmotic minipump, or by intramuscular infusion. Formulations can be provided in unit-dose or multi-dose sealed containers, such as ampoules and vials. In even more particular embodiments, the pharmaceutical compositions are for intracerebroventricular or intravenous administration, more particularly for systemic intravenous administration.

[0074] In another embodiment of the first, second, third, and fourth aspects, optionally in combination with any of the embodiments provided above and below, the polypeptide for use according to the first aspect, the nucleic acid sequence for use according to the second aspect, the genetic construct for use according to the third aspect, or the expression vector for use according to the fourth aspect is administered in combination with another active agent. Suitable active agents for administration in combination with the compounds of the invention include, but are not limited to, vitamin A, berberine, α-ketoglutarate, resveratrol, caffeine, 5-aminoimidazole-4-carboxamido-ip-d-ribonucleoside, 2-deoxyglucose, apigenin, metformin, quercetin, and rosaglitazone.

[0075] In embodiments of the above aspects, optionally in combination with any of the embodiments provided above or below, the subject is a healthy subject. In embodiments of the above aspects, optionally in combination with any of the embodiments provided above or below, the subject does not suffer from an age-related disease. "Age-related disease" refers to a disorder or disease in which aging is a significant risk factor.

[0076] In embodiments of the above aspects, optionally in combination with any of the embodiments provided above or below, the subject is a mammal. In more particular embodiments, the mammal is a dog, cat, horse, cow, pig, rabbit, sheep, rodent, non-human primate, or human. In even more particular embodiments, the mammal is a human.

[0077] In embodiments of the above aspect, optionally in combination with any of the embodiments provided above or below, the subject is selected from the group consisting of a young subject, an adult subject, and an elderly subject. More particularly, the subject is a human and is selected from the group consisting of a young subject, an adult subject, and an elderly subject.

[0078] In embodiments of the above aspect, optionally in combination with any of the embodiments provided above or below, increasing lifespan comprises an increase in lifespan of at least 5%, at least 10%, at least 15%, or at least 19% as compared to the expected lifespan of a subject of the same or similar species.

[0079] In another embodiment of the above aspect, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered for a specific period of time or for a chronic treatment period, which is an extended period including the entire life of the subject. Within the treatment period, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered on a specific time schedule. In further embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered one, two, three, or four times per day. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered once per day. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered twice per day. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered in the morning and evening. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered one, two, three, or four times per week. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered once per week. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered 1, 2, 3, or 4 times per month. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered once per month. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered for at least 3 months every year. In some embodiments, the polypeptide, nucleic acid sequence, gene construct, or expression vector is administered for 1 month every 6 months.

[0080] As mentioned above, in a sixth aspect the present invention provides a method for extending the lifespan of a subject, the method comprising administering to the subject a polypeptide consisting of the sequence of SEQ ID NO: 1, or a variant thereof consisting of a sequence which is at least 85% identical to SEQ ID NO: 1, or a nucleic acid sequence encoding the polypeptide or variant thereof. Embodiments of the first and second aspects, particularly those relating to the sequence and nucleic acid sequences of the polypeptide or variant thereof, are intended to apply to this sixth aspect as well.

[0081] In certain embodiments of the sixth aspect, the method is a non-therapeutic method.

[0082] In embodiments of the sixth aspect of the invention, the nucleic acid sequence is comprised in a genetic construct operably linked to an expression promoter. In more particular embodiments, the genetic construct is comprised in an expression vector. The embodiments of the third and fourth aspects relating to expression constructs, vectors, and subjects are intended to also apply to the sixth aspect of the invention.

[0083] In an embodiment of the sixth aspect, the subject is a healthy subject. In another embodiment, the subject does not suffer from an age-related disease. In another embodiment, the subject is selected from the group consisting of a young subject, an adult subject, and an elderly subject. More particularly, the subject is a human and is selected from the group consisting of a young subject, an adult subject, and an elderly subject.

[0084] As noted above, the present invention also provides, in a seventh aspect, the use of a polypeptide or a nucleic acid encoding same for extending the lifespan of a subject. All embodiments of the above aspects are intended to apply to this seventh aspect as well.

[0085] Throughout the specification and claims, the word "comprise" and variations of this word are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise" also includes the example of "consisting of." Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the present invention. The following examples and drawings are provided by way of illustration and are not intended to be limiting of the present invention. Reference signs placed in parentheses in connection with the drawings and in the claims are intended solely to enhance the comprehension of the claims and should not be construed as limiting the scope of the claims. Furthermore, the present invention covers all possible combinations of the specific and preferred embodiments described herein.

[0086] For reasons of completeness, the various aspects of the invention are set out in the following numbered clauses.

[0087] 1. A polypeptide consisting of the sequence of SEQ ID NO: 1, or a variant thereof consisting of a sequence which is at least 85% identical to SEQ ID NO: 1, for use in extending the lifespan of a subject.

[0088] 2. A polypeptide for use according to claim 1, wherein said polypeptide consists of the sequence SEQ ID NO: 1 or a variant thereof consisting of a sequence that is at least 88% identical to SEQ ID NO: 1.

[0089] 3. A polypeptide for use according to claim 1 or 2, wherein said polypeptide consists of the sequence SEQ ID NO: 1 or a variant thereof consisting of a sequence that is at least 98% identical to SEQ ID NO: 1.

[0090] 4. A polypeptide for use according to any one of items 1 to 3, wherein the polypeptide consists of SEQ ID NO: 1 or SEQ ID NO: 2.

[0091] 5. A nucleic acid sequence encoding a polypeptide or variant thereof as defined in any one of clauses 1 to 4 for use in extending the lifespan of a subject.

[0092] 6. A genetic construct comprising the nucleic acid sequence of paragraph 5 operably linked to an expression promoter, for use in extending the lifespan of a subject.

[0093] 7. An expression vector comprising the genetic construct of paragraph 6, for use in extending the lifespan of a subject.

[0094] 8. An expression vector for use according to item 7, which is a viral vector.

[0095] 9. An expression vector for use according to paragraph 8, which is an adeno-associated virus of a serotype selected from the group consisting of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, PHPeB, and 9P31.

[0096] 10. A polypeptide for use according to any one of clauses 1 to 4, a nucleic acid sequence for use according to clause 5, a genetic construct for use according to clause 6 or an expression vector for use according to any one of clauses 7 to 9, wherein the subject is a mammal, in particular a human.

[0097] 11. A polypeptide for use according to any one of paragraphs 1 to 4 and 10, a nucleic acid sequence for use according to paragraphs 5 and 10, a genetic construct for use according to paragraphs 6 and 10, or an expression vector for use according to any one of paragraphs 7 to 10, wherein the subject is a healthy subject.

[0098] 12. A polypeptide for use according to any one of clauses 1 to 4 and 10 to 11, a nucleic acid sequence for use according to any one of clauses 5 and 10 to 11, a genetic construct for use according to any one of clauses 6 and 10 to 11, or an expression vector for use according to any one of clauses 7 to 11, administered in the form of a pharmaceutical composition, together with at least one pharmaceutically acceptable excipient, diluent or carrier.

[0099] 13. A polypeptide for use according to any one of paragraphs 1 to 4 and 10 to 12, a nucleic acid sequence for use according to paragraphs 5 and 10 to 12, a genetic construct for use according to paragraphs 6 and 10 to 12, or an expression vector for use according to any one of paragraphs 7 to 12, administered in combination with another active agent.

[0100] 14. A non-therapeutic method for extending the lifespan of a subject, comprising the step of administering to the subject a polypeptide consisting of the sequence of SEQ ID NO: 1 or a variant thereof consisting of a sequence that is at least 85% identical to SEQ ID NO: 1, or a nucleic acid sequence encoding said polypeptide or variant thereof.

[0101] 15. Use of a polypeptide consisting of the sequence of SEQ ID NO: 1, or a variant thereof consisting of a sequence that is at least 85% identical to SEQ ID NO: 1, or a nucleic acid sequence encoding said polypeptide or said variant, for extending the lifespan of a subject.

[0102] [Example] Materials and Methods Animal housing C57BL / 6J male (n = 48) and female mice (n = 48) were purchased from Charles River. The animals were randomly divided into three groups per sex. Two of these were treated with a null control vector (SEQ ID NO: 10) (n = 16) or an s-KL expression vector (SEQ ID NO: 9) (n = 16) when they were 7 months of age. The third group was treated with an s-KL expression vector (SEQ ID NO: 9) (n = 16) when they were 12 months of age. The majority of these animals were followed throughout their lifespan to study health and viability, and a second subset of four animals / group was randomly selected and euthanized when they were 24 months of age to study viral vector function.

[0103] Mice were allowed free access to food and water and maintained under standard temperature conditions (22 ± 2°C) and a 12-h light / dark cycle (300 lux / 0 lux). Mice were checked periodically for overall health until natural death or euthanasia. The euthanasia protocol was cervical dislocation, administered by a blinded veterinarian at the animal housing facility when the animal was deemed to have reached the endpoint criteria.

[0104] Treatment Generation and Administration Gene therapy treatment consisted of an expression cassette under the control of the CAG promoter containing either a control null sequence or the secreted isoform of the mouse α-KL gene (SEQ ID NO: 2).

[0105] Two adeno-associated viral vectors, serotype 9 (AAV9), independently containing these constructs were generated according to the triple transfection method described in Piedra JX et al., 2015. To transduce as many mouse tissues as possible, animals were simultaneously administered intracerebroventricularly and intravenously. Intracerebroventricular stereotactic injection of AAV vectors was performed as previously described (Masso A. et al., "Secreted αKlotho isoform protects against age-dependent memory deficits," Mol Psychiatry, 2018, Vol. 23(9), pp. 1937-1947). Briefly, treatments were administered into the right hemisphere at coordinates of -0.2 mm anterior-posterior, -2 mm dorsoventral, and +1 mm medial-lateral from the bregma. The vector dose was 1 x 10 in 6 μl. 11 Intravenous injections were performed using 4 × 10 viral genomes / animal, diluted with NaCl 0.9% to a final volume of 200 μL, using an ultramicropump (World Precision Instruments) at a rate of 0.5 μl / min. 11 The dose consisted of 100 viral genomes / animal and was manually injected into the lateral tail vein of the mice using a syringe.

[0106] Serum biochemical analysis Blood samples were obtained by decapitation of deeply anesthetized animals using SST serum collection tubes (BD microtainer). Blood was allowed to stand at room temperature for 5 minutes and then placed on ice. Serum was isolated by centrifugation of the tubes at 3000 rpm for 10 minutes for 15 minutes, and finally aliquoted and kept frozen at -80°C. KL serum levels were measured using a mouse KL-specific ELISA kit (IBL) according to the manufacturer's instructions.

[0107] Gene expression Total RNA isolation was performed using TRIsure™ Reagent according to the manufacturer's instructions (Bioline Reagent). The tissue used for RNA extraction was liver. Samples were homogenized using a TissueLyzer LT sample disruption device (QIAGEN). RNA quantity and purity were measured using a NanoDrop™ 1000 spectrophotometer (Thermo Scientific). RNA reverse transcription was performed using the iScript™ Advanced cDNA Synthesis Kit (Bio-Rad). Gene expression was analyzed by real-time quantitative PCR (RT-qPCR) using a Bio-Rad CFX-384 PCR instrument at the Analysis and Photodocumentation Service of the Universitat Autónoma de Barcelona according to the manufacturer's instructions. Each reaction contained 25 ng of cDNA, 7.5 μL of iTaq™ Universal SYBR Green Supermix (Bio-Rad), and a primer concentration of 0.2 nM, with a final reaction volume of 15 μL. The primers used are listed in Table 1.

[0108] [Table 1]

[0109] statistical analysis Statistical analysis and graphical presentation were performed using GraphPad Prism version 8 (GraphPad Software). Statistical differences between groups were analyzed using a two-tailed unpaired Student's t-test when comparing two groups, or one-way analysis of variance (ANOVA), followed by Tukey's as a post-hoc analysis for comparing all treatment groups. Longevity curves were presented as Kaplan-Meier longevity curves, and statistical differences were analyzed using the log-rank (Mantel-Cox) statistical test. Data were presented as the mean ± standard error of the mean (SEM). Statistical differences were accepted when p-values ​​were ≤ 0.05.

[0110] result s-KL treatment efficiently increased s-KL protein concentrations Viral vector administration was performed by continuous intravenous and intracerebroventricular injection (Fig. 1a). Of the 96 treated animals, one died immediately after the intervention. To evaluate viral vector function, a randomly selected subset of four animals from each group was euthanized at 24 months of age. s-KL gene expression was studied in the liver, as this organ is transfected after AAV9 serotype injection and is the main secretory organ in adult animals (Fig. 1b). s-KL cDNA expression was significantly increased in all KL-treated groups, higher in males than in females, and higher in the 12MO (12-month-old) group compared with 6MO (6-month-old)-treated animals. Furthermore, efficient protein production and secretion into the bloodstream was confirmed by ELISA (Fig. 1c). At both 6 and 12 months of age, significantly higher KL protein levels were detected in serum in treated mice compared with animals treated with null-containing AAV9. Again, concentrations were much higher in males than in females. In males, this was also significantly increased in 12MO males compared to the 6MO group, showing twice as high s-KL concentrations.

[0111] s-KL treatment extended the lifespan of treated mice Body weight is an important variable of health status. In males, mean body weight decreased at 14 months of age in the null group, 15 months of age in the s-KL 6MO group, and was delayed until 20 months of age in the s-KL 12MO group (Fig. 2a).

[0112] As shown in Figure 2b, s-KL treatment increased longevity (i.e., lifespan). Median survival was 24.6, 25.8, and 28.3 months for null-treated, sKL-6MO, and sKL-12MO, respectively, representing a statistical difference of 15% increase in life expectancy for s-KL 12MO versus null-treated animals.

[0113] Finally, the animals' overall longevity (i.e., maximum lifespan) was also monitored, and the results are shown in Kaplan-Meier survival curves (Figure 2c). Overall longevity was 26.3, 29.8, and 31.5 months for the null treatment, s-KL 6MO, and s-KL 12MO, respectively, representing a 19.7% increase. Comparison of the overall survival curves of the groups yielded p-values ​​of 0.08 (comparing null 6MO to s-KL 6MO) and 0.005 (comparing null 6MO to s-KL 12MO) in the long-rank Mantel-Cox test.

[0114] All these results clearly indicate that s-KL administration may be a useful therapy for extending the life span of animals.

[0115] List of References Altschul et al., “Basic local alignment search tool”, 1990, J. Mol. Biol, vol. 215, pp. 403-410. International Publication No. 2017085317 Masso A. et al., “Secreted αKlotho isoform protects against age-dependent memory deficits” Mol Psychiatry, 2018, vol. 23(9), pp. 1937-1947 Piedra, J. et al., “Development of a rapid, robust, and universal picogreen-based method to titer adeno-associated vectors”, 2015, Human Gene Therapy Methods, vol. 26(1), pp. 35-42

Claims

1. 1. A method for extending the lifespan of a subject, comprising administering to the subject a polypeptide consisting of the sequence of SEQ ID NO: 1, or a variant thereof consisting of a sequence that is at least 85% identical to SEQ ID NO: 1, or a nucleic acid sequence encoding the polypeptide or variant thereof.

2. 2. The method of claim 1, wherein the polypeptide consists of the sequence of SEQ ID NO: 1 or a variant thereof consisting of a sequence that is at least 88% identical to SEQ ID NO:

1.

3. 3. The method of claim 1 or 2, wherein the polypeptide consists of the sequence of SEQ ID NO: 1 or a variant thereof consisting of a sequence that is at least 98% identical to SEQ ID NO:

1.

4. The method of any one of claims 1 to 3, wherein the polypeptide consists of SEQ ID NO: 1 or SEQ ID NO:

2.

5. The method according to any one of claims 1 to 4, wherein the subject is a mammal, in particular a human.

6. The method of any one of claims 1 to 5, wherein the subject does not suffer from an age-related disease.

7. The method of any one of claims 1 to 6, wherein the subject is a healthy subject.

8. The method of any one of claims 1 to 7, wherein the subject is selected from the group consisting of a young subject, an adult subject, and an elderly subject.

9. 9. The method of any one of claims 1 to 8, wherein extending lifespan comprises an increase in lifespan of at least 5%, at least 10%, at least 15%, or at least 19% as compared to the expected lifespan of a subject of the same or similar species.

10. The method of any one of claims 1 to 9 for increasing maximum lifespan.

11. The method according to any one of claims 1 to 10, which is a non-therapeutic method.

12. Use of a polypeptide consisting of the sequence of SEQ ID NO: 1, or a variant thereof consisting of a sequence that is at least 85% identical to SEQ ID NO: 1, or a nucleic acid sequence encoding said polypeptide or variant thereof, for extending the lifespan of a subject.

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