Telomerase reverse transcriptase therapy for renal fibrosis and its non-human animals

Abstract

The present invention provides compositions and methods useful for the treatment and prevention of conditions associated with short telomere length, particularly renal fibrosis.

Description

[Technical Field] 【0001】 The present invention relates to the fields of molecular biology, biotechnology, and medicine. More specifically, to compositions and methods useful for treating non-human animals and conditions associated with short telomere length. More specifically, to compositions and methods useful for treating conditions associated with renal fibrosis associated with short telomere length. [Background technology] 【0002】 Telomeres are specialized structures at the ends of chromosomes that play a role in protecting chromosome ends from DNA repair and degradation. Mammalian telomeres consist of TTAGGG repeats linked by a multiprotein complex known as shelterin. Since shortened telomeres are associated with many diseases, the minimum length of TTAGGG repeats and the integrity of the shelterin complex are necessary for telomere protection. These structures are essential for chromosomal integrity by preventing telomere fusion and telomere fragility. Telomere length is controlled by telomerase, a ribonucleoprotein enzyme that can de novo-add telomere sequences to telomeres. Telomerase is a cellular reverse transcriptase (TERT, telomerase reverse transcriptase; also known as TP2, TRT, EST2, TCS1, hEST2) that can compensate for telomere shortening by de novo-adding TTAGGG repeats to chromosome ends using the relevant RA component as a template (Terc, telomerase RNA component). Telomerase is expressed in most adult stem cell compartments, but this is insufficient to maintain telomere length, as evidenced by the fact that telomere shortening occurs with age in most human and mouse tissues. 【0003】 Telomere sequences are spontaneously lost during every cell division (known as the end replication problem), and somatic cells either express telomerase at very low levels or not express shortened telomeres at all throughout their lifespan. When telomeres become significantly shortened, they lose their protective function, triggering a persistent DNA damage response at the telomere, which subsequently leads to a cellular senescence response (Harley et al., 1990, Flores et al., 2008). 【0004】 The accumulation of short telomeres is a prominent feature of aging. Mutations in telomerase or telomere-binding proteins result in telomere shortening or dysfunction, characterized by loss of tissue regenerative capacity and fibrous lesions, and are the origin of human lesions known as “telomere syndromes,” including several cases of aplastic anemia, congenital keratosis, and pulmonary fibrosis. Adeno-associated virus (AAV)-based telomerase gene therapy has been found to be beneficial in extending the period of health in wild-type mice under normal physiological aging conditions. Adult and aged mice were subjected to AAV9-mTERT gene therapy to broadly express the catalytic subunit of mouse telomerase (mTERT). The period of health in TERT-treated mice was significantly increased, and aging was slowed, as indicated by several physiological parameters (glucose and insulin resistance, osteoporosis, neuromuscular coordination, rotarod, etc.). Furthermore, their mean lifespan increased by 24% and 13%, respectively, in adult and aged mice compared to the control group. A single intravenous administration of AAV9-TERT in adult mice resulted in an increase in telomere length in peripheral blood cells (Bernardes de Jesus et al., 2012). These results highlight the importance of mouse models for studying specific disease conditions and potential therapeutic approaches. 【0005】 Chronic kidney disease (CKD) is a highly fatal disorder whose incidence is increasing due to demographic aging. Renal fibrosis, characterized by fibroblast activation and excessive production and deposition of the extracellular matrix (ECM), is a major determinant of end-stage renal disease, leading to renal parenchymal destruction, inflammatory and fibrotic responses, and decreased renal function. Short telomeres are thought to contribute to renal fibrosis. However, the role of short telomeres in renal fibrosis remains poorly understood, partly due to the lack of suitable mouse models. Furthermore, it is unknown whether telomerase-deficient mice with short telomeres develop renal fibrosis or require further injury to contribute to the disease. In the past, we have developed mouse models that develop aplastic anemia and pulmonary fibrosis associated with short telomeres. These mouse models demonstrate the role of short telomeres in the origin of these diseases and point to potential therapeutic strategies. 【0006】 In this invention, the inventors have developed a suitable mouse model for studying renal fibrosis associated with short telomeres, thereby enabling them to investigate whether telomerase activation may be an effective treatment for attenuating renal fibrosis associated with short telomeres. 【0007】 This invention provides compositions and methods useful for the treatment and prevention of renal fibrosis associated with short telomeres. [Brief explanation of the drawing] 【0008】 [Figure 1]A mouse model of renal fibrosis is associated with short telomeres. a. Schematic diagram of the experimental approach. Gradual doses of FA (0, 50, 100, and 125 mg kg-1 body weight (BW)) were administered on day 0 to either 8-9 week old Tert+ / + or G3 Tert- / - mice. Blood samples were collected for analysis one and two weeks after FA administration. Mice were euthanized on day 14. b. Macroscopic appearance of the kidney at the endpoint in Tert+ / +, FA-treated Tert+ / +, G3 Tert- / -, and FA-treated G3 Tert- / - mice. c, d. Serum creatinine (c) and BUN (d) levels in untreated and FA-treated Tert+ / + mice as well as G3 Tert- / - mice. e shows representative images and quantifications of Masson staining and PAS+D staining in Tert+ / + mice, FA-treated Tert+ / + mice, G3 Tert- / - mice, and FA-treated G3 Tert- / - mice at the endpoint. Two-way analysis of variance (ANOVA) with the post-hoc Bonferroni test was used for statistical analysis. Data are shown as mean ± sem. The number of mice analyzed for each genotype (n) is shown. *P≦0.05, **P≦0.01, and ***P≦0.001. NS, not significant. [Figure 2] Severe renal impairment in telomerase-deficient mice treated with a sublethal dose of folic acid. a. Schematic diagram of the experimental approach. A dose of 125 mg / kg-1 of folic acid was administered to 8-9 week old Tert+ / + and G3 Tert- / - mice. b. Urinary albumin to creatinine ratio (UACR). c. Macroscopic appearance of kidneys in untreated and FA-treated Tert+ / + and G3 Tert- / - mice. d. Representative images and quantifications of Masson trichrome staining, Sirius red staining, and PAS+D staining in untreated and FA-treated Tert+ / + kidneys and G3 Tert- / - kidneys. One-way ANOVA by post-hoc Tukey test and two-way ANOVA by post-hoc Bonferroni test were used for statistical analysis. Data are shown as mean ± sem. The number of mice analyzed for each genotype is shown. **P≦0.01 and ***P≦0.001. [Figure 3] Telomerase-deficient mice exhibit collagen deposition and activated myofibroblasts in the kidney after sublethal doses of folate. a. Representative images and quantifications of α-SMA, fibronectin, type VI collagen, and biimmunofluorescence for α-SMA and vimentin in Tert+ / + mice, FA-treated Tert+ / + mice, G3 Tert- / - mice, and FA-treated G3 Tert- / - mice. b. Biimmunofluorescence for α-SMA and Ki67 in Tert+ / + mice, FA-treated Tert+ / + mice, G3 Tert- / - mice, and FA-treated G3 Tert- / - mice. White arrows indicate α-SMA+Ki67+ cells. c. Relative mRNA expression of Acta2, Vim, Col1a1, Col3a1, Col4a1, and Fn1 in Tert+ / +, FA-treated Tert+ / +, G3 Tert- / -, and FA-treated G3 Tert- / - mice 14 days after FA administration. Statistical analysis was performed using one-way ANOVA with the Post-Hoc Tukey test. Data are presented as mean ± sem. The number of mice analyzed for each genotype is shown. **P ≤ 0.01 and ***P ≤ 0.001. Vh, vehicle. [Figure 4] Increased renal apoptosis and aging in telomerase-deficient mice after sublethal doses of folate. a-d, Representative images and quantifications of immunohistochemical staining of CC3(a), p21(b), p53(c), and γ-H2AX(d) in Tert+ / +, FA-treated Tert+ / +, G3 Tert- / -, and FA-treated G3 Tert- / - mice. Inset: Amplified images. e, Representative images and quantifications of mean total nuclear telomere length by Q-FISH analysis in Tert+ / + mice, FA-treated Tert+ / + mice, G3 Tert- / - mice, and FA-treated G3 Tert- / - mice. Amplified images are shown below. auf, fluorescence in arbitrary units. Statistical significance was determined by one-way ANOVA with post-hoc Tukey test. *P≦0.05, **P≦0.01, and ***P≦0.001. Data are shown as mean ± sem. This shows the number of mice analyzed for each genotype. [Figure 5]Short telomeres induce tubular damage and immune infiltration in the kidney. a. Relative expression of Havcr1, Lcn2, and Emr1 in Tert+ / +, FA-treated Tert+ / +, G3 Tert- / -, and FA-treated G3 Tert- / - mice 14 days after low-dose FA administration. b. Representative images and quantification of F4 / 80, CD3e, CD4, and CD8a immunohistochemical staining. Inset: Amplified images. One-way ANOVA with post-hoc Tukey test was used for statistical analysis. Data are shown as mean ± sem. Number of mice analyzed per genotype is shown. **P≦0.01 and ***P≦0.001. [Figure 6]Hyperactivation of EMT and TGFβ signaling pathways in telomerase-deficient mice. a, b, gene expression data obtained by RNA-seq from kidney samples of 10-week-old Terc+ / + and G3 Tert- / - mice untreated or treated with FA at a dose of 125 mg kg-1 body weight. Mice were euthanized 14 days after treatment. Samples were analyzed by GSEA to determine significantly enriched gene sets. GSEA plots comparing EMT (a) and TGFβ signaling pathways (b) for untreated Tert+ / + mice vs. untreated G3 Tert- / - mice, FA-treated Tert+ / + vs. FA-treated G3 Tert- / - mice, FA-treated Terc+ / + vs. untreated Tert+ / + mice, and FA-treated G3 Tert- / - vs. untreated G3 Tert- / - mice. Horizontal bars from red to blue represent a ranked list. Genes located in the central region of the bars indicate small differences in gene expression between pairwise comparisons. The red ends of the bars indicate genes with higher expression levels, and the blue ends indicate genes with lower expression levels. The red and blue arrows indicate upregulation and downregulation of pathways in pairwise comparisons, respectively. The false detection rate (FDR) is shown. The samples correspond to the kidneys of four independent Tert+ / +, G3 Tert- / -, and FA-treated G3 Tert- / - mice, as well as three FA-treated Tert+ / + mice. c shows the relative expression of Tgfb1, Snail1, Snail2, Twist1, Zeb1, Zeb2, Loxl2, Cdh1, and Smad3 in Tert+ / +, FA-treated Tert+ / +, G3 Tert- / -, and FA-treated G3 Tert- / - mice 14 days after administration of a sublethal FA dose. d shows representative images and quantifications of E-cadherin and phosphorylated SMAD3 (p-SMAD3) immunohistochemical staining. Inset: Amplified p-SMAD3 stained image. One-way ANOVA with Posthock Tukey's test was used for statistical analysis. Data are shown as mean ± sem. The number of mice analyzed for each genotype is shown. *P≦0.05, **P≦0.01, and ***P≦0.001. [Figure 7]Trf1 deletion induces renal fibrosis. a. Schematic diagram of the experimental approach. b. Representative images and quantifications of Masson trichrome staining, Sirius red staining, and SMA staining in TRF1+ / + mice and TRF1flox / flox mice. c. Relative expression of TRF1 and mesenchymal markers Acta2 and Fn1 in TRF1+ / + and TRF1flox / flox mice. d. Relative expression of EMT markers Tgfb1, Snail1, Snail2, Twist1, Zeb1, and Zeb2 in TRF1+ / + mice and TRF1flox / flox mice. A two-tailed t-test was used for statistical analysis. Data are shown as mean ± sem. The number of mice analyzed for each genotype is shown. *P≦0.05, **P≦0.01, and ***P≦0.001. [Figure 8] TERT activation rescued the EMT phenotype in vitro. a, b. Relative mRNA mTERT expression (a) and telomere length analysis (b) in wild-type and G3 Tert- / - PTCs transduced with either an empty (null) vector or an mTert-containing vector. c. Immunofluorescence of E-cadherin, SMA, and Snail1 / Slug in PTCs of 10-11 week old Tert+ / + and G3 Tert- / - PTCs transduced with either an empty (null) vector or an mTert-containing vector. Twenty micrographs were taken for each condition. d. Relative mRNA expression of Cdh1, Acta2, Vim, Col3a1, Col4a1, Tgfb1, Snail1, Snail2, and Zeb1. Data are shown as mean ± sem. The number of mice analyzed for each genotype is shown. One-way ANOVA post-hoc Tukey test was used for statistical analysis. *P≦0.05, **P≦0.01, and ***P≦0.001. [Figure 9]Telomerase-deficient mice with short telomeres do not spontaneously develop renal fibrosis. a. Scheme of the experimental approach. b-f. Representative images and quantifications of Masson's trichrome (b), Sirius red (c), PAS-diastase (d), SMA (e), and E-cadherin (f) in Tert+ / + and G3Tert- / - mice. g-h. Representative images and quantifications of p21 (g) and CC3 (h) immunohistochemical staining in Tert+ / + and G3Tert- / - mice. Insets show amplified images. A two-tailed t-test was used for statistical analysis. Data are shown as mean + / - SEM. The number of mice analyzed for each genotype is shown. [Figure 10] Blood parameters. Serum creatinine (a) and blood urea nitrogen (BUN) (b) levels in untreated and FA-treated Tert+ / + and G3Tert- / - mice. Blood samples were collected on days 2, 7, and 14. Mice were sacrificed on day 14. a) Two-way ANOVA with post-hoc Bonferroni test was used for statistical analysis. Data are shown as mean + / - SEM. The number of mice analyzed for each genotype is shown. *p≦0.05;**p≦0.01;***p≦0.001. [Figure 11] The effect of short telomeres on cell cycle regulators. Relative expression of CCnd1, CCnd2, CCnb1, and CCne1 in Tert+ / + mice, FA-treated Tert+ / + mice, G3Tert- / - mice, and FA-treated G3Tert- / - mice 14 days after administration of low-dose FA. One-way Anova was used for statistical analysis. Data are shown as mean + / - SEM. The number of mice analyzed for each genotype is shown. *p≦0.05;**p≦0.01;***p≦0.001. [Figure 12] The effect of shorter telomeres on EMT-related genes. a. Gene expression data obtained by RNA-seq from kidney samples of 7-week-old and 47-week-old Terc+ / + mice and G3 Tert- / - mice. b. Gene set enrichment analysis (GSEA) plot for the EMT pathway. The false discovery rate (FDR) is shown. Samples correspond to the kidneys of five independent Tert+ / + mice and G3 Tert- / - mice. [Figure 13] Effects of shorter telomeres on renal precursor genes. a. Relative expression of Sox-9, Wt-1, Pax-2, Sall2, Acvr2b, and Klotho in Tert+ / +, FA-treated Tert+ / +, G3Tert- / -, and FA-treated G3Tert- / - mice 14 days after low-dose FA administration. b. Representative images and quantification of Sox-9 immunohistochemical staining. Insets indicate amplified images. One-way Anova was used for statistical analysis. Data are shown as mean + / - SEM. Number of mice analyzed per genotype is shown. *p≦0.05;**p≦0.01;***p≦0.001. [Figure 14] The effect of shorter telomeres on Notch target genes. Relative expression of Notch1, 2, 3, Jagged1, and Tfam in Tert+ / + mice, FA-treated Tert+ / + mice, G3Tert- / - mice, and FA-treated G3Tert- / - mice 14 days after low-dose FA administration. One-way Anova was used for statistical analysis. Data are shown as mean + / - SEM. The number of mice analyzed for each genotype is shown. *p≦0.05;**p≦0.01;***p≦0.001. [Figure 15] TERT activation rescued the EMT phenotype in vitro. a, b, bright-field microscopic images of proximal tubular cell (PTC) cultures at 8 (a) and 14 days after transduction with either an empty vector or an mTert-containing vector (b). c, representative images of immunofluorescence for E-cadherin, SMA, and Tgfβ1. Twenty micrographs were taken from each condition. [Modes for carrying out the invention] 【0009】 Brief description of the invention In one embodiment, the present invention provides a nucleic acid vector comprising a coding sequence for telomerase reverse transcriptase (TERT) for use in treating renal fibrosis associated with the presence of short telomeres. Preferably, TERT is coded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Preferably, TERT is coded by a nucleic acid sequence consisting solely of the sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Preferably, TERT comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Preferably, TERT consists solely of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Preferably, the nucleic acid sequence encoding TERT is operably ligated to a regulatory sequence that drives the expression of the coding sequence. Preferably, the vector is a non-integrated vector. Preferably, the vector is ribonucleic acid (RNA), preferably messenger RNA. 【0010】 Preferably, the vector is an adeno-associated virus-based non-integrated vector. Preferably, the vector is an adeno-associated virus-based vector derived from serotype 9 adeno-associated virus (AAV9). Preferably, the capsid of the adeno-associated virus-based vector is made of the capsid protein of serotype 9 adeno-associated virus (AAV9), with internal terminal repeat sequences corresponding to serotype 2 adeno-associated virus flanking both ends of the nucleic acid sequence contained in the capsid. Preferably, the nucleic acid contained in the capsid contains a fragment encoding an amino acid sequence encoding TERT. Preferably, the vector contains a regulatory sequence which is a constitutive promoter, preferably a cytomegalovirus (CMV) promoter. 【0011】 In a further embodiment, the present invention provides a non-human animal characterized by exhibiting a pathological condition of renal fibrosis, which can be obtained when a sublethal dose of folic acid is administered to the non-human animal, the germ cells of which are heritably inactivated in both alleles of the Tert gene. 【0012】 Preferably, the animal is a mammal, preferably a rodent. The sublethal dose of folic acid is preferably up to 200 mg / kg body weight, preferably 125 mg / kg body weight. 【0013】 Preferably, folic acid is administered intraperitoneally. Preferably, folic acid is administered to animals when they are 4 to 10 weeks old, preferably 6 to 8 weeks old. Preferably, folic acid is administered as a single dose. 【0014】 Preferably, non-human animals whose germ cells contain heritable inactivation of both alleles of the Tert gene (Tert- / -) are third generation (G3) of the Tert- / - lineage. 【0015】 Detailed description of the invention The role of short telomeres in aplastic anemia and pulmonary fibrosis has been found to be crucial, primarily for the development of appropriate mouse models, which has paved the way for the development of potential therapeutic strategies for these diseases. However, other complex diseases, such as renal fibrosis, lack appropriate animal models, hindering the study of the role of short telomeres in these pathological conditions. 【0016】 In this study, the inventors aimed to investigate the involvement of short telomeres in renal fibrosis. To this end, it was necessary to develop a suitable animal model. The inventors first developed a wild-type mouse, and then a G3 telomere model that had been reared for three generations (G3), and therefore G3 Tert - / - We analyzed the kidneys of mice lacking the telomerase catalytic subunit Tert (Figure 9a). However, we found that the wild-type (Tert + / + ) Mice and 8-9 week old G3 Tert - / - We found that both mice exhibited remarkably normal renal histology, with no evidence of glomerular or tubular defects, and no fibrosis or collagen fiber accumulation (Figure 9b, c). Similarly, mice lacking the telomerase catalytic subunit Tert did not have increased tubular damage or loss of brush borders in the kidney. - / - Matching the normal kidney of a mouse, Tert+ / + Compared with the mouse (Figs. 1e - h), no differences were found in the presence of activated fibroblasts, apoptosis, senescence, or the expression of E - cadherin (a marker of EMT associated with tissue fibrosis). From these results, it was concluded that telomerase - deficient mice themselves do not spontaneously develop renal fibrosis, and the question was raised as to whether mice with short telomeres require additional factors to contribute to the development of this pathological condition. In this regard, since the incidence of renal fibrosis increases with age, the inventors also questioned whether renal fibrosis could be caused by a combination of molecular and cellular senescence events such as the presence of short telomeres together with exogenous injury to the kidney. 【0017】 Folic acid (FA) induces interstitial fibrosis, but only at high doses. Intraperitoneal administration of a high dose of folic acid (250 μg / g BW) to mice rapidly induces folic acid crystals accompanied by tubular necrosis in the acute phase (1 - 14 days) and patchy interstitial fibrosis in the chronic phase (28 - 42 days). However, lower doses of FA were not found to induce renal fibrosis in wild - type mice. Considering this, the inventors aimed to study the effect of a sub - lethal dose of FA in Tert + / + and G3 Tert - / - mice and its contribution to the development of renal fibrosis associated with short telomeres. For this purpose, the inventors first subjected 8 - 9 - week - old Tert + / + and G3 Tert - / - mice to increasing doses of FA (50, 100, 125, and 250 mg kg−1 body weight; Fig. 1a) and selected the highest dose of FA that did not induce renal fibrosis in wild - type mice. The results showed that 125 mg kg−1 was the maximum tolerated dose of FA that did not cause lethality in wild - type mice with normal telomere length (Fig. 1). The inventors then examined whether this dose of FA + / +Although not sufficient to induce renal fibrosis in mice, we investigated whether it could synergistically induce renal fibrosis in telomerase-deficient mice by shortening telomeres. After a series of tests, we found that telomerase-deficient mice treated with a sublethal dose of FA exhibited all the prominent features of the human disease, including severe renal impairment, as indicated by elevated blood levels of creatinine and urea. 【0018】 Furthermore, to evaluate the contribution of dysfunctional telomeres to the induction of renal fibrosis, the inventors used a second model of telomere dysfunction by deleting TRF1, one of the components of the Shertelin telomere protective complex. In particular, the inventors used a mouse model in which treatment with tamoxifen resulted in the deletion of TRF1 in all kidney cells (Figure 7). The results showed that mice with dysfunctional telomeres due to TRF1 deletion spontaneously developed renal fibrosis, highlighting the importance of proper telomere function in protecting against fibrous lesions. 【0019】 Overall, the present invention provides a suitable mouse model for studying telomere length-related renal fibrosis. This mouse model is an important tool for understanding the role of short, dysfunctional telomeres and resulting DNA damage in molecular events associated with fibrosis, particularly renal fibrosis. They also enable the development of gene therapy approaches aimed at correcting the accumulation of very short telomeres associated with the condition. 【0020】 In consideration of the above, the present invention, in a first aspect, provides compositions and methods useful for subjects requiring treatment and prevention of conditions associated with short telomere length, particularly renal fibrosis. “Conditions associated with short telomere length” are characterized by the accumulation of very short telomeres. In certain embodiments, conditions associated with short telomere length are characterized by mutations in one or more genes involved in telomere maintenance. Specific examples of such genetically based conditions include, but are not limited to, renal fibrosis, preferably telomere-shortened renal fibrosis. Short telomeres exacerbate the epithelial-to-mesenchymal transition (EMT) program in the kidney and thus promote pathological scarring of renal tissue, i.e., fibrosis. Telomere-shortened renal fibrosis is characterized by the presence of short / dysfunctional telomeres due to mutations in genes involved in telomere maintenance, with those encoding telomerase complex proteins (i.e., TERT, TERC, NOP10, DKC1, NHP2) being the most frequently mutated. Both functional telomerase complexes and shelltelin proteins, along with proper telomere sealing structures, are necessary for the maintenance and capping of chromosome ends, respectively. 【0021】 In some embodiments, renal fibrosis is characterized primarily by the abnormal production and deposition of extracellular matrix (ECM) proteins in the renal interstitium, leading to structural damage, impaired renal function, and ultimately end-stage renal disease (ESRD). Clinical features of patients with renal fibrosis include, among others, swelling of the ankles, feet, or hands, shortness of breath, fatigue, blood in the urine, insomnia, itchy skin, muscle cramps, and headaches. Renal fibrosis is a direct consequence of the kidney's limited ability to regenerate after injury. In some embodiments, renal fibrosis is characterized by fibroblast activation and excessive production and deposition of extracellular matrix (ECM), leading to destruction of the renal parenchyma, inflammatory and fibrotic responses, and decreased renal function. Note that renal fibrosis is synonymous with renal fibrosis. 【0022】 Accordingly, the present invention provides compositions and methods for treating subjects suffering from short telomere length, preferably symptoms associated with renal fibrosis, which require such treatment, comprising administering to a patient an agent that increases the patient's telomere length. The present invention also relates to methods for preventing such conditions. "Prevent," "prevent," or "prevention," or any other similar terms include, but are not limited to, reducing, mitigating, or improving the risk of a symptom, disorder, condition, or disease, and protecting an animal from a symptom, disorder, condition, or disease. Prevention may be applied or administered prophylactically. "Treat," "treat," or "cure," or any other similar terms include, but are not limited to, inhibiting, slowing, stopping, reducing, improving, or reversing the progression or severity of an existing symptom, clinical sign, disorder, condition, or disease. Cure may be applied or administered therapeutically. 【0023】 "Subjects requiring treatment" as used herein refers to subjects suffering from a condition exhibiting the early onset of a pathology resulting from the incomplete regenerative capacity of kidney tissue. In certain embodiments, subjects requiring treatment exhibit a fibrotic reaction, preferably a renal fibrotic reaction, and / or impaired renal function. In some embodiments, subjects requiring treatment present with a disease or condition that implies or ultimately leads to renal fibrosis, such as glomerulosclerosis, interstitial fibrosis, diabetes mellitus, hypertension, infectious glomerulonephritis, nephrovasculitis, ureteral obstruction, genetic alterations, autoimmune diseases, or any combination thereof. Preferably, subjects requiring treatment suffer from renal fibrosis, preferably renal fibrosis associated with telomere shortening. The terms "patient" and "subject" are considered synonymous herein and refer to mammals. In certain embodiments, the patient is a rodent, primate, human, ungulate, cat, dog, mouse, rat, rabbit, pig, horse, sheep, cattle, domestic cat or dog, or other domestic pet or livestock mammal. In a preferred embodiment, the patient or the person requiring it is a human being. 【0024】 Compositions and methods In one embodiment, the active ingredient prevents the degradation of chromosome ends. In one embodiment, the drug increases the activity of telomerase reverse transcriptase (TERT). In one embodiment, the treatment method is a gene therapy method that includes administering a nucleic acid vector containing the coding sequence of telomerase reverse transcriptase (TERT) to a patient. 【0025】 In certain embodiments, the TERT sequence used in a gene therapy vector is derived from the same species as the target. For example, gene therapy in humans is performed using a human TERT sequence. In one embodiment, TERT is encoded by a nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 (human TERT variants 1 and 2), or by an active fragment or functional equivalent of SEQ ID NO: 1 or SEQ ID NO: 3. The polypeptide sequence encoded by SEQ ID NO: 1 is shown in SEQ ID NO: 2. The polypeptide encoded by SEQ ID NO: 3 is shown in SEQ ID NO: 4. As used herein, “functional equivalent” refers to a polypeptide having TERT activity or a nucleic acid molecule encoding a polypeptide having TERT activity. Functional equivalents may exhibit 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100%, or more activity compared to TERT encoded by SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, TERT is encoded by a nucleic acid sequence containing or consisting solely of the sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Functional equivalents may exist artificially or naturally. For example, naturally occurring variants of TERT sequences in a population fall within the range of functional equivalents. TERT sequences derived from other species also fall within the scope of the term “functional equivalent,” particularly the mouse TERT sequence shown in SEQ ID NO: 5. In certain embodiments, a functional equivalent is a nucleic acid having a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identical to SEQ ID NO: 1 or SEQ ID NO: 3. In further embodiments, a functional equivalent is a polypeptide having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identical to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, TERT contains or consists solely of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. For functional equivalents, sequence identity should be calculated along the entire length of the nucleic acid.Functional equivalents may include one or more nucleotide insertions, deletions, and / or substitutions, e.g., 2, 3, 4, 5, 10, 15, 20, 30 or more, when compared to SEQ ID NO: 1 or SEQ ID NO: 3. The term “functional equivalent” also includes nucleic acid sequences encoding TERT polypeptides that have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity with the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, but show little homology to the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 due to genetic code degeneracy. Sequence identity can be calculated by any one of the various methods in the art, e.g., BLAST and variations of these alignment programs. 【0026】 As used herein, the term “active fragment” refers to a polypeptide having TERT activity, or a nucleic acid molecule encoding a polypeptide that has TERT activity but is a fragment of the nucleic acid shown in SEQ ID NO: 1 or SEQ ID NO: 3, or an amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. Active fragments may be of any size, as long as TERT activity is retained. Fragments have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, and 100% identity with SEQ ID NOs: 1-4 along the alignment length between the shorter fragment and SEQ ID NOs: 1-4. 【0027】 Fusion proteins containing these fragments may be included in nucleic acid vectors necessary to carry out the present invention. For example, an additional 5, 10, 20, 30, 40, 50, or even 100 amino acid residues from the polypeptide sequence or homologous sequence may be included in either or both of the C-terminus and / or N-terminus without impairing the polypeptide fragment's ability to fold correctly and exhibit biological activity. 【0028】 In one embodiment, the therapeutic method is a gene therapy method, and / or the nucleic acid vector used is a gene therapy vector. Gene therapy methods and vectors are well known in the art and generally involve targeting and delivering nucleic acids encoding therapeutically active proteins. Nucleic acids can be delivered in several ways and forms, including delivery of naked deoxyribonucleic acid (DNA) such as plasmids or minicircles, delivery of ribonucleic acid (RNA) such as messenger RNA, use of liposomes or cationic polymers or other engineered nanoparticles containing nucleic acids, or viral vectors that capture nucleic acids. 【0029】 In further embodiments, gene therapy is achieved using stable transformation of organisms with inducible expression systems. Suitable inducible expression systems are known in the art and include CRE-LOX recombinase-based systems suitable for use in mice and tetracycline-regulated systems that can be used for the treatment of human subjects. 【0030】 In one embodiment, the gene therapy vector is a ribonucleic acid (RNA) vector containing or consisting solely of the coding sequence for telomerase reverse transcriptase (TERT) as defined above. In some embodiments, the RNA nucleic acid vector is delivered in a naked form, i.e., unshielded RNA. In some other embodiments, the RNA nucleic acid vector is modified to protect it from nuclease degradation. Such modifications include chemical modifications to the RNA structure, such as those made at the 2' position, phosphate bonds, and / or nucleic acid bases. Other modifications include encapsulation of the RNA nucleic acid vector into nanoparticles, polymers, endosomes, lipids and lipid-like particles, or other delivery vectors such as viruses. Nanoparticle encapsulation of RNA physically protects the nucleic acid from degradation and, depending on the specific chemistry, can assist in cellular uptake and endosomal evasion. Polymers such as poly(β-aminoesters), poly-L-lysine, polyamidoamines, and polyethyleneimines, as well as naturally occurring polymers such as chitosan, have all been applied to RNA delivery. A chemically well-defined delivery method involves directly conjugating bioactive ligands, such as N-acetylgalactosamine, cholesterol, vitamin E, antibodies, and peptides, into RNA that allows them to enter the target cell. 【0031】 Preferably, the RNA nucleic acid vector is a single-stranded RNA molecule, more preferably a messenger RNA (mRNA) vector. The delivery of therapeutic mRNA vectors has been facilitated by maximizing the translation and stability of the mRNA vector and preventing the development of its immunostimulatory activity and in vivo delivery techniques. In some embodiments, mRNA vectors are modified to increase in vivo delivery efficacy. In some embodiments, the mRNA vector is modified to include the incorporation of a 5' cap and / or 3' poly(A) tail for efficient translation and half-life extension of mature mRNA. Cap analogues such as 120-150 bp ARCA (anti-reverse cap analogues) and poly(A) tails can also be used. Novel types of cap analogues with resistance to RNA decapping complexes, such as 1,2-dithiodiphosphate-modified caps, are also included. So-called codon optimization also promotes better efficacy of protein synthesis and limits mRNA destabilization by rare codons. Similarly, manipulation of the 3' and 5' untranslated regions (UTRs) containing sequences involved in the recruitment of RNA-binding proteins (RBPs) can increase the level of protein products. N1-methyl-pseudridine base modifications are commonly used to mask mRNA immunostimulatory activity. 【0032】 In one embodiment, the regulatory sequence operably ligated to the TERT coding sequence is a cytomegalovirus promoter (CMV), but other suitable regulatory sequences are known to those skilled in the art. In another embodiment, the regulatory sequence operably ligated to the TERT coding sequence is a kidney-specific promoter. In a preferred embodiment, the coding sequence of the telomerase reverse transcriptase (TERT) gene contained in an RNA nucleic acid vector is operably ligated to CMV or a kidney-specific promoter, and the RNA nucleic acid vector further comprises a poly(A) sequence positioned at the end of the coding sequence of the telomerase reverse transcriptase (TERT) gene. 【0033】 In one embodiment, the gene therapy vector is a viral vector. Viral gene therapy vectors are well known in the art. Examples of vectors include embedded and non-embedded vectors based on retroviruses, adenoviruses (AdV), adeno-associated viruses (AAV), antiviruses, poxviruses, alphaviruses, and herpesviruses. It appears particularly advantageous to use non-embedded viral vectors such as AAV. In one embodiment, this is because non-embedded vectors do not cause any permanent genetic modification. Secondly, the vector targets adult tissue, avoiding placing the target under the influence of constitutive telomerase expression from the early stages of development. Furthermore, non-embedded vectors effectively incorporate safety mechanisms to avoid the overgrowth of TERT-expressing cells. Once cells begin to proliferate rapidly, they lose the vector (and consequently, telomerase expression). 【0034】 Specific examples of suitable non-integrated vectors include those based on adenoviruses (AdV), particularly gutless adenoviruses, adeno-associated viruses (AAVs), integrase-deficient lentiviruses, poxviruses, alphaviruses, and herpesviruses. Preferably, the non-integrated vectors used in the present invention are adeno-associated virus-based non-integrated vectors similar to naturally occurring adeno-associated virus particles. 【0035】 Adeno-associated virus (AAV) vectors are emerging as one of the best vectors for many gene transduction applications due to their many desirable properties, including the ability to efficiently transduce a wide range of tissues, low immunogenicity, and an excellent safety profile, with no toxicity observed in many preclinical models. AAV vectors can transduce postmittal cells and maintain long-term gene expression (up to several years) in both small and large animal models of disease. The safety and efficacy of AAV gene transduction have been extensively studied in humans, with promising results in the liver, muscle, CNS, and retina. 【0036】 AAV preferentially targets postmittal tissues that are considered more resistant to cancer than highly proliferative tissues. Examples of adeno-associated virus-based non-integrated vectors include vectors based on any AAV serotype, namely AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and pseudotyped AAV. Tissue specificity is determined by the capsid serotype. Pseudotyping and capsid engineering of AAV vectors to modify their targeting range are likely to be important for their use in therapeutics. 【0037】 AAV2 is the best-characterized serotype for gene transfer studies in both human and experimental models. AAV2 exhibits innate tropism towards skeletal muscle, neurons, vascular smooth muscle cells, and hepatocytes. Therefore, AAV2 is a good choice of vector for targeting these tissues, particularly when treating conditions associated with one of these tissues using the methods or vectors of the present invention. For example, the treatment of neuromuscular degeneration can thus target skeletal muscle and / or neurons. 【0038】 Newly isolated serotypes such as AAV7, AAV8, and AAV9 have been successfully employed in preclinical studies, enabling long-term expression of therapeutic genes depending on the target tissue and route of administration. Furthermore, the use of non-human serotypes such as AAV8 and AAV9 may be useful in overcoming these immunological responses in subjects, and clinical trials have just begun (ClinicalTrials.gov Identifier:NCT00979238). 【0039】 Overall, these promising data suggest that AAV vectors are a useful tool for treating human diseases with a high safety and efficient profile. 【0040】 The selection of broadly directional adeno-associated viruses, such as those derived from serotype 9 adeno-associated virus (AAV9), is particularly advantageous when treating conditions associated with short telomere length. AAV9 viruses exhibit efficient transduction across a wide range of tissues, with high directionality to the liver, heart, and skeletal muscle, thus enabling the beneficial effects of gene therapy to be achieved in more tissues. Furthermore, AAV9 vectors possess the unique ability to cross the blood-brain barrier upon intravenous injection in adult mice and cats, and to target the brain (Foust et al. Nature biotechnology 2009). 【0041】 One aspect of the present invention provides a system in which the capsid (the part of the virus that determines the virulence) of an adeno-associated virus system vector is made from the capsid protein of serotype 9 adeno-associated virus (AAV9). In one embodiment of the viral vector for use in the present invention, the polynucleotide sequence packed into the capsid is adeno-associated virus, preferably a serotype 2 internal terminal repeat (ITR) that presents a coding sequence located between ITRs. As described above, the nucleic acid preferably codes for a functional TERT polypeptide. In one embodiment, the regulatory sequence operably ligated to the TERT coding sequence is a cytomegalovirus promoter (CMV), but other suitable regulatory sequences are known to those skilled in the art. In another embodiment, the regulatory sequence operably ligated to the TERT coding sequence is a kidney-specific promoter. 【0042】 When treating conditions associated with short telomere length, it is advantageous to target the treatment to the affected tissue. Therefore, the selection of the AAV serotype for the capsid protein of a gene therapy vector may be based on the desired site of gene therapy. If the target tissue is skeletal muscle, for example, in the treatment of loss of neuromuscular coordination, AAV1 and AAV6-based viral vectors can be used. Both of these serotypes are more efficient in muscle transfection than other AAV serotypes. 【0043】 Alternatively, other viral vectors can be used in this invention. Any vector suitable for use in gene therapy can be used in this invention. Heilbronn & Weger (2010) Handb Exp Pharmacol. 197:143-70 provides a review of viral vectors useful for gene therapy. 【0044】 As discussed above, a vector containing a telomerase reverse transcriptase (TERT) coding sequence suitable for use in gene therapy is a key point for carrying out the present invention. A suitable gene therapy vector comprises any type of particle containing a polynucleotide fragment encoding a telomerase reverse transcriptase (TERT) protein, operably linked to a regulatory element such as a promoter that enables the expression of a functional TERT protein exhibiting telomerase reverse transcriptase activity in target cells. Preferably, TERT is encoded by the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or is an active fragment or functional equivalent of TERT. The term gene therapy vector, provided that the TERT coding sequence and its linked regulatory element are inserted into a plasmid, includes, within its scope, plasmids or minicircles, i.e., naked DNA molecules such as circular DNA molecules that do not contain bacterial DNA sequences, as well as more complex systems, such as a viral particle having a structure that includes at least one capsid and at least one polynucleotide sequence, and having a size that allows the polynucleotide sequence to be filled into the capsid in a manner similar to that of the natural genome of the virus from which the capsid originates. The polynucleotide sequence must include a region into which the TERT coding sequence and its linked regulatory elements are inserted, so that when a viral particle infects a cell, the telomerase reverse transcriptase protein can be expressed from that polynucleotide sequence. 【0045】 In one embodiment, a gene therapy vector suitable for use in the present invention is a non-integrated vector, such as an adeno-associated virus-based non-integrated vector. For the purposes of the present invention, the selection of non-integrated vectors appears to be particularly advantageous because they do not cause any permanent genetic modification. Furthermore, as mentioned above, such vectors incorporate safety mechanisms to avoid the overgrowth of TERT-expressing cells, which would lose the vector if the cells began to proliferate rapidly. 【0046】 Adeno-associated virus-based vectors derived from serotype 9 adeno-associated virus (AAV9) are preferred because they can achieve beneficial effects in a wider range of tissues (see above). In one particularly preferred embodiment, the regulatory sequence operably ligated to the TERT coding sequence is a cytomegalovirus promoter (CMV). The nucleic acid sequence encoding TERT is operably ligated to a regulatory sequence that drives the expression of the coding sequence. As used herein, the term “regulatory element” means a nucleic acid sequence that acts as a promoter, i.e., regulates the expression of a nucleic acid sequence operably ligated to a promoter. Such “regulatory elements” or “promoters” can control the expression of the ligated nucleic acid sequence either constitutively or inductively. The regulatory sequence may be a constitutive promoter. An example of a regulatory sequence that is a constitutive promoter is the cytomegalovirus (CMV) promoter. 【0047】 The expression of TERT after gene therapy according to the present invention lasts for several months to several years. In one embodiment of the present invention, the subject is treated once. In an alternative embodiment, the subject is treated first, and then again when the TERT expression level has decreased by about 50% to the expression level obtained immediately after treatment. Treatment may be repeated as needed, for example, annually, or every 5 or 10 years, using the same or an alternative vector to maintain the reduction of age-related impairment. When administering a second or subsequent dose, it may be necessary to use a different gene therapy vector; for example, when using an AAV-based vector, the second and subsequent doses may be a vector having a capsid derived from a different serotype than the one used for the first dose. 【0048】 The therapeutic method of the present invention has the effect of treating and / or preventing conditions associated with short telomere length. Accordingly, in a further embodiment, the present invention relates to a gene therapy method or the use of the above nucleic acid vector for use in the treatment or prevention of renal fibrosis, preferably renal or fibrosis associated with short telomere length, in subjects requiring it. 【0049】 The effectiveness of treatment for conditions associated with short telomere length can be measured by various methods known in the art. In one embodiment, the effectiveness of treatment is measured by the increase in lifespan of treated patients with the condition associated with short telomere length compared to the expected lifespan of untreated patients with the same condition. In certain embodiments, lifespan is extended by 5%, 10%, 15%, 20%, or more, relative to the expected lifespan of patients with the same condition. In one embodiment, the effectiveness of treatment is measured by delayed or prophylactic renal failure in treated patients with the condition associated with short telomere length compared to the expected onset renal failure in untreated patients with the same condition. In certain embodiments, the delay in the onset of bone marrow failure in treated patients with the condition associated with short telomere length is extended by 5%, 10%, 15%, 20%, or more, relative to the expected onset of renal failure in untreated patients with the same condition. 【0050】 In one embodiment, the effectiveness of treatment is measured by the increase in the overall fitness of treated patients with a condition associated with short telomere length, preferably renal fibrosis, compared to the overall fitness of untreated patients with the same condition. Overall fitness can be determined by measuring physical attributes associated with a particular condition. Thus, an increase in overall fitness can be determined by a decrease in physical attributes associated with a particular condition, as shown by the treated patient. In one embodiment, the increase in overall fitness is measured by determining the estimated glomerular filtration rate (eGFR) and / or albuminuria (ACR). A glomerular filtration rate (GFR) of 60 or higher is within the normal range. A GFR of less than 60 may indicate kidney disease. A GFR of 15 or lower may indicate renal failure. 【0051】 A standardized albumin / creatinine ratio (ACR) test indicates the presence of albumin in the urine. A normal amount of albumin in urine is less than 30 mg / g. A level greater than 30 mg / g may indicate kidney disease, even if the glomerular filtration rate (GFR) is above 60. In certain embodiments, the glomerular filtration rate (GFR) of treated patients increases by 5%, 10%, 15%, 20%, or more compared to the GFR of untreated patients with the same condition. 【0052】 The effectiveness of treatment can also be measured by directly determining the telomere length in a sample taken from the patient. Telomere length can be measured, for example, in a sample taken from the patient using standard hybridization techniques such as fluorescence in situ hybridization (FISH), quantitative fluorescence in situ hybridization (Q-FISH), or high-throughput quantitative fluorescence in situ hybridization (HT Q-FISH). Suitable samples for telomere analysis include blood, urine, or tissue biopsies such as kidney biopsies. 【0053】 In certain embodiments, samples can be taken from patients receiving treatment throughout the course of treatment to determine both the absolute telomere length and the rate of telomere shortening over the course of treatment. Samples may be taken daily or at longer intervals during the course of treatment. In one embodiment, samples may be taken once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, or more. Telomere length comparisons can be measured by comparing the percentage of short telomeres in samples taken from patients. In one embodiment, the percentage of short telomeres is the percentage of telomeres that exhibit an intensity below the average intensity of the sample, as measured by in situ hybridization techniques (e.g., FISH or Q-FISH). In embodiments, the percentage of short telomeres is the percentage of telomeres that exhibit an intensity 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, or lower than the average intensity of the sample. In one particular embodiment, the proportion of short telomeres is the proportion of telomeres that exhibit an intensity 50% or more lower than the average intensity of the sample. 【0054】 In another embodiment, the proportion of short telomeres is the proportion of telomeres of a certain length, for example, 8kb, 7kb, 6kb, 5kb, or shorter. In one embodiment, the proportion of short telomeres is the proportion of telomeres of 8kb or less. In another embodiment, the proportion of short telomeres is the proportion of telomeres of 7kb or less. In another embodiment, the proportion of short telomeres is the proportion of telomeres of 6kb or less. In another embodiment, the proportion of short telomeres is the proportion of telomeres of 5kb or less. In another embodiment, the proportion of short telomeres is the proportion of telomeres of 4kb or less. In another embodiment, the proportion of short telomeres is the proportion of telomeres of 3kb or less. 【0055】 In one embodiment, the effectiveness of the treatment is measured by a reduction in the percentage of short telomeres in samples taken from treated patients suffering from a condition associated with short telomere length, preferably renal fibrosis, compared to a control sample. In one embodiment, the percentage of short telomeres in samples taken from treated patients is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, or more compared to a control sample. In one embodiment, the control sample is a sample taken from the same patient before treatment or a sample taken in the early stages of treatment. In another embodiment, the control sample is a sample taken from a patient suffering from the same condition but not being offered treatment. 【0056】 In a further embodiment, the present invention is applied to a subject by administering a pharmaceutical composition comprising an effective amount of any one of the gene therapy vectors conforming to the present invention described above. 【0057】 A "pharmaceutical composition" is intended to include a combination of an activator and an inactive or active carrier that makes the composition suitable for diagnostic or therapeutic use in vitro, in vivo, or ex vivo. 【0058】 "Composition" is intended to mean a combination of an active agent and another compound or composition that is inactive (e.g., a detectable active substance or label) or active. "Effective dose" is an amount sufficient to produce a beneficial or desired result. An effective dose may be administered in one or more doses, applications, or administrations. 【0059】 These typically contain components in addition to the active ingredient (such as a gene therapy vector), for example, one or more pharmaceutical carriers and / or excipients. 【0060】 The composition is generally administered to the target in aqueous form. However, prior to administration, the composition may be in a non-aqueous form. For example, some viral vectors are manufactured in aqueous form, then filled and dispensed, and administered in aqueous form, while other viral vectors are lyophilized during manufacturing and reconstituted into aqueous form at the time of use. Therefore, the composition of the present invention can be dried, such as in a lyophilized formulation. The composition may contain preservatives such as thiomersal or 2-phenoxyethanol. However, it is preferable that the composition is substantially free of mercury materials, such as thiomersal (i.e., less than 5 μg / m³). 【0061】 To control isotonicity, it is preferable to include physiological salts such as sodium salts. Sodium chloride (NaCl) is preferred, and it can be present at 1 to 20 mg / ml, for example, about 10+2 mg / ml. Other possible salts include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dihydrate, magnesium chloride, and calcium chloride. 【0062】 The composition generally has a weight osmolality of 200 mOsm / kg to 400 mOsm / kg, preferably 240 to 360 mOsm / kg, and more preferably within the range of 290 to 310 mOsm / kg. 【0063】 The composition may contain one or more buffers. Typical buffers and specifications include phosphate buffer; Tris buffer; borate buffer; succinate buffer; histidine buffer (especially containing aluminum hydroxide adjuvant); or citrate buffer. The buffer is typically contained in the range of 5 to 20 mM. 【0064】 The composition may contain materials for a single dose or materials for multiple doses (i.e., a "multi-dose" kit). The inclusion of a preservative is preferred in the multi-dose configuration. Alternatively (or in addition to) including a preservative in the multi-dose composition, the composition may be housed in a container having a sterile adapter for removing materials. 【0065】 The compositions of the present invention for use in humans are typically administered in doses of about 0.5 ml, but half a dose (i.e., about 0.25 ml) may be administered to children. 【0066】 Similar to the therapeutic methods described herein, the present invention also provides nucleic acid sequences encoding TERT for therapeutic use. The present invention also provides nucleic acid vectors containing a telomerase reverse transcriptase (TERT) coding sequence for therapeutic use, and gene therapy vectors containing a telomerase reverse transcriptase (TERT) coding sequence for therapeutic use. In particular, the therapeutic methods may treat or prevent conditions associated with short telomere length. As described in the therapeutic methods, the TERT nucleic acid sequence may be the sequence listed in SEQ ID NO: 1 or SEQ ID NO: 3, or a fragment or functional equivalent thereof. The TERT protein may have the sequence described in SEQ ID NO: 2 or SEQ ID NO: 4, or a fragment or functional equivalent thereof. 【0067】 The following embodiments also derive from the first aspect and are included herein. 【0068】 1. A method for treating a patient having renal fibrosis associated with short telomere length, comprising administering a nucleic acid vector containing a coding sequence for telomerase reverse transcriptase (TERT) to the patient. 【0069】 2. The method according to 1, wherein the TERT is encoded by a nucleic acid sequence that includes a sequence that is at least 90% identical to the sequence of sequence number 1 or sequence number 3. 【0070】 3. The method according to 1 or 2, wherein the TERT is encoded by a nucleic acid sequence containing the sequence of sequence number 1 or sequence number 3. 【0071】 4. The method according to any one of 1 to 3, wherein the TERT is encoded by a nucleic acid sequence consisting only of the sequence of sequence number 1 or sequence number 3. 【0072】 5. The method according to any one of 1 to 4, wherein the TERT contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. 【0073】 6. The method according to any one of 1 to 5, wherein the TERT comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. 【0074】 7. The method according to any one of 1 to 6, wherein the TERT consists only of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. 【0075】 8. The method according to any one of 1 to 7, wherein the nucleic acid sequence encoding the TERT is operably linked to a regulatory sequence that drives the expression of the coding sequence. 【0076】 9. The method according to any one of 1 to 8, wherein the vector is a non-embedded vector. 【0077】 10. The method according to any one of 1 to 9, wherein the vector is an adeno-associated virus-based non-integrated vector or RNA vector. 【0078】 11. The method according to any one of 1 to 10, wherein the vector is an adeno-associated virus-based vector derived from serotype 9 adeno-associated virus (AAV9). 【0079】 12. The method according to 11, wherein the capsid of the adeno-associated virus-based vector is made of the capsid protein of serotype 9 adeno-associated virus (AAV9), and internal terminal repeat sequences corresponding to serotype 2 adeno-associated virus are adjacent to both ends of the nucleic acid sequence contained in the capsid. 【0080】 13. The method according to 12, wherein the nucleic acid contained in the capsid contains a fragment encoding an amino acid sequence encoding TERT. 【0081】 14. The method according to any one of 1 to 13, wherein the vector comprises a regulatory sequence that is a constitutive promoter. 【0082】 15. The method according to 14, wherein the regulatory sequence is a cytomegalovirus (CMV) promoter. 【0083】 16. The method according to any one of 1 to 15, wherein the renal fibrosis associated with short telomere length is characterized by mutations in one or more genes involved in telomere maintenance. 【0084】 Non-human knockout animal models In a second aspect of the present invention, the present invention provides a non-human knockout animal model characterized by exhibiting a pathological condition of renal fibrosis, the non-human animal being obtained or can be obtained when a sublethal dose of folic acid is administered to a non-human knockout animal comprising inactivation of at least one, preferably both, alleles of the Tert gene. "Inactivation of at least one, preferably both, alleles of the Tert gene" means, as herein, a process by which the sequence of one or both alleles of the Tert gene is altered, thereby causing the allele(s) to lose their biological function. The alteration of the allele(s) sequence may be by inserting nucleotides into the allele(s) or by partially or completely deleting nucleotides in the allele(s). 【0085】 Specifically, non-human knockout animal models have a knockout mutation in the Tert gene, resulting in the knockout of Tert. As used herein, the term “knockout animal” refers to a non-human animal, preferably a mammal, having one or more genetic modifications that result in the inactivation of the Tert gene, one or preferably both alleles of the gene. Such knockout mutations in one or both alleles of the Tert gene may be present in the germ cells, somatic cells, or both of an animal (i.e., knockout mutations in the Tert gene are present in all animal cells). “Germ cells” is used herein to mean germ cells or gametes (eggs and sperm). “Somatic cells” is used herein to mean cells that form the body of a multicellular organism other than germ cells or gametes. 【0086】 In a preferred embodiment, the non-human knockout animal is a rodent, such as a mouse or rat. The founder of the male animal is called G0, and the first, second, and third generations are called G1, G2, and G3, respectively. Preferably, the non-human knockout animal is Tert - / - The third generation (G3) or later generations (G4, G5, etc.) of the knockout (know-out) animal lineage. Preferably, the non-human animal model is G3 Tert - / - It's a mouse. "G3 Tert - / - In this specification, the expression "Tert gene inactivation" refers to a third-generation animal, preferably a mouse, in which both alleles of the Tert gene are inactivated, preferably the inactivation is hereditary, i.e., present in the germ cells of the animal. - / - To produce animal colonies, heterozygous animals Tert + / - By crossing them, homozygous animals Tert - / - These are produced for further phenotypic determination. Knockout techniques are well known in this field. 【0087】 Genes for the production of knockout animals can be introduced using embryonic cells at various developmental stages. Different methods are used depending on the developmental stage of the embryonic cells. Such transfected embryonic stem (ES) cells can then implant in the embryo after their introduction into the blastocyst stage embryo and contribute to the germ cells of the resulting chimeric animal. If the knockout gene provides a means for such selection before introducing transfected ES cells into the blastocyst, the transfected ES cells can be subjected to various selection protocols to enrich the proportion of ES cells incorporating the knockout gene. Alternatively, PCR can be used to screen for ES cells incorporating the knockout. 【0088】 Furthermore, knockout genes can also be introduced into non-human animals using retroviral infection. Developing non-human embryos can be cultured in vitro up to the blastocyst stage. During this period, blastomeres can be targeted for retroviral infection. Efficient infection of blastomeres can be achieved by enzymatic treatment to remove the zona pellucida. The viral vector system used to introduce the knockout is typically a replication-deficient retrovirus containing the knockout. Transfection is easily and efficiently achieved by culturing blastomeres on a monolayer of virus-producing cells. Alternatively, infection can be performed at a later stage. The virus or virus-producing cells can be injected into the blastocoel. Since integration occurs only in a subset of cells that form the knockout animal, most primordia are mosaics of the knockout gene. Furthermore, founders can generally contain various retroviral insertions of the knockout gene at different positions in the genome that separate into offspring. In addition, the knockout gene can be introduced into the germline by intrauterine retroviral infection of mid-term embryos. Further means of using retroviruses or retroviral vectors to produce knockout animals known to those skilled in the art include microinjecting retroviral particles or retrovirus-producing cells treated with mitomycin C into the perivitelline space of a fertilized egg or early embryo. 【0089】 Once the emergent autologous cells are produced, they can be bred, inbred, uninbred, or crossbred to produce colonies of a particular animal. Knockout animals are screened and evaluated to select animals with the desired phenotype. Initial screening can be performed, for example, using Southern blotting or PCR techniques to analyze animal tissue to confirm that the knockout gene has occurred. Know-out animals can be identified by evaluating the level of TERT mRNA expression in the tissues of knockout animals using techniques including, but not limited to, Northern blotting, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR) of tissue samples obtained from animals. Suitable tissue samples can be immunocytochemically evaluated using antibodies or tags such as EGFP that are specific to the Tert protein. Knockout non-human mammals can be further characterized to identify animals with phenotypes useful in the present invention. In particular, knockout non-human mammals in which one or both Tert alleles are inactivated can be screened according to the presence of renal pathology or fibrosis when administered a sublethal dose of FA. 【0090】 As shown in the following example, G3 Tert - / -Non-human knockout mice become a non-human animal model of renal fibrosis only when administered a sublethal dose of FA. Therefore, it should be noted that a second embodiment of the non-human animal model, characterized by knockout of at least one, preferably both, alleles of the gene encoding the telomerase catalytic subunit Tert, is further characterized by exhibiting a pathological state of renal fibrosis upon treatment with a sublethal dose of folic acid. “Sublethal dose” as used herein refers to a dose that does not induce renal fibrosis in wild-type mice. In some embodiments, the sublethal dose is less than 250 mg / kg body weight. Preferably, the dose of FA administered to non-human animals is in the range of 50–200 mg / kg, more preferably 50–175 mg / kg, 50–150 mg / kg, 100–150 mg / kg, and most preferably about 125 mg / kg body weight. Preferably, the dose of FA is up to 200 mg / kg body weight, more preferably up to 125 mg / kg body weight. 【0091】 The non-human knockout animal model of renal fibrosis is further characterized by elevated blood creatinine and BUN levels upon administration of a sublethal dose of FA. Furthermore, the non-human knockout animal model exhibits increased renal fibrosis as detected by Masson's trichrome staining and increased renal tubular damage as determined by increased PAS+D staining, as shown in the following examples. 【0092】 In some embodiments, the FA dose is administered once, preferably at the start of the study. In some embodiments, the FA is administered several times. In some other embodiments, the FA is administered once daily over several days (e.g., once daily, once every two days, once a week, once every two weeks, or once a month). In some embodiments, the FA dose is administered systemically, preferably intraperitoneally or intravenously. 【0093】 The non-human knockout animals administered with FA to create the non-human animal model of the second embodiment are 4 to 10 weeks old, preferably 6 to 8 weeks old. 【0094】 In a third aspect, the present invention provides a non-human knockout animal model for use in studying and / or evaluating renal fibrosis associated with the presence of short telomeres, the non-human knockout animal being as defined in the second aspect or any of its embodiments. 【0095】 In a fourth aspect, the present invention provides a method for creating a non-human knockout animal model for evaluating renal fibrosis associated with the presence of short telomeres, the method comprising: i) A third generation (G3 Tert) as defined in either of the second embodiment or its embodiments. - / - A step to provide a non-human animal, preferably a rodent, characterized in that one, preferably both, alleles of the gene encoding the telomerase catalytic subunit Tert, derived from a non-human animal, are knocked out, and ii) a step of administering at least one sublethal dose of folic acid to the non-human animal i), as defined in the second embodiment or any of its embodiments. 【0096】 In a fifth aspect, the present invention provides a non-human knockout animal model for use in evaluating renal fibrosis associated with the presence of short telomeres, the non-human animal being obtained according to the method of the fourth aspect or any of its embodiments. 【0097】 In a sixth aspect, the present invention provides a method for evaluating renal fibrosis associated with the presence of short telomeres, the method comprising the use of a non-human animal model as defined in the second aspect or any of its embodiments. 【0098】 In a seventh aspect, the present invention provides a non-human animal model, as defined in the second aspect or any of its embodiments, for use in a method for screening compounds to improve, treat or prevent renal fibrosis associated with the presence of short telomeres. In a preferred embodiment, this method is: i) A step of administering the compound to a non-human animal model of renal fibrosis associated with the presence of short telomeres as defined in the second embodiment or any of its embodiments, ii) A step of detecting or evaluating the symptoms of renal fibrosis, and iii) A step of comparing the symptoms of renal fibrosis with those of an animal group that was not administered the compound, and selecting a compound that improved the symptoms, and / or iv) A step of comparing the symptoms of renal fibrosis before and after administration of the test compound. 【0099】 In the eighth aspect, the present invention provides a screening method for test compounds for renal fibrosis associated with the presence of short telomeres, wherein the test compound is administered to a non-human knockout animal model as defined in the second aspect or any embodiment thereof, any change in life expectancy or symptoms associated with renal fibrosis associated with the presence of short telomeres is measured and evaluated, and the test compound that improves such change is determined to have a therapeutic effect. A particular embodiment of the screening method of the seventh or eighth aspect includes the step of contacting tissues, organs or cells derived from the non-human knockout animal model with the test compound, measuring and evaluating changes in such tissues, organs or cells to determine which test compounds cause improvement and therefore have a therapeutic effect. 【0100】 In the seventh or eighth aspect, a test compound found to have a therapeutic effect on renal fibrosis associated with the presence of short telomeres, which can be obtained by the screening method disclosed herein, can be used as a treatment for patients who have or are at risk of developing renal fibrosis associated with the presence of short telomeres. In one embodiment of the screening method, a case of a non-human animal model of the second aspect or any embodiment thereof is evaluated in comparison to a case of a wild-type mouse or a non-human animal of the second aspect that has not been treated with the test compound. 【0101】 In a ninth aspect, the present invention relates to the use of a non-human animal model as defined in the second aspect or any of its embodiments in a method for screening test compounds for renal fibrosis associated with the presence of short telomeres. In a preferred embodiment, this method is i) A step of administering the compound to a non-human animal model of renal fibrosis associated with the presence of short telomeres as defined in the second embodiment or any of its embodiments, ii) A step of detecting or evaluating the symptoms of renal fibrosis, and iii) A step of comparing the symptoms of renal fibrosis with those of an animal group that was not administered the compound, and selecting a compound that improved the symptoms, and / or iv) The procedure includes a step of comparing the symptoms of renal fibrosis before and after administration of the test compound and selecting the compound that improved the symptoms. 【0102】 It should be noted that the embodiments and definitions included in the second aspect of the present invention also apply to the third, fourth, fifth, sixth, seventh, eighth, and ninth aspects. 【0103】 Sequence List Sequence ID 1 caggcagcgc tgcgtcctgc tgcgcacgtg ggaagccctg gccccggcca cccccgcgat gccgcgcgct ccccgctgcc gagccgtgcg ctccctgctg cgcagccact accgcgaggt gctgccgctg gccacgttcg tgcggcgcct ggggccccag ggctggcggc tggtgcagcg cggggacccg gcggctttcc gcgcgctggt ggcccagtgc ctggtgtgcg tgccctggga cgcacggccg ccccccgccg ccccctcctt ccgccaggtg tcctgcctga aggagctggt ggcccgagtg ctgcagaggc tgtgcgagcg cggcgcgaag aacgtgctgg ccttcggctt cgcgctgctg gacggggccc gcgggggccc ccccgaggcc ttcaccacca gcgtgcgcag ctacctgccc aacacggtga ccgacgcact gcgggggagc ggggcgtggg ggctgctgct gcgccgcgtg ggcgacgacg tgctggttca cctgctggca cgctgcgcgc tctttgtgct ggtggctccc agctgcgcct accaggtgtg cgggccgccg ctgtaccagc tcggcgctgc cactcaggcc cggcccccgc cacacgctag tggaccccga aggcgtctgg gatgcgaacg ggcctggaac catagcgtca gggaggccgg ggtccccctg ggcctgccag ccccgggtgc gaggaggcgc gggggcagtg ccagccgaag tctgccgttg cccaagaggc ccaggcgtgg cgctgcccct gagccggagc ggacgcccgt tgggcagggg tcctgggccc acccgggcag gacgcgtgga ccgagtgacc gtggtttctg tgtggtgtca cctgccagac ccgccgaaga agccacctct ttggagggtg cgctctctgg cacgcgccac tcccacccat ccgtgggccg ccagcaccac gcgggccccc catccacatc gcggccacca cgtccctggg acacgccttg tcccccggtg tacgccgaga ccaagcactt cctctactcc tcaggcgaca aggagcagct gcggccctcc ttcctactca gctctctgag gcccagcctg actggcgctc ggaggctcgt ggagaccatc tttctgggtt ccaggccctg gatgccaggg actccccgca ggttgccccg cctgccccag cgctactggc aaatgcggcc cctgtttctg gagctgcttg ggaaccacgc gcagtgcccc tacggggtgc tcctcaagac gcactgcccg ctgcgagctg cggtcacccc agcagccggt gtctgtgccc gggagaagcc ccagggctct gtggcggccc ccgaggagga ggacacagac ccccgtcgcc tggtgcagct gctccgccag cacagcagcc cctggcaggt gtacggcttc gtgcgggcct gcctgcgccg gctggtgccc ccaggcctct ggggctccag gcacaacgaa cgccgcttcc tcaggaacac caagaagttc atctccctgg ggaagcatgc caagctctcg ctgcaggagc tgacgtggaa gatgagcgtg cgggactgcg cttggctgcg caggagccca ggggttggct gtgttccggc cgcagagcac cgtctgcgtg aggagatcct ggccaagttc ctgcactggc tgatgagtgt gtacgtcgtc gagctgctca ggtctttctt ttatgtcacg gagaccacgt ttcaaaagaa caggctcttt ttctaccgga agagtgtctg gagcaagttg caaagcattg gaatcagaca gcacttgaag agggtgcagc tgcgggagct gtcggaagca gaggtcaggc agcatcggga agccaggccc gccctgctga cgtccagact ccgcttcatc cccaagcctg acgggctgcg gccgattgtg aacatggact acgtcgtggg agccagaacg ttccgcagag aaaagagggc cgagcgtctc acctcgaggg tgaaggcact gttcagcgtg ctcaactacg agcgggcgcg gcgccccggc ctcctgggcg cctctgtgct gggcctggac gatatccaca gggcctggcg caccttcgtg ctgcgtgtgc gggcccagga cccgccgcct gagctgtact ttgtcaaggt ggatgtgacg ggcgcgtacg acaccatccc ccaggacagg ctcacggagg tcatcgccag catcatcaaa ccccagaaca cgtactgcgt gcgtcggtat gccgtggtcc agaaggccgc ccatgggcac gtccgcaagg ccttcaagag ccacgtctct accttgacag acctccagcc gtacatgcga cagttcgtgg ctcacctgca ggagaccagc ccgctgaggg atgccgtcgt catcgagcag agctcctccc tgaatgaggc cagcagtggc ctcttcgacg tcttcctacg cttcatgtgc caccacgccg tgcgcatcag gggcaagtcc tacgtccagt gccaggggat cccgcagggc tccatcctct ccacgctgct ctgcagcctg tgctacggcg acatggagaa caagctgttt gcggggattc ggcgggacgg gctgctcctg cgtttggtgg atgatttctt gttggtgaca cctcacctca cccacgcgaa aaccttcctc aggaccctgg tccgaggtgt ccctgagtat ggctgcgtgg tgaacttgcg gaagacagtg gtgaacttcc ctgtagaaga cgaggccctg ggtggcacgg cttttgttca gatgccggcc cacggcctat tcccctggtg cggcctgctg ctggataccc ggaccctgga ggtgcagagc gactactcca gctatgcccg gacctccatc agagccagtc tcaccttcaa ccgcggcttc aaggctggga ggaacatgcg tcgcaaactc tttggggtct tgcggctgaa gtgtcacagc ctgtttctgg atttgcaggt gaacagcctc cagacggtgt gcaccaacat ctacaagatc ctcctgctgc aggcgtacag gtttcacgca tgtgtgctgc agctcccatt tcatcagcaa gtttggaaga accccacatt tttcctgcgc gtcatctctg acacggcctc cctctgctac tccatcctga aagccaagaa cgcagggatg tcgctggggg ccaagggcgc cgccggccct ctgccctccg aggccgtgca gtggctgtgc caccaagcat tcctgctcaa gctgactcga caccgtgtca cctacgtgcc actcctgggg tcactcagga cagcccagac gcagctgagt cggaagctcc cggggacgac gctgactgcc ctggaggccg cagccaaccc ggcactgccc tcagacttca agaccatcct ggactgatgg ccacccgccc acagccaggc cgagagcaga caccagcagc cctgtcacgc cgggctctac gtcccaggga gggaggggcg gcccacaccc aggcccgcac cgctgggagt ctgaggcctg agtgagtgtt tggccgaggc ctgcatgtcc ggctgaaggc tgagtgtccg gctgaggcct gagcgagtgt ccagccaagg gctgagtgtc cagcacacct gccgtcttca cttccccaca ggctggcgct cggctccacc ccagggccag cttttcctca ccaggagccc ggcttccact ccccacatag gaatagtcca tccccagatt cgccattgtt cacccctcgc cctgccctcc tttgccttcc acccccacca tccaggtgga gaccctgaga aggaccctgg gagctctggg aatttggagt gaccaaaggt gtgccctgta cacaggcgag gaccctgcac ctggatgggg gtccctgtgg gtcaaattgg ggggaggtgc tgtgggagta aaatactgaa tatatgagtt tttcagtttt gaaaaaaa கார்க்கை அக்க்கியுக்க்கு3 caggcagcgc tgcgtcctgc tgcgcacgtg ggaagccctg gccccggcca cccccgcgat gccgcgcgct ccccgctgcc gagccgtgcg ctccctgctg cgcagccact accgcgaggt gctgccgctg gccacgttcg tgcggcgcct ggggccccag ggctggcggc tggtgcagcg cggggacccg gcggctttcc gcgcgctggt ggcccagtgc ctggtgtgcg tgccctggga cgcacggccg ccccccgccg ccccctcctt ccgccaggtg tcctgcctga aggagctggt ggcccgagtg ctgcagaggc tgtgcgagcg cggcgcgaag aacgtgctgg ccttcggctt cgcgctgctg gacggggccc gcggggcccc ccccgaggcc ttcaccacca gcgtgcgcag ctacctgccc aacacggtga ccgacgcact gcgggggagc ggggcgtggg ggctgctgct gcgccgcgtg ggcgacgacg tgctggttca cctgctggca cgctgcgcgc tctttgtgct ggtggctccc agctgcgcct accaggtgtg cgggccgccg ctgtaccagc tcggcgctgc cactcaggcc cggcccccg cacacgctag tggaccccga aggcgtctgg gatgcgaacg ggcctggaac catagcgtca gggaggccgg ggtccccctg ggcctgccag ccccgggtgc gaggaggcgc gggggcagtg ccagccgaag tctgccgttg cccaagaggc ccaggcgtgg cgctgcccct gagccggagc ggacgcccgt tgggcagggg tcctgggccc acccgggcag gacgcgtgga ccgagtgacc gtggtttctg tgtggtgtca cctgccagac ccgccgaaga agccacctct ttggagggtg cgctctctgg cacgcgccac tcccacccat ccgtgggccg ccagcaccac gcgggccccc catccacatc gcggccacca cgtccctggg acacgccttg tcccccggtg tacgccgaga ccaagcactt cctctactcc tcaggcgaca aggagcagct gcggccctcc ttcctactca gctctctgag gcccagcctg actggcgctc ggaggctcgt ggagaccatc tttctgggtt ccaggccctg gatgccaggg actccccgca ggttgccccg cctgccccag cgctactggc aaatgcggcc cctgtttctg gagctgcttg ggaaccacgc gcagtgcccc tacggggtgc tcctcaagac gcactgcccg ctgcgagctg cggtcacccc agcagccggt gtctgtgccc gggagaagcc ccagggctct gtggcggccc ccgaggagga ggacacagac ccccgtcgcc tggtgcagct gctccgccag cacagcagcc cctggcaggt gtacggcttc gtgcgggcct gcctgcgccg gctggtgccc ccaggcctct ggggctccag gcacaacgaa cgccgcttcc tcaggaacac caagaagttc atctccctgg ggaagcatgc caagctctcg ctgcaggagc tgacgtggaa gatgagcgtg cgggactgcg cttggctgcg caggagccca ggggttggct gtgttccggc cgcagagcac cgtctgcgtg aggagatcct ggccaagttc ctgcactggc tgatgagtgt gtacgtcgtc gagctgctca ggtctttctt ttatgtcacg gagaccacgt ttcaaaagaa caggctcttt ttctaccgga agagtgtctg gagcaagttg caaagcattg gaatcagaca gcacttgaag agggtgcagc tgcgggagct gtcggaagca gaggtcaggc agcatcggga agccaggccc gccctgctga cgtccagact ccgcttcatc cccaagcctg acgggctgcg gccgattgtg aacatggact acgtcgtggg agccagaacg ttccgcagag aaaagagggc cgagcgtctc acctcgaggg tgaaggcact gttcagcgtg ctcaactacg agcgggcgcg gcgccccggc ctcctgggcg cctctgtgct gggcctggac gatatccaca gggcctggcg caccttcgtg ctgcgtgtgc gggcccagga cccgccgcct gagctgtact ttgtcaagga caggctcacg gaggtcatcg ccagcatcat caaaccccag aacacgtact gcgtgcgtcg gtatgccgtg gtccagaagg ccgcccatgg gcacgtccgc aaggccttca agagccacgt ctctaccttg acagacctcc agccgtacat gcgacagttc gtggctcacc tgcaggagac cagcccgctg agggatgccg tcgtcatcga gcagagctcc tccctgaatg aggccagcag tggcctcttc gacgtcttcc tacgcttcat gtgccaccac gccgtgcgca tcaggggcaa gtcctacgtc cagtgccagg ggatcccgca gggctccatc ctctccacgc tgctctgcag cctgtgctac ggcgacatgg agaacaagct gtttgcgggg attcggcggg acgggctgct cctgcgtttg gtggatgatt tcttgttggt gacacctcac ctcacccacg cgaaaacctt cctcaggacc ctggtccgag gtgtccctga gtatggctgc gtggtgaact tgcggaagac agtggtgaac ttccctgtag aagacgaggc cctgggtggc acggcttttg ttcagatgcc ggcccacggc ctattcccct ggtgcggcct gctgctggat acccggaccc tggaggtgca gagcgactac tccagctatg cccggacctc catcagagcc agtctcacct tcaaccgcgg cttcaaggct gggaggaaca tgcgtcgcaa actctttggg gtcttgcggc tgaagtgtca cagcctgttt ctggatttgc aggtgaacag cctccagacg gtgtgcacca acatctacaa gatcctcctg ctgcaggcgt acaggtttca cgcatgtgtg ctgcagctcc catttcatca gcaagtttgg aagaacccca catttttcct gcgcgtcatc tctgacacgg cctccctctg ctactccatc ctgaaagcca agaacgcagg gatgtcgctg ggggccaagg gcgccgccgg ccctctgccc tccgaggccg tgcagtggct gtgccaccaa gcattcctgc tcaagctgac tcgacaccgt gtcacctacg tgccactcct ggggtcactc aggacagccc agacgcagct gagtcggaag ctcccgggga cgacgctgac tgccctggag gccgcagcca acccggcact gccctcagac ttcaagacca tcctggactg atggccaccc gcccacagcc aggccgagag cagacaccag cagccctgtc acgccgggct ctacgtccca gggagggagg ggcggcccac acccaggccc gcaccgctgg gagtctgagg cctgagtgag tgtttggccg aggcctgcat gtccggctga aggctgagtg tccggctgag gcctgagcga gtgtccagcc aagggctgag tgtccagcac acctgccgtc ttcacttccc cacaggctgg cgctcggctc caccccaggg ccagcttttc ctcaccagga gcccggcttc cactccccac ataggaatag tccatcccca gattcgccat tgttcacccc tcgccctgcc ctcctttgcc ttccaccccc accatccagg tggagaccct gagaaggacc ctgggagctc tgggaatttg gagtgaccaa aggtgtgccc tgtacacagg cgaggaccct gcacctggat gggggtccct gtgggtcaaa ttggggggag gtgctgtggg agtaaaatac tgaatatatg agtttttcag ttttgaaaaa aa Sequence No. 4 sequence number 5 gtgggaggcc catcccggcc ttgagcacaa tgacccgcgc tcctcgttgc cccgcggtgc gctctctgct gcgcagccga taccgggagg tgtggccgct ggcaaccttt gtgcggcgcc tggggcccga gggcaggcgg cttgtgcaac ccggggaccc gaagatctac cgcactttgg ttgcccaatg cctagtgtgc atgcactggg gctcacagcc tccacctgcc gacctttcct tccaccaggt gtcatccctg aaagagctgg tggccagggt tgtgcagaga ctctgcgagc gcaacgagag aaacgtgctg gcttttggct ttgagctgct taacgaggcc agaggcgggc ctcccatggc cttcactagt agcgtgcgta gctacttgcc caacactgtt attgagaccc tgcgtgtcag tggtgcatgg atgctactgt tgagccgagt gggcgacgac ctgctggtct acctgctggc acactgtgct ctttatcttc tggtgccccc cagctgtgcc taccaggtgt gtgggtctcc cctgtaccaa atttgtgcca ccacggatat ctggccctct gtgtccgcta gttacaggcc cacccgaccc gtgggcagga atttcactaa ccttaggttc ttacaacaga tcaagagcag tagtcgccag gaagcaccga aacccctggc cttgccatct cgaggtacaa agaggcatct gagtctcacc agtacaagtg tgccttcagc taagaaggcc agatgctatc ctgtccccgag agtggaggag ggaccccaca ggcaggtgct accaacccca tcaggcaaat catgggtgcc aagtcctgct cggtcccccg aggtgcctac tgcagaagaa gatttgtctt ctaaaaa ggtgtctgac ctgagtctct ctgggtcggt gtgcttaaa caaagccca gctccacatc tctgctgtca ccaccccgcc aaaatgcctt tcagctcagg ccatttattg agaccagaca tttcctttac tccaggggag atggccaaga gcgtctaaac ccctcattcc tactcagcaa cctccagcct aacttgactg gggccaggag actggtggag atcatctttc tgggctcaag gcctaggaca tcaggaccac tctgcaggac acaccgtcta tcgcgtcgat actggcagat gcggcccctg ttccaacagc tgctggtgaa ccatgcagag tgccaatatg tcagactcct caggtcacat tgcaggtttc gaacagcaaa ccaacaggtg acagatgcct tgaacaccag cccaccgcac ctcatggatt tgctccgcct gcacagcagt ccctggcagg tatatggttt tcttcgggcc tgtctctgca aggtggtgtc tgctagtctc tggggtacca ggcacaatga gcgccgcttc tttaagaact taagaagtt catctcgttg gggaatacg gcaagctatc actgcaggaa ctgatgtgga agatgaaagt agaggattgc cactggctcc gcagcagccc ggggaaggac cgtgtccccg ctgcagagca ccgtctgagg gagaggatcc tggctacgtt cctgttctgg ctgatggaca catacgtggt acagctgctt aggtcattct tttacatcac agagagcaca ttccagaaga acaggctctt cttctaccgt aagagtgtgt ggagcaagct gcagagcatt ggagtcaggc aacaccttga gagagtgcgg ctacgggagc tgtcacaaga ggaggtcagg catcaccagg acacctggct agccatgccc atctgcagac tgcgcttcat ccccaagccc aacggcctgc ggcccattgt gaacatgagt tatagcatgg gtaccagagc tttgggcaga aggaagcagg cccagcattt cacccagcgt ctcaagactc tcttcagcat gctcaactat gagcggacaa aacatcctca ccttatgggg tcttctgtac tgggtatgaa tgacatctac aggacctggc gggcctttgt gctgcgtgtg cgtgctctgg accagacacc caggatgtac tttgttaagg cagatgtgac cggggcctat gatgccatcc cccagggtaa gctggtggag gttgttgcca atatgatcag gcactcggag agcacgtact gtatccgcca gtatgcagtg gtccggagag atagccaagg ccaagtccac aagtccttta ggagacaggt caccaccctc tctgacctcc agccatacat gggccagttc cttaagcatc tgcaggattc agatgccagt gcactgagga actccgttgt catcgagcag agcatctcta tgaatgagag cagcagcagc ctgtttgact tcttcctgca cttcctgcgt cacagtgtcg taaagattgg tgacaggtgc tatacgcagt gccagggcat cccccagggc tccagcctat ccaccctgct ctgcagtctg tgtttcggag acatggagaa caagctgttt gctgaggtgc agcgggatgg gttgctttta cgttttgttg atgactttct gttggtgacg cctcacttgg accaagcaaa aaccttcctc agcaccctgg tccatggcgt tcctgagtat gggtgcatga taaacttgca gaagacagtg gtgaacttcc ctgtggagcc tggtaccctg ggtggtgcag ctccatacca gctgcctgct cactgcctgt ttccctggtg tggcttgctg ctggacactc agactttgga ggtgttctgt gactactcag gttatgccca gacctcaatt aagacgagcc tcaccttcca gagtgtcttc aaagctggga agaccatgcg gaacaagctc ctgtcggtct tgcggttgaa gtgtcacggt ctatttctag acttgcaggt gaacagcctc cagacagtct gcatcaatat atacaagatc ttcctgcttc aggcctacag gttccatgca tgtgtgattc agcttccctt tgaccagcgt gttaggaaga acctcacatt ctttctgggc atcatctcca gccaagcatc ctgctgctat gctatcctga aggtcaagaa tccaggaatg acactaaagg cctctggctc ctttcctcct gaagccgcac attggctctg ctaccaggcc ttcctgctca agctggctgc tcattctgtc atctacaaat gtctcctggg acctctgagg acagcccaaa aactgctgtg cgggaagctc ccagaggcga caatgaccat ccttaaagct gcagctgacc cagccctaag cacagacttt cagaccattt tggactaacc ctgtctcctt ccgctagatg aacatg TIFF0007874179000001.tif247170TIFF0007874179000002.tif38170 【0104】 Although the invention described herein is explained in some detail as an example and illustration to clarify its meaning, the explanation and illustration should not be construed as limiting the scope of the invention. All patent and scientific literature disclosures cited herein are expressly incorporated in their entirety by reference. [Examples] 【0105】 Example 1: Method Handling of Mice and Animals As previously described, 63 Tert heterozygous mice were backcrossed to a C57BL / 6 background with >98% Tert + / -Mice are crossbred to produce first-generation (G1) homozygous Tert - / - Knockout mice were created. G3 Tert - / - The mouse, G2 Tert - / - Prepared by continuous rearing of mice. Tert in a pure C57BL / 6 background. + / + and G3 Tert - / - Male and female mice were treated with FA (F7876; Sigma-Aldrich). Male and female G3 Tert - / - Mice (7, 27, and 47 weeks old) were euthanized, and their renal phenotype was analyzed for signs of fibrosis and RNA-seq analysis. 【0106】 Trf1 lox / lox Mouse 64 was crossed with a mouse expressing CreERT2 recombinase driven by the tamoxifen-inducible ubiquitin C (UBC) promoter 65, and Trf1 lox / lox ;hUBC-CreERT2 or TRF1 + / + hUBC-CreERT2 mice were created. These mice were given a long-term tamoxifen-containing diet as an ad libitum, starting at 10 weeks of age. 【0107】 All mice were generated and maintained in a 12-hour light / dark cycle under specific pathogen-free conditions at the Spanish National Cancer Research Centre (CNIO) animal facility. The mice were housed in plastic cages with bedding made of wood shavings or chipped wood, with free access to food and water, as recommended by the Federation of European Laboratory Animal Science Associations. All animal procedures were approved by the CNIO-ISCIII Ethics Committee for Research and Animal Welfare (CBA 03_2019 v2). 【0108】 Folic acid dose titration and inoculation Vehicle (0.2 ml of 0.3 mmol l -1NaHCO3) or different doses of the vehicle alone (50, 100, 125, and 250 mg / kg) -1 A single intraperitoneal injection of FA (body weight) is administered to 6-8 week old Tert + / + Mouse and G3 Tert - / - Mice were administered the drug. Blood was collected on days 7 and 14 and analyzed using the VetScan Comprehensive Diagnostic Profile kit. Mice were euthanized 14 days after FA injection, and kidneys were collected from FA-treated or vehicle-treated animals and analyzed for renal fibrosis by immunohistochemistry. 【0109】 125 mg kg -1 A single intraperitoneal injection of FA at body weight or a low dose of 0.3M NaHCO3 (200ul) is administered to 6-8 week old Tert + / + Mouse and G3 Tert - / - Mice were inoculated. Blood samples were collected on days 2, 7, and 14 and analyzed using the VetScan Comprehensive Diagnostic Profile kit. Mice were euthanized on day 14, and their kidneys were perfused with cold PBS and recovered. 【0110】 Morphological analysis Kidneys were fixed with 4% formaldehyde and embedded in paraffin. Paraffin sections (5 μm thick) were stained with Masson's trichrome, Sirius Red, and PAS+D using standard procedures. The percentage of fibrotic areas was quantified using the National Institutes of Health (NIH) ImageJ program. 【0111】 primary cell culture The first generation of PTC mice were raised to Tert's dens at 10-11 weeks of age, as previously described. + / + and G3 Tert - / -mTert-pBabe-puro was isolated from mice. mTert-pBabe-puro was donated by M. Alvarez and J. Bidwell (Addgene plasmid number 36413). The retroviral plasmid vector (mTert-pBabe-puro) and packaging plasmids (PLC Eco and pCMV-VSV-G) were co-transfected into packaging cell line 293T. After 48 hours, the viral supernatant was collected, centrifuged to remove cell debris, filtered through a 0.45 μm filter (Millipore), and used to extract the Tert + / + and G3 Tert - / - The mice were infected with isolated PTCs derived from mice. From day 10 to day 14, 2 μg ml was administered. -1 Stable cell lines were selected using puromycin. Cells were collected for RNA extraction and immunofluorescence. 【0112】 Immunohistochemical staining and immunofluorescence staining Kidney tissue was fixed with 4% formaldehyde and embedded in paraffin. Paraffin-embedded kidney tissue sections 5-7 μm thick were subjected to immunohistochemical staining. Primary antibodies (and their dilutions) were rat monoclonal for p21 (HUGO-291H / B5; CNIO pathology core; 1:400), rat monoclonal for p53 (POE316A / E9; CNIO pathology core; 1:400), rat monoclonal for p21 (HUGO-291H / B5; CNIO pathology core; 1:400), rat monoclonal for CD8a (AM-OTO94A; CNIO pathology core), mouse monoclonal for phosphohistone H2AX (Ser139; 05-636; Millipore; 1:400), and mouse monoclonal for E-cadherin (610182; BD These included rat monoclonal for F4 / 80 (MCA497; AbD serotype; 1:400), rabbit polyclonal activated caspase-3 (9661; Cell Signaling Technology; 1:400), rabbit monoclonal for CD3e (99940; Cell Signaling Technology; 1:400), rabbit monoclonal for CD4 (25229; Cell Signaling Technology; 1:400), rabbit polyclonal for type VI collagen (ab6588; Abcam; 1:400), rabbit polyclonal for fibronectin (ab2413; Abcam; 1:400), and rabbit polyclonal for Sox9 (AB5535; Millipore; 1:400). 【0113】 Immunofluorescence was performed using mouse monoclonal antibody against α-SMA-Cy3 (C6198; Sigma; 1:400), rabbit monoclonal antibody against vimentin (5741; Cell Signaling Technology; 1:200), rabbit monoclonal antibody against Ki67 (12202; Cell Signaling Technology; 1:400), rabbit polyclonal TGFβ (3711S; Cell Signaling Technology; 1:200), rabbit polyclonal SNAIL+SLUG (ab180714; Abcam; 1:200), and rat monoclonal E-cadherin (DECMA-1; ab11512, Abcam; 1:200). Images were obtained using a confocal superspectrometer (Leica TCS-SP5). The percentage of positively stained areas by immunohistochemistry and immunofluorescence was quantified using NIH ImageJ (v1.52n). 【0114】 Gene expression analysis and real-time PCR testing Total RNA was isolated from kidney tissue and PTCs using TRIzol reagent (Takara) according to the manufacturer's instructions. cDNA was synthesized using 1 μg of total RNA, cDNA synthesis mix (BioMake), and oligo-dT primers. Gene expression was measured by real-time PCR assay (BioMake) and 7900HT real-time PCR system (Applied Biosystems). The relative amount of mRNA to the internal control was 2 ΔCT Calculate as follows, where ΔCT = ΔCT 実験 -ΔCT 対照 The genes and primers are listed in Table 2 (F: forward primer; R: reverse primer). 【0115】 Table 2: qPCR primers used in this study (sequence (5'-3')) TIFF0007874179000003.tif248170TIFF0007874179000004.tif189170 【0116】 For the RNA-seq experiment, a total RNA sample (300 ng) was used. The RNA quality score was an average of 6.3 (range 4.7-8.0) when assayed on a PerkinElmer LabChip analyzer. The sequencing library was prepared using the QuantSeq 3'mRNA-Seq Library Prep Kit (FWD) (Lexogen; 015) for Illumina, according to the manufacturer's instructions. Library generation was initiated by reverse transcription with oligo-dT priming, followed by second-strand synthesis from random primers using DNA polymerase. Primers from both steps contained Illumina-compatible sequences. The cDNA library was purified, applied to an Illumina flow cell for cluster generation, and sequenced on an Illumina NextSeq 550 (using the v2.5 reagent kit) according to the manufacturer's protocol. Read adapters and poly(A) tails were removed using the command "bbduk.sh" as recommended by Lexogen. The processed reads were analyzed using Nextpresso pipeline 67 as follows: The sequencing quality was checked using FastQC v0.11.7 (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ). Reads were aligned to the mouse reference genome (GRCm38) using TopHat (v1.0.0) 68 with Bowtie (v 2.0.10) 69 and SAMtools (v0.1.19) 70 (--library type fr-secondstrand in TopHat), allowing for 3 mismatches and 20 multihits. Read counts were obtained using HTSeq-count (v 0.6.1) 71 with mouse gene annotation from GENCODE (GRCm38; vM20 Ensembl 95). GSEA was performed on several gene signatures on a pre-ranked gene list with 1,000 gene set permutations using GSEAPreranked 72. Only gene sets with significant enrichment levels (FDR q value < 0.25) were examined. 【0117】 Telomere analysis Q-FISH determination of paraffin-embedded tissue sections was performed as previously described41. After deparaffinization, the tissues were fixed in 4% formaldehyde for 5 minutes, washed three times in PBS for 5 minutes each, and incubated in pepsin solution (0.1% porcine pepsin, Sigma; 0.01M HCl, Merck) at 37°C for 15 minutes. After another wash and fixation as described above, the slides were dehydrated in a series of ethanol solutions (70%, 90%, and 100%; 5 minutes each). After air-drying for 10 minutes, 30 L of telomere probe mix (10 mM Tris-Cl (pH 7), 25 mM MgCl2, 9 mM citrate, 82 mM Na2HPO4, 70% deionized formamide (Sigma), 0.25% blocking reagent (Roche), and 0.5 μg ml) were added. -1 Telomere PNA probe (Panagene) was added to each slide. Coverslips were added, and the slides were heated to 85°C. The slides were incubated for 3 minutes, then incubated at room temperature in a humid chamber in the dark for a further 2 hours. The slides were washed twice for 15 minutes each with 10 mM Tris-Cl (pH 7) and 70% formamide containing 0.1% BSA, while shaking vigorously, then washed three times for 5 minutes each with 0.08% TBS containing Tween 20, and then bathed in a DAPI bath (4 μg ml in PBS). -1 After incubation in Sigma, the samples were mounted in Vectashield medium (Vector). Confocal images were acquired using a Leica SP5-MP confocal microscope as stacks totaling 5 μm at 1 μm intervals, and maximal projection was performed using LAS-AF software. Telomere signal intensity was quantified using Definiens software. 【0118】 Statistics and Reproducibility Although no statistical methods were used to predetermine the sample size, our sample size is similar to those previously reported.4,46 Mice were randomly assigned to groups, and researchers were blinded to group assignments. Our sample size corresponds to the number of mice used in each experiment, as shown. Quantitative analysis of immunohistochemical staining was performed on the entire scanned kidney section, including 10–15 regions. 10–20 images were collected from each individual for immunofluorescence analysis. A normal data distribution was assumed, but this was not formally tested. No data points or mice were excluded from the analysis. Results of statistical analysis are expressed as mean ± sem. In Prism (GraphPad), statistical analysis of immunohistochemical / immunofluorescence quantification and qPCR and Q-FISH analysis was performed using one-way ANOVA with the post-hoc Tukey test. Blood and urine parameters were analyzed using two-way ANOVA with the post-hoc Bonferroni test. Statistical significance was defined as P<0.05. 【0119】 Example 2: Results The mouse model of renal fibrosis is associated with short telomeres. To investigate the involvement of short telomeres in renal fibrosis, the inventors first analyzed the kidneys of wild-type mice and mice lacking the telomerase catalytic subunit Tert, and bred these for three generations (G3), identifying very short telomeres, i.e., G3 Tert. - / - The presence of mouse 24 was induced (Figure 9a). Wild-type mice (Tert) aged 8-9 weeks were used. + / + ) Mouse and G3 Tert - / - Both mice exhibited remarkably normal renal histology, and as assessed by Masson's trichrome staining and Sirius red staining, respectively, there was no evidence of glomerular or tubular defects, nor fibrosis or collagen fiber accumulation (Figure 9b, c). Similarly, the inventors did not detect increased renal tubular damage or brush border loss when measured by periodate Schiff with diastase staining (PAS+D; Figure 9d). Also, G3 Tert - / -Consistent with a remarkably normal mouse kidney, the inventors have identified the presence of activated fibroblasts determined by the expression of smooth muscle actin (α-SMA); apoptosis determined by the expression of cleavage caspase-3 (CC3); aging as determined by the expression of p21 cell cycle inhibitors; or Tert of the same age. + / + No differences were observed in the expression of E-cadherin (a marker of EMT associated with histiofibrosis) compared to mice (Figures 9e-h). 【0120】 The incidence of renal fibrosis increases with age, and therefore, it is likely caused by a combination of molecular and cellular aging events, such as the presence of short telomeres 14, along with exogenous damage to the kidney. Therefore, the inventors then proposed 250 mg kg -1 It has been previously reported that high doses of FA can induce renal fibrosis. + / + and G3 Tert - / - The kidneys of mice were challenged.27 Fat-induced nephropathy is widely used to study interstitial renal fibrosis.4,27,28 In particular, intraperitoneal administration of FA to mice leads to the rapid appearance of FA crystals in the renal tubules, followed by severe nephrotoxicity. 【0121】 For this purpose, the inventors first used 8-9 week old Tert + / + and G3 Tert - / - Mice were given escalating doses of FA (50, 100, 125, and 250 mg / kg) -1 The highest dose of FA that did not induce renal fibrosis in wild-type mice was selected based on body weight (Figure 1a): 50, 100, and 125 mg / kg. -1 Wild-type mice treated with body weight appeared normal and showed no signs of renal pathology, as determined by normal kidney appearance (Figure 1b), normal creatinine and blood urea nitrogen (BUN) levels in the blood (Figures 1c, d), and the absence of fibrosis as indicated by Masson's trichrome staining (Figure 1e) and the absence of tubular injury as indicated by PAS+D staining (Figure 1e). In contrast, 250 mg kg -1The majority of wild-type mice (75%) treated with a body-weight FA dose died on day 2 post-treatment due to FA-induced acute renal failure (data not shown). Therefore, 125 mg / kg -1 However, this was the maximum tolerable dose of FA that did not cause death in wild-type mice with normal telomere length. The inventors believe that this dose of FA is effective against Tert + / + Although not sufficient to induce renal fibrosis in mice, we hypothesized that it could induce renal fibrosis in telomerase-deficient mice by synergistically acting with short telomeres. 【0122】 In fact, the inventors of the G3 Tert - / - Mice were treated with the same dose of FA, and 125 mg kg -1 Even after doses, we observed that the kidneys exhibited a pale color suggestive of renal fibrosis (Figure 1b). In fact, G3 Tert treated with escalating doses of FA... - / - Mice: 125 mg kg -1 The FA dose showed elevated serum creatinine (Figure 1c) and BUN (Figure 1d) levels, renal failure, and increased fibrosis detected by Masson's trichrome staining (Figure 1e), as well as increased renal tubular damage determined by increased PAS+D staining (Figure 1e). 125 mg kg -1 FA doses less than G3 Tert - / - The fibrous phenotype was not induced in mice (Figure 1e). Therefore, for further experiments, we administered 125 mg kg -1 I selected the FA dosage based on body weight. 【0123】 Severe renal impairment in telomerase-deficient mice treated with a sublethal dose of folic acid. To address the role of short telomeres in renal fibrosis, the inventors of the present invention have developed a method for 8-9 week old telomeres. + / + and G3 Tert - / - In mice, 125 mg kg does not induce fibrosis in wild-type mice. -1The mice were administered FA by body weight (Figure 2a). Blood was collected from the submandibular vein on days 2, 7, and 14. On day 14, the mice were euthanized and their kidneys were collected for further analysis. As expected, FA-treated G3 Tert - / - Kidneys derived from mice appeared paler in color and more damaged compared to wild-type kidneys treated similarly (Figure 2b, c). To confirm kidney damage, we analyzed urinary parameters and FA-treated Tert + / + Mouse and untreated Tert + / + Mouse and G3 Tert - / - Compared to mice, FA treatment G3 Tert - / - In mice, a significant increase in 24-hour urinary albumin excretion and urinary albumin-to-creatinine ratio was observed (Figure 2b). 【0124】 The inventors also examined BUN and creatinine in all mouse cohorts. High doses of FA are known to induce transient increases in BUN and creatinine levels 48 hours after injection, followed by subsequent renal dysfunction with interstitial fibrosis.29 However, low doses of FA (125 mg kg) -1 Two days after the injection of body weight, untreated and FA-treated wild-type mice (Tert + / + Both ) showed normal BUN and creatinine levels, which indicated normal renal function (Figure 10a, b). Consistently, the inventors found that low doses of FA (125 mg kg) were effective. -1 Wild-type mice treated with body weight (Tert + / + No decrease in mobility or increase in mortality was observed in untreated G3 Tert. - / - The mice showed normal creatinine and BUN levels, as well as normal survival rates, on day 2. As expected, FA treatment G3 Tert - / - Mice showed significant increases in both BUN and creatinine levels on days 2, 7, and 14, with creatinine levels gradually increasing from day 2 onward, and renal dysfunction occurring as early as two days after FA administration (Figure 10a, b). + / + and G3 Tert - / -Additional biochemical parameters in the blood of the mice indicated kidney injury (Table 1). Alkaline phosphatase and amylase levels are generally elevated in patients with CKD and those requiring dialysis30, and are indicators of kidney damage31. Kidney dysfunction can lead to hyperamylasemia32. On days 7 and 14, FA-treated G3 Tert - / - mice were FA-treated Tert + / + mice and untreated Tert + / + mice as well as G3 Tert - / - showed significantly higher levels of alkaline phosphatase and amylase compared to the levels in G3 Tert mice (Table 1). Hypercalcemia33, hyperphosphatemia34, hypernatremia35 and hyperkalemia36 are common complications in patients with CKD, especially those with end-stage renal disease. We observed increases in calcium, phosphorus and sodium levels in FA-treated G3 Tert - / - mice on days 2, 7 and 14, but the potassium level increased only on day 14 compared to FA-treated Tert + / + mice and untreated Tert + / + mice as well as G3 Tert - / - mice (Table 1). As indicators of tubular function, we measured blood glucose, globulin, total protein and albumin levels. On days 2, 7 and 14, blood glucose, globulin and total protein levels were significantly elevated in FA-treated G3 Tert - / - mice compared to FA-treated Tert + / + mice and untreated Tert + / + mice as well as G3 Tert - / - mice, but albumin did not change (Table 1). 【0125】 Table 1: Blood parameters of mice on days 2, 7 and 14 after FA. Data are presented as mean ± standard deviation of the mean. TIFF0007874179000005.tif150170 【0126】 Telomerase-deficient mice show collagen deposition and activated myofibroblasts in the kidney after sub-lethal doses of folic acid It has previously been shown that FA-induced kidney injury coincides with the development of segmental interstitial fibrotic lesions approximately 2 weeks after treatment37. Consistent with this, on day 14 after treatment, the inventors observed areas of interstitial fibrosis in the kidneys of FA-treated G3 Tert - / - mice, but not in G3 Tert - / - , Tert + / + nor FA-treated Tert + / + mice (Figure 2c), which also indicated that short telomeres become sensitive to renal fibrosis after sub-pathological doses of FA that do not induce fibrosis in wild-type mice. FA-treated G3 Tert - / - mice also developed significant tubulointerstitial injury, as shown by increased PAS+D staining, including tubular dilation, atrophy, and loss of epithelial differentiation, compared to normal kidney histology in FA-treated Tert + / + mice, untreated Tert + / + mice, and G3 Tert - / - mice (Figure 2c). Next, the inventors performed immunohistochemistry for α-SMA, fibronectin, and type VI collagen, as well as double immunofluorescence using α-SMA and vimentin, to detect interstitial myofibroblasts (Figure 3a). Again, the inventors detected only increased staining for α-SMA, fibronectin, type VI collagen, and vimentin in FA-treated G3 Tert + / + mice compared to undetectable staining in FA-treated Tert + / + mice, untreated Tert - / - mice, and G3 Tert - / - mice (Figure 3a). 【0127】 EMT involves a shift from apical-basal polarity in epithelial cells to anterior-posterior polarity in mesenchymal cells, as well as the expression of mesenchymal markers such as fibroblast-specific protein-1, vimentin, N-cadherin, and α-SMA. These changes induce enhanced migratory ability, increased invasiveness, increased resistance to apoptosis, and increased production of ECM components.38 FA Therapeutic G3 Tert - / - To demonstrate that the proliferating cells observed in mice were actually EMT cells and not tubular cells, the inventors performed double immunofluorescence staining for Ki67 and α-SMA. The inventors also performed untreated G3 Tert - / - Compared to mice, FA treatment G3 Tert - / - In mice, 36% of proliferating cells are myofibroblasts (α-SMA). + Ki67 + ) was found to be (Figure 3b). Quantitative PCR (qPCR) was used to determine the mRNA levels of key fibrous genes, including Acta2 (encoding α-SMA), Vim (encoding vimentin), Col1a1, Col3a1, and Col4a1 (encoding type I collagen alpha-1, -3, and -4 strands, respectively), and Fn1 (encoding fibronectin 1), in FA treatment G3 Tert. - / - An increase in fibrosis was observed in mice, but not in similarly treated wild-type mice or untreated mice. These genes are related to FA treatment G3 Tert - / - In the kidneys of mice, FA treatment Tert + / + Mouse and untreated Tert + / + Mouse and G3 Tert - / - Compared to mice, the control was significantly improved (Figure 3c). 【0128】 Increased renal apoptosis and aging in telomerase-deficient mice after sublethal doses of folate. We have shown that short telomeres induce a persistent DNA damage response at the chromosome ends, leading to cell cycle arrest or apoptosis.39,40 In particular, short telomeres induce p53 and p21 cell cycle inhibitors.22,41 Interestingly, p21 is known to be an activator of TGFβ37, thus providing a potential mechanism by which short telomeres may contribute to the activation of the EMT program even in the absence of fibrosis. 【0129】 In this regard, the inventors have found that G3 Tert treated with FA - / - We investigated whether increased renal dysfunction and fibrosis in mice are accompanied by increased cellular senescence or apoptosis, two well-known cellular responses to telomere dysfunction associated with aging.14 We investigated whether FA therapy is associated with increased FA therapy Tert + / + Mouse and untreated Tert + / + Mouse and G3 Tert - / - Compared to undetectable levels in mice, G3 Tert - / - In mice, we observed significant increases in the levels of CC3 (an apoptosis marker), p21 and p53 cell cycle inhibitors, and γ-H2AX (a DNA damage marker) (Figures 4a-d). Our model suggests that these events increase the turnover of renal cells and lead to telomere shortening. In support of this, we observed increased transcription levels of cell cycle regulators encoding the genes CCnd1, Ccnd2, Ccnb1, and Ccne1 (Figure 11a), suggesting that a marked pro-fibrotic phenotype can lead to G2 / M arrest. Finally, we found that G3 telomere shortening was induced in kidney sections by direct telomere quantitative fluorescence in situ hybridization (Q-FISH) compared to wild-type mice. - / - We confirmed the presence of shorter telomeres in the kidneys of mice (Figure 4e). Treatment with low doses of FA induced clear telomere shortening in both genotypes (Figure 4e). 【0130】 Short telomeres are susceptible to tubular damage and immune infiltration in the kidneys. Next, the inventors decided to study the pathway by which short telomeres induce kidney injury. Kidney injury molecule-1 (KIM-1, also known as hepatitis A virus cell receptor (HAVCR) 1; encoded by the gene Havcr1) is undetectable in healthy kidneys but is greatly induced after injury42 and is a type 1 transmembrane protein localized to the apical surface of viable proximal tubular cells (PTCs)43. Inflammation has been proposed as a significant promoting factor of renal fibrosis42, and the neutrophil gelatinase-associated lipocalin (NGAL) gene product (lipocalin-2 (Lcn2) or siderocalin)44 is induced during acute kidney injury (AKI)44. Therefore, the inventors used Havcr1 and Lcn2 as biomarkers for CKD resulting from tubulointerstitial injury. The inventors found that Havcr1 and Lcn2 are related to FA treatment Tert - / - Mouse and control, untreated Tert + / + Mouse and G3 Tert - / - Compared to baseline expression in mice, FA-treated G3 Tert + / + We found that these were upregulated by 134 and 206 times, respectively, in mice (Figure 5a). Next, we studied the expression of molecules involved in immune infiltration, which is also related to histofibrosis. We found that FA treatment G3 Tert - / - In mice (Figure 5a), a 16-fold upregulation of Emr1 mRNA expression (the mouse gene encoding the F4 / 80 antigen) was observed. The inventors also examined the expression of the pan-macrophage marker F4 / 80 and T-cell markers (CD3e, CD4, and CD8a) by immunohistochemistry. The inventors developed FA treatment for G3 Tert - / - Increased macrophage colony formation (F4 / 80 positive area) and increased T cells in mouse kidneys were observed with FA treatment. + / + Mouse or untreated Tert + / + and G3 Tert - / - We observed the results in comparison with the kidneys of mice (Figure 5b). 【0131】 Activation of epithelial-mesenchymal transition-related pathways in G3 Tert- / - mice Macrophage recruitment in histofibrosis is known to be accompanied by TGFβ and is also important in the EMT process associated with renal fibrosis4546. Therefore, we next sought to determine whether the presence of short telomeres is associated with changes in the expression of genes involved in EMT. For this purpose, we investigated untreated or low-dose FA (125 mg kg) -1 A 10-week-old Tert (body weight) was treated. + / + Mouse and G3 Tert - / - RNA sequencing (RNA-seq) was performed in mouse kidneys to study the expression of EMT and EMT-related pathways, including TGFβ signaling, which is a major environmental stimulus that induces EMT in adult epithelium. (Untreated G3 Tert) - / - Untreated Tert in mice + / + Gene set enrichment analysis (GSEA) of mice showed that the EMT (normalized enrichment score (NES) = 3.37) and TGFβ (NES = 1.9) pathways were upregulated in telomerase-deficient mice compared to wild-type mice (Figure 6a, b), suggesting that shorter telomeres may contribute to higher basal activation of several EMT genes. Upregulation of EMT gene transcription was observed in FA-treated animals compared to untreated animals. + / + Animal and FA treatment G3 Tert - / - This was more pronounced in animals (NES>6). These findings were observed in untreated young Tertian at 7 weeks of age. + / + Mouse vs. G3 Tert - / - This was confirmed in mice, as well as older mice (47 weeks old; Figure 12a, b). Therefore, short telomeres are associated with changes in the expression levels of some, but not all, of the genes involved in EMT in renal epithelial cells, and are therefore not sufficient to activate the classical EMT program or induce fibrosis on their own. Interestingly, treatment with FA was found to affect both genotypes, Tert + / + and G3 Tert - / - This induced enrichment of the EMT pathway and TGFβ pathway. This enrichment was observed in FA treatment G3 Tert. - / - In the kidneys, FA treatment Tert + / +The levels were higher compared to the kidney (NES = 2.42), thus supporting the idea of ​​higher EMT activation in response to FA in telomerase-deficient mice (Figure 6a, b). 【0132】 Upregulation of key EMT transcription factors in telomerase-deficient mice with short telomeres. TGFβ1-induced EMT is mediated by ZEB1 and SNAIL in a Smad-dependent manner.45 TWIST, SNAIL, and ZEB1 are transcription factors that regulate the EMT transcription program. Activation of Twist, Snail, or Zeb1 is sufficient to induce the mesenchymal phenotype4. Therefore, we decided to determine the mRNA expression levels of Tgfb1, Snail1, Snail2, Twist1, Zeb1, and Zeb2 by reverse transcription-PCR (RT-PCR). We compared FA-treated G3 Tert with other mouse cohorts. - / - A 32-fold increase in Tgfb1 expression was observed in mice (Figure 6c). Snail1 and Snail2 also showed increased expression of FA-treated G3 Tert - / - In mice, control was improved by a 6-fold increase (Figure 6c). Twist1 was improved by a 7-fold increase, while Zeb1 and Zeb2 were improved in FA treatment G3 Tert. - / - In mice, these were upregulated fourfold and fivefold, respectively (Figure 6c). Therefore, since all major EMT players were upregulated in the kidneys of telomerase-deficient mice with short telomeres administered a sublethal dose of FA compared to wild-type mice administered the same dose, it is suggested that short telomeres may contribute to EMT changes after FA treatment. Lysyl oxidase (LOXL2) is necessary and sufficient for suppressing hypoxia of E-cadherin, which mediates cell transformation and induces EMT47. Loxl2 expression is associated with FA-treated G3 Tert - / -In mice, it was upregulated 20-fold (Figure 6c). One of the characteristics of EMT is the downregulation of E-cadherin isoforms such as Cdh1 and the expression of mesenchymal markers45. Consistent with this, the expression of E-cadherin Cdh1 was determined by both mRNA levels and immunohistochemistry in FA-treated G3 Tert compared to other mouse cohorts. - / - In mice, it was downregulated by a factor of two (Figure 6c, d). SMAD3 is an important effector in TGFβ signaling. We have shown that Smad3 is involved in FA treatment G3 Tert - / - We found that it was controlled twice as much in mice (Figure 6c, d). 【0133】 To understand how progenitor cells and stem cells may be affected by short telomeres during FA-induced renal fibrosis, we tested the expression of sex-determining region Y-box 9 (Sox9), Wilms tumor (Wt1), pair box 2 (Pax2), Sport-like transcription factor 2 (Sall2), activin A receptor 2B (Acvr2b, a Tgfb superfamily member), and Klotho (Kl). SOX9 is known to be important in kidney development in mouse48 and human49 studies and is required for renal fibrosis50. It precedes the expression of Wt1, Pax2, and NOTCH signaling in the epithelium. Wt1 is required to maintain the transition between mesenchymal and epithelial cell states and to induce mesenchymal-to-epithelial transformation (MET), playing a crucial role in the progression of nephropogenesis51. The nuclear transcription factor PAX2 is associated with MET and is required for renal cell differentiation52. Reexpression of Wt1 and Pax2 in tubular epithelial cells plays a crucial role in promoting EMT, and silencing Pax2 and Wt1 may have therapeutic value in preventing or reversing renal fibrosis.53 In this regard, we have found that Tert exposed to FA compared with all other groups - / -Significant increases in transcriptional levels of Sox9, Pax2, Wt1, and Acvr2b expression were observed in mice, suggesting that under these conditions, short telomeres sensitized the kidney and underwent EMT (Figure 13a). Sox9 immunostaining was observed in untreated and treated telomeres. - / - Mouse and G3 Tert + / + Compared to mice, FA treatment G3 Tert - / - It was significantly increased in mice (Figure 13b). Sall2 is negatively regulated by Wt1 (see 54), and klotho (Kl) is an anti-aging protein mainly produced in the kidney 55 that regulates telomerase activity 56. Low Kl levels may be a pathological intermediate of worsening kidney injury. Interestingly, we found that Sall2 and Kl expression are associated with FA treatment G3 Tert - / - We found that it is downregulated in mice (Figure 13a). 【0134】 Sustained expression of the NOTCH signaling pathway in epithelium leads to interstitial fibrosis.57 NOTCH is a potent regulator of SNAIL1 and SNAIL2 (see 58). Therefore, the inventors analyzed the expression of Notch receptors (Notch1, Notch2, and Notch3), Notch ligand Jagged 1 (Jag1), and mitochondrial transcription factor A (Tfam) as direct Notch targets important for renal function.59 The inventors developed a FA treatment for Tert - / - Mouse and untreated Tert + / + Mouse and G3 Tert - / - Compared to mice, FA treatment G3 Tert + / + In mice, we found a threefold increase in Notch1 and Notch2, and a fourfold increase in Notch3 and Jag1 (Figure 14). The inventors also found that FA treatment Tert - / - Mouse and untreated Tert + / + Mouse and G3 Tert - / - Compared to mice, FA treatment G3 Tert + / + We found a five-fold upward control of Tfam in mice (Figure 14). 【0135】 Trf1 deletion-induced telomere dysfunction induces renal fibrosis associated with EMT activation. To evaluate the contribution of dysfunctional telomeres to the induction of renal fibrosis, we used a second model of telomere dysfunction by deleting TRF1, one of the components of the Shertelin telomere protective complex. In particular, we used the 60 mouse model previously described by us in which treatment with tamoxifen resulted in the deletion of TRF1 in all kidney cells (Figure 7). The tamoxifen diet was maintained until the humane endpoint, and TRF1 deletion was confirmed by RT-qPCR (Figure 7a). Mice deficient in TRF1 showed TRF1 + / + Compared to mice, the mice developed renal fibrosis, as indicated by increased collagen deposition (Mason's trichrome) and fibrous lesions (Sirius red staining) (Figure 7b), as well as activation of myofibroblasts (SMA staining; Figure 7b). The inventors also developed Trf1 + / + Compared to mice, TRF1 flox / flox In mice, we observed a 3- to 4-fold increase in mesenchymal markers (Acta2 and Fn1) and a 2- to 6-fold increase in EMT markers (Tgfb1, Snail1, Snail2, Twist1, Zeb1, Zeb2) (Figure 7c, d), suggesting that dysfunctional telomeres may also contribute to EMT-related transcriptional changes. 【0136】 Telomerase overexpression rescues EMT changes associated with short telomeres. To further investigate the contribution of short telomeres to the activation of the EMT program in the kidney, the inventors investigated whether short telomeres could be rescued by the expression of a catalytic subunit of telomerase or TERT61. For this purpose, the inventors first used 10-11 week old Tert + / + Mouse and G3 Tert - / - Kidney epithelial cells, particularly PTCs, were isolated from mice. PTCs were transduced with either an empty vector or a vector expressing Tert (Methods). Telomerase overexpression was confirmed by qPCR (Figure 8a). Consistent with telomerase overexpression were observed in wild-type cells and G3 Tert cells.- / - Both cells showed a significant increase in telomere length after 1 week of culture (Figure 8b). Before transduction, on day 8 of culture, G3 Tert - / - The cells exhibited a transparent mesenchymal morphology, indicated by a spindle shape not observed in wild-type cells, and this showed a cobblestone-like morphology (Figure 15a). G3 Tert transduced with an empty vector. - / - The cells exhibited a complete myofibroblast phenotype characterized by E-cadherin loss, enhanced SMA staining, and positivity for Snail1 and TGFβ markers (Figure 8c and Figure 15b, c), suggesting that 100% of the cells underwent EMT. In contrast, control Tert cells transduced with an empty vector... + / + The cells showed a mixture of epithelial cells (E-cadherin-positive, Snail1-negative, TGFβ-negative, and SMA-negative cells) and myofibroblasts (E-cadherin-negative, Snail1-positive, TGFβ-positive, and SMA-positive cells; Figure 8c and Figure 15b, c), showing some evidence of EMT. Interestingly, mTERT was transduced into Tert + / + The cells showed evidence of MET, recovery of the cobblestone phenotype in culture, expression of E-cadherin, and phenotypic reversal including loss of expression of SMA, TGFβ, and Snail1 (Figures 8c, d and 15b, c). Importantly, G3 Tert cells transduced to mTERT - / - The cells also showed a reversal of the mesenchymal phenotype, with loss of SMA, TGFβ, and Snail1 expression (Figure 8c and Figure 15b, c). In fact, G3 Tert cells transduced to mTERT - / - The cells were transduced with an empty vector (Figure 8d) into G3 Tert - / -Compared to cells, the cells showed decreased expression of many ECM (Acta2, Vim, Col3a1, and Col4a1) and EMT (Tgfb1, Snail1, Snail2, and Zeb1) genes, and the recovery of epithelial features during TERT expression and telomere elongation was confirmed. Therefore, our results indicate that mTERT overexpression and telomere elongation are sufficient to rescue the EMT phenotype and restore the MET phenotype in renal tubular cells. This culture system provides a powerful tool for investigating TERT-driven EMT. 【0137】 Example 3: Discussion EMT is a cell plasticity process in which the epithelial layer loses its integrity simultaneously with the loss of cell polarity and intercellular interactions mediated by the loss of E-cadherin 3. The resulting cells express mesenchymal characteristics, including the expression of vimentin and α-SMA. EMT is regulated by several transcription factors (EMT-TFs) and plays an important role both during normal development3 and in pathological conditions such as cancer and various histofibrotic diseases including renal fibrosis4,62. 【0138】 Since both cancer and histiofibrosis are associated with aging, understanding how known molecular and cellular mechanisms of aging14 may influence EMT-TF expression and the origin of EMT pathological processes is particularly relevant. 【0139】 The accumulation of short, dysfunctional telomeres associated with cell division during an organism's lifespan is considered one of the major features of aging, as it causes persistent DNA damage sufficient to impair the regenerative capacity of adult stem cell compartments.10,13 In fact, accelerated telomere shortening due to telomerase deficiency in both mice24,25 and humans8 results in premature loss of tissue regenerative capacity, including the development of histofibrosis, one of which is most commonly pulmonary fibrosis8,22. 【0140】 Here, the inventors created a mouse model of renal fibrosis associated with short telomeres by challenging telomerase-deficient mice with short telomeres to a low sublethal dose of FA, a kidney-damaging agent that does not induce fibrosis in similarly treated wild-type controls. Telomerase-deficient mice treated with a sublethal dose of FA exhibit all the prominent features of the human disease, including severe renal dysfunction, as indicated by elevated blood levels of creatinine and urea. Furthermore, mice with dysfunctional telomeres due to a deletion of the telotelin protein, Trf1, spontaneously develop renal fibrosis, highlighting the importance of proper telomere function in protecting fibrous lesions. Therefore, the novel mouse model created here is an excellent tool for understanding the role of short, dysfunctional telomeres and the resulting DNA damage in molecular events associated with fibrosis. 【0141】 Interestingly, the inventors found that while short telomeres lead to altered expression of genes involved in EMT, these alterations were not sufficient to induce renal fibrosis. These alterations were further exacerbated in telomerase-deficient mice treated with FA, which develops renal fibrosis. This is supported by increased expression of the Snail1, Snail2, Zeb1, Zeb2, and Twist1 transcription factors, which cause epithelial cells to transform into myofibroblasts. 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Claims

[Claim 1] A composition for use in treating renal fibrosis associated with the presence of short telomeres, comprising a nucleic acid vector containing the coding sequence of telomerase reverse transcriptase (TERT). [Claim 2] The composition according to claim 1, wherein TERT is encoded by a nucleic acid sequence containing the sequence of sequence number 1. [Claim 3] The composition according to claim 1 or 2, wherein TERT comprises the amino acid sequence of SEQ ID NO:

2. [Claim 4] The composition according to claim 1 or 2, wherein the nucleic acid sequence encoding TERT is operably linked to a regulatory sequence that drives the expression of the coding sequence. [Claim 5] The composition according to claim 1 or 2, wherein the vector is a non-embedded vector. [Claim 6] The composition according to claim 1 or 2, wherein the nucleic acid vector is ribonucleic acid (RNA). [Claim 7] The composition according to claim 1 or 2, wherein the vector is an adeno-associated virus-based non-embedded vector. [Claim 8] The composition according to claim 7, wherein the vector is an adeno-associated virus-based vector derived from serotype 9 adeno-associated virus (AAV9). [Claim 9] The composition according to claim 8, wherein the capsid of the adeno-associated virus-based vector is made of the capsid protein of serotype 9 adeno-associated virus (AAV9), and internal terminal repeats corresponding to serotype 2 adeno-associated virus are adjacent to both ends of the nucleic acid sequence contained in the capsid. [Claim 10] The composition according to claim 9, wherein the nucleic acid contained in the capsid comprises a fragment encoding an amino acid sequence encoding TERT. [Claim 11] The composition according to claim 1 or 2, wherein the vector comprises a regulatory sequence that is a constitutive promoter.

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