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

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 is in the field of molecular biology, biotechnology and medicine. More particularly, the present invention relates to compositions and methods useful for treating non-human animals and conditions associated with short telomere length. More particularly, the present invention relates 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 serve to protect the chromosome ends from DNA repair and degradation activities. Mammalian telomeres consist of TTAGGG repeats bound by a multiprotein complex known as shelterin. Since shortened telomeres are associated with many diseases, a minimum length of TTAGGG repeats and the integrity of the shelterin complex are required for telomere protection. These structures are essential for chromosome integrity by preventing telomere fusion and telomere fragility. Telomere length is controlled by the ribonucleoprotein enzyme telomerase, which can de novo add telomeric sequences onto 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 addition of TTAGGG repeats to chromosome ends by using an associated RA component as a template (Terc, telomerase RNA component). Although telomerase is expressed in most adult stem cell compartments, this is not sufficient 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 naturally lost with every cell division (known as the end-replication problem), and somatic cells express telomerase at very low levels or not at all and shorten telomeres throughout life. When telomeres become significantly shortened, they lose their protective function and a persistent DNA damage response is triggered at the telomeres, which subsequently results in a cellular senescence response (Harley et al., 1990, Flores et al., 2008).

[0004] Accumulation of short telomeres is a hallmark of aging. Mutations in telomerase or telomere-binding proteins result in shortening or dysfunction of telomeres, which are characterized by loss of tissue regenerative capacity and fibrotic lesions and are the origin of human pathologies known as "telomere syndromes", including aplastic anemia, dyskeratosis congenita and some cases of pulmonary fibrosis. Adeno-associated virus (AAV)-based telomerase gene therapy was found to be beneficial in extending health span in the context of normal physiological aging in wild-type mice. Adult and aged mice were subjected to AAV9-mTERT gene therapy to broadly express the catalytic subunit of mouse telomerase (mTERT). Health span of TERT-treated mice was significantly increased and aging slowed down as indicated by several physiological parameters (glucose and insulin resistance, osteoporosis, neuromuscular coordination, rotarod, etc.). Moreover, their average life span was increased by 24% and 13% in adult aged mice, respectively, compared to the control group. A single intravenous administration of AAV9-TERT in adult mice resulted in increased telomere length in peripheral blood cells (Bernardes de Jesus et al., 2012). These results highlight the importance of mouse models for the study of specific disease conditions and potential therapeutic approaches.

[0005] Chronic kidney disease (CKD) is a disorder with a high mortality rate and its incidence is increasing due to the demographic aging phenomenon. Renal fibrosis is characterized by fibroblast activation and excessive production and deposition of extracellular matrix (ECM), which is a major determinant of end-stage renal disease leading to destruction of renal parenchyma, inflammatory and fibrotic responses, and reduced renal function. It is believed that short telomeres may contribute to renal fibrosis. However, the role of short telomeres in renal fibrosis remains poorly understood, in part due to the lack of appropriate mouse models. Furthermore, it is not known whether telomerase-deficient mice with short telomeres will develop renal fibrosis or require further insults to contribute to 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 the present invention, we developed a suitable mouse model to study renal fibrosis associated with short telomeres, which allows us to investigate whether telomerase activation could be an effective treatment to attenuate renal fibrosis associated with short telomeres.

[0007] The present invention provides compositions and methods useful for the treatment and prevention of short telomere-associated renal fibrosis. [Brief description of the drawings]

[0008] [Figure 1]Mouse models of renal fibrosis are associated with short telomeres. a, Schematic of the experimental approach. Increasing 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. One and two weeks after FA administration, blood samples were taken for analysis. Mice were euthanized on day 14. b. Gross appearance of kidneys at endpoint in Tert+ / +, FA-treated Tert+ / +, G3 Tert- / - and FA-treated G3 Tert- / - mice. c, d, Blood creatinine (c) and BUN (d) levels in untreated and FA-treated Tert+ / + and G3 Tert- / - mice. e shows representative images and quantification of Masson's and PAS+D staining in Tert+ / +, FA-treated Tert+ / +, G3 Tert- / - and FA-treated G3 Tert- / - mice at endpoint. Two-way analysis of variance (ANOVA) with post-hoc Bonferroni test was used for statistical analysis. Data are shown as mean ± sem. Number of mice analyzed per genotype (n) is indicated. *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001. NS, not significant. [Diagram 2] Severe renal dysfunction in telomerase-deficient mice treated with sublethal doses of folic acid. a, Schematic of the experimental approach. A FA dose of 125 mg kg-1 of body weight 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 quantification of Masson's trichrome, Sirius red and PAS+D staining in untreated and FA-treated Tert+ / + and G3 Tert- / - kidneys. Statistical analysis used one-way ANOVA with post-hoc Tukey's test and two-way ANOVA with post-hoc Bonferroni test. Data are shown as mean ± sem. Number of mice analyzed per genotype is indicated. **P ≤ 0.01 and ***P ≤ 0.001. [Diagram 3] Telomerase-deficient mice show collagen deposition and activated myofibroblasts in the kidney after sublethal doses of folic acid. a. Representative images and quantification of α-SMA, fibronectin, collagen VI and double immunofluorescence for α-SMA and vimentin in Tert+ / +, FA-treated Tert+ / +, G3 Tert- / - and FA-treated G3 Tert- / - mice. b. Double immunofluorescence for α-SMA and Ki67 in Tert+ / +, FA-treated Tert+ / +, G3 Tert- / - 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 at 14 days after FA administration. One-way ANOVA with post-hoc Tukey's test was used for statistical analysis. Data are presented as mean ± sem. Number of mice analyzed per genotype is indicated. **P ≤ 0.01 and ***P ≤ 0.001. Vh, vehicle. [Figure 4] Increased renal apoptosis and senescence in telomerase-deficient mice after a sublethal dose of folic acid. a–d, Representative images and quantification of immunohistochemical staining for 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 quantification of mean total nuclear telomere length from Q-FISH analysis in Tert+ / +, FA-treated Tert+ / +, G3 Tert− / − 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. The number of mice analyzed per genotype is indicated. [Diagram 5]Short telomeres induce tubular injury 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 administration of low dose FA. b, Representative images and quantification of F4 / 80, CD3e, CD4 and CD8a immunohistochemical staining. Inset: amplified image. For statistical analysis, one-way ANOVA with post-hoc Tukey test was used. Data are presented as mean ± sem. Number of mice analyzed per genotype is indicated. **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 of kidney samples from 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+ / + vs. untreated G3 Tert- / - mice, FA-treated Tert+ / + vs. untreated Tert+ / + mice, FA-treated Terc+ / + vs. untreated Tert+ / + mice, and FA-treated G3 Tert- / - vs. untreated G3 Tert- / - mice. Red to blue horizontal bars represent ranked lists. Genes located in the central region of the bars show small differences in gene expression between pairwise comparisons. The red ends of the bars are genes showing higher expression levels, and the blue ends of the bars are genes showing lower expression levels. Red and blue arrows indicate up- and down-regulation of pathways in pairwise comparisons, respectively. False discovery rates (FDRs) are indicated. Samples correspond to kidneys of four independent Tert+ / +, G3 Tert- / - and FA-treated G3 Tert- / - mice and three FA-treated Tert+ / + mice. c, 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, Representative images and quantification of E-cadherin and phosphorylated SMAD3 (p-SMAD3) immunohistochemical staining. Inset: amplified p-SMAD3 staining images. Statistical analysis used one-way ANOVA with post-hoc Tukey's test. Data are shown as mean ± sem. Number of mice analyzed per genotype is indicated. *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001. [Figure 7]Trf1 deletion induces renal fibrosis. a, Schematic of the experimental approach. b. Representative images and quantification of Masson's trichrome, Sirius red and SMA staining in Trf1+ / + 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+ / + and Trf1flox / flox mice. Two-tailed t-test was used for statistical analysis. Data are presented as mean ± sem. Number of mice analyzed per genotype is indicated. *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 empty (null) or mTert-containing vectors. c, Immunofluorescence of E-cadherin, SMA and Snail1 / Slug in PTCs of 10-11 week-old Tert+ / + and G3 Tert- / - PTCs transduced with either empty (null) or mTert-containing vectors. Twenty photomicrographs were taken for each condition. d, Relative mRNA Cdh1, Acta2, Vim, Col3a1, Col4a1, Tgfb1, Snail1, Snail2 and Zeb1 expression. Data are shown as mean ± sem. Number of mice analyzed per genotype is indicated. One-way ANOVA post-hoc Tukey's 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 quantification 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 quantification of p21 (g) and CC3 (h) immunohistochemical staining in Tert+ / + and G3Tert− / − mice. Insets show amplified images. Two-tailed t-tests were used for statistical analysis. Data are presented as mean + / - SEM. Number of mice analyzed per genotype is indicated. [Figure 10] Blood parameters. Blood 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 presented as mean + / - SEM. Number of mice analyzed per genotype is indicated. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. [Figure 11] Effect of short telomeres on cell cycle regulators. Relative expression of CCnd1, CCnd2, CCnb1 and CCne1 in Tert+ / +, FA-treated Tert+ / +, G3Tert- / - and FA-treated G3Tert- / - mice 14 days after administration of low dose FA. One-way Anova was used for statistical analysis. Data are presented as mean + / - SEM. Number of mice analyzed per genotype is indicated. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. [Figure 12] Effects of shorter telomeres on EMT-related genes. a, Gene expression data obtained by RNA-seq of kidney samples from 7- and 47-week-old Terc+ / + and G3 Tert- / - mice. b, Gene set enrichment analysis (GSEA) plot for EMT pathways. False discovery rates (FDR) are shown. Samples correspond to kidneys from 5 independent Tert+ / +, G3 Tert- / - mice. [Figure 13] Effect 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 administration of low doses of FA. b. Representative images and quantification of Sox-9 immunohistochemical staining. Insets show amplified images. One-way Anova was used for statistical analysis. One-way Anova was used for statistical analysis. Data are presented as mean + / - SEM. Number of mice analyzed per genotype is indicated. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. [Figure 14] Effect of shorter telomeres on Notch target genes. Relative expression of Notch1, 2, 3, Jagged1 and Tfam in Tert+ / +, FA-treated Tert+ / +, G3Tert- / - and FA-treated G3Tert- / - mice 14 days after administration of low dose FA. One-way Anova was used for statistical analysis. Data are presented as mean + / - SEM. Number of mice analyzed per genotype is indicated. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. [Figure 15] TERT activation rescued the EMT phenotype in vitro. a, b, Microscopic brightfield images of proximal tubule cell (PTC) cultures at days 8 (a) and 14 after transduction with either empty vector or mTert-containing vector (b). c, Representative images of immunofluorescence for E-cadherin, SMA and Tgfβ1. Twenty micrographs were taken from each condition. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Brief description of the invention In one aspect, the present invention provides a nucleic acid vector comprising a coding sequence of telomerase reverse transcriptase (TERT) for use in treating renal fibrosis associated with the presence of short telomeres. Preferably, TERT is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO:1 or SEQ ID NO:3. Preferably, TERT is encoded by a nucleic acid sequence consisting of only 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 of only the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. Preferably, the nucleic acid sequence encoding TERT is operably linked to a regulatory sequence that drives expression of the coding sequence. Preferably, the vector is a non-integrating vector. Preferably, the vector is a ribonucleic acid (RNA), preferably a messenger RNA.

[0010] Preferably, the vector is an adeno-associated virus-based non-integrating 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), and the nucleic acid sequence contained in the capsid is flanked on both ends by internal terminal repeat sequences corresponding to serotype 2 adeno-associated virus. Preferably, the nucleic acid contained in the capsid comprises a fragment encoding an amino acid sequence encoding TERT. Preferably, the vector comprises a regulatory sequence that is a constitutive promoter, preferably a cytomegalovirus (CMV) promoter.

[0011] In a further aspect, the present invention provides a non-human animal characterized in that it exhibits a pathological condition of renal fibrosis, which is obtained or obtainable when a sublethal dose of folic acid is administered to the non-human animal, whose germ cells comprise a heritable inactivation of both alleles of the Tert gene.

[0012] Preferably, the animal is a mammal, preferably a rodent. The sublethal dose of folic acid is preferably a dose of up to 200 mg / kg body weight, preferably a dose of 125 mg / kg body weight.

[0013] Preferably, the folic acid is administered intraperitoneally. Preferably, the folic acid is administered when the animal is 4 to 10 weeks old, preferably 6 to 8 weeks old. Preferably, the folic acid is administered once.

[0014] Preferably, the non-human animal, whose germ cells contain a heritable inactivation of both alleles of the Tert gene (Tert- / -), is the third generation (G3) of a Tert- / - strain.

[0015] Detailed Description of the Invention The role of short telomeres in aplastic anemia and pulmonary fibrosis has been found to be important mainly due to the development of suitable mouse models, which paved the way for the development of potential therapeutic strategies for these diseases. However, other complex diseases such as renal fibrosis lack suitable animal models, which hinders the study of the role of short telomeres in these pathologies.

[0016] In this study, we aimed to study the involvement of short telomeres in renal fibrosis. To do so, we needed to develop a suitable animal model. We first developed wild-type mice, as well as mice bred for three generations (G3), thus showing the involvement of short telomeres in renal fibrosis. - / - We analyzed kidneys from mice lacking the telomerase catalytic subunit Tert (Fig. 9a). However, we found that wild-type (Tert + / + ) mice and 8-9 week-old G3 Tert - / - We found that both G3 and G4 mice exhibited remarkably normal kidney histology, with no evidence of glomerular or tubular defects and no fibrosis or accumulation of collagen fibers (Fig. 9b, c). Similarly, mice lacking the telomerase catalytic subunit Tert did not have increased tubular injury or loss of brush borders in the kidney. Also, G3 Tert - / - Consistent with normal kidneys in mice, age-matched Tert+ / + No differences were found in the presence of activated fibroblasts, apoptosis, senescence, or expression of E-cadherin (a marker of EMT associated with tissue fibrosis) compared to telomerase-deficient mice (Figure 1e-h). These results led to the conclusion that telomerase-deficient mice do not spontaneously develop renal fibrosis per se, raising the question of whether mice with short telomeres require further factors to contribute to the development of this pathology. In this regard, since the incidence of renal fibrosis increases with age, we also asked whether renal fibrosis could be caused by a combination of molecular and cellular senescence events such as the presence of short telomeres together with extrinsic damage to the kidney.

[0017] Folic acid (FA) induces interstitial fibrosis, but only at high doses. Intraperitoneal administration of high doses of folic acid (250ug / g BW) to mice rapidly induces folate crystallization 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. With this in mind, we performed a 5-day FA study in 8-9 week-old Tert mice. + / + and G3 Tert - / - We aimed to study the effect of sublethal doses of FA in mice and its contribution to the development of renal fibrosis associated with short telomeres. To this end, we first investigated the effect of sublethal doses of FA in mice at 8–9 weeks of age. + / + and G3 Tert - / - Mice were subjected to increasing doses of FA (50, 100, 125 and 250 mg kg body weight; Fig. 1a) and the highest dose of FA that did not induce renal fibrosis in wild-type mice was selected. The results showed that 125 mg kg was the maximum tolerated dose of FA that did not cause lethality in wild-type mice with normal telomere length (Fig. 1). We then demonstrated that this dose of FA reduced the Tert + / +We investigated whether FA, which is not sufficient to induce renal fibrosis in mice, could synergize with short telomeres in telomerase-deficient mice to induce renal fibrosis. After conducting a series of tests, we found that telomerase-deficient mice treated with a sublethal dose of FA exhibited all the hallmarks of the human disease, including severe renal dysfunction, as indicated by elevated blood levels of creatinine and urea.

[0018] To further 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 shelterin telomere protection complex. In particular, we used a mouse model in which treatment with tamoxifen led to the deletion of Trf1 in all kidney cells (Figure 7). The results showed that mice with dysfunctional telomeres due to deletion of Trf1 spontaneously developed renal fibrosis, highlighting the importance of proper telomere function in protecting against fibrotic lesions.

[0019] Overall, the present invention provides a suitable mouse model suitable for studying renal fibrosis associated with telomere length.The mouse model is an important tool for understanding the role of short and dysfunctional telomeres and the resulting DNA damage in the molecular events associated with fibrosis, especially renal fibrosis.They also allow the development of gene therapy approaches aimed at correcting the accumulation of very short telomeres associated with the condition.

[0020] In view of the above, the present invention provides, in a first aspect, compositions and methods useful for subjects in need of 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 renal fibrosis associated with telomere shortening. Short telomeres exacerbate the epithelial-to-mesenchymal transition (EMT) program in the kidney, thus promoting pathological scarring of kidney tissue, i.e., fibrosis. Renal fibrosis associated with telomere shortening is characterized by the presence of short / dysfunctional telomeres due to mutations in genes associated with telomere maintenance, with those encoding proteins of the telomerase complex (i.e., TERT, TERC, NOP10, DKC1, NHP2) being the most frequently mutated. Both a functional telomerase complex and proper telomere sealing structure by shelterin proteins are required for the maintenance and capping of chromosome ends, respectively.

[0021] In some embodiments, renal fibrosis is characterized by abnormal production and deposition of extracellular matrix (ECM) proteins, primarily in the renal interstitium, leading to structural damage, impaired renal function, and ultimately end-stage renal disease (ESRD). Clinical features of patients suffering from 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 result of the kidney's limited ability to regenerate after injury. In some embodiments, renal fibrosis is characterized by activation of fibroblasts and excessive production and deposition of extracellular matrix (ECM), leading to destruction of renal parenchyma, inflammatory and fibrotic responses, and reduced renal function. It should be noted that renal fibrosis is synonymous with renal fibrosis.

[0022] Thus, the present invention provides compositions and methods for treating a subject suffering from and in need of a condition associated with short telomere length, preferably renal fibrosis, comprising administering to the patient an agent that increases the patient's telomere length. The present invention also relates to a method for preventing the condition. "Prevent", "preventing" or "prevention" or any other similar term includes, but is not limited to, reducing, reducing, or ameliorating the risk of a condition, disorder, condition, or disease, as well as protecting an animal from a condition, disorder, condition, or disease. Prevention can be applied or administered prophylactically. "Treat", "treating" or "treatment" or any other similar term includes, but is not limited to, inhibiting, slowing, stopping, reducing, ameliorating, or reversing the progression or severity of an existing symptom, clinical sign, disorder, condition, or disease. Treatment can be applied or administered therapeutically.

[0023] A "subject in need thereof" herein refers to a subject suffering from a condition that exhibits early onset of pathology due to defective regenerative capacity of kidney tissue. In certain embodiments, the subject in need of said treatment exhibits a fibrotic response, preferably a renal fibrotic response, and / or reduced renal function. In some embodiments, the subject in need of said treatment exhibits a disease or condition that suggests or ultimately leads to renal fibrosis, such as glomerulosclerosis, renal interstitial fibrosis, diabetes, hypertension, infectious glomerulonephritis, renal vasculitis, ureteral obstruction, genetic alterations, autoimmune disease, or any combination thereof. Preferably, the subject in need of said treatment suffers from renal fibrosis, preferably renal fibrosis associated with telomere shortening. The terms "patient" or "subject" are considered synonymous herein and refer to a mammal. In certain embodiments, the patient is a rodent, primate, human, ungulate, cat, dog, mouse, rat, rabbit, pig, horse, sheep, cow, domestic cat or dog, or other domestic pet or livestock mammal. In a preferred embodiment, the patient or subject in need thereof is a human.

[0024] Compositions and methods In one embodiment, the agent prevents degradation of chromosome ends. In one embodiment, the agent increases the activity of telomerase reverse transcriptase (TERT). In one embodiment, the method of treatment is a gene therapy method comprising administering to the patient a nucleic acid vector comprising a coding sequence for telomerase reverse transcriptase (TERT).

[0025] In certain embodiments, the TERT sequence used in the gene therapy vector is derived from the same species as the subject. 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 is 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. A functional equivalent 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 an embodiment, TERT is encoded by a nucleic acid sequence that includes or consists of the sequence of SEQ ID NO:1 or SEQ ID NO:3. A functional equivalent may be artificial or naturally occurring. For example, naturally occurring variants of the TERT sequence in a population are within the scope of functional equivalents. TERT sequences from other species are also within the scope of the term "functional equivalent", in particular the mouse TERT sequence shown in SEQ ID NO:5. In certain embodiments, a functional equivalent is a nucleic acid having a nucleotide sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity with SEQ ID NO:1 or SEQ ID NO:3. In further embodiments, a functional equivalent is a polypeptide having an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity with SEQ ID NO:2 or SEQ ID NO:4. In certain embodiments, TERT comprises or consists of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. In the case of functional equivalents, the sequence identity should be calculated along the entire length of the nucleic acid.A functional equivalent may include one or more, e.g., 2, 3, 4, 5, 10, 15, 20, 30 or more, nucleotide insertions, deletions and / or substitutions when compared to SEQ ID NO:1 or SEQ ID NO:3. The term "functional equivalent" also encompasses nucleic acid sequences encoding TERT polypeptides having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% sequence identity with the sequence shown in SEQ ID NO:2 or SEQ ID NO:4, but which show little or no homology with the nucleic acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3 due to the degeneracy of the genetic code. Sequence identity may be calculated by any one of a variety of methods in the art, including, for example, BLAST and variations of these alignment programs.

[0026] As used herein, the term "active fragment" refers to a nucleic acid molecule encoding a polypeptide having TERT activity, or a fragment of a nucleic acid having TERT activity but which is set forth in SEQ ID NO:1 or SEQ ID NO:3, or the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4. An active fragment can be of any size so long as TERT activity is retained. A fragment has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 100% identity to SEQ ID NO:1-4 along the length of the alignment between the shorter fragment and SEQ ID NO:1-4.

[0027] Fusion proteins containing these fragments can be included in the nucleic acid vectors necessary to practice the invention. For example, an additional 5, 10, 20, 30, 40, 50 or even 100 amino acid residues from the polypeptide sequence or a homologous sequence can be included at either or both the C-terminus and / or N-terminus without impairing the ability of the polypeptide fragment 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 delivering a nucleic acid that codes for a therapeutically active protein to a subject.Nucleic acid can be delivered in several ways and forms, including delivery of naked deoxyribonucleic acid (DNA), such as plasmid or minicircle, delivery of ribonucleic acid (RNA), such as messenger RNA, using liposomes or cationic polymers or other engineered nanoparticles that contain nucleic acid, or viral vectors that entrap nucleic acid.

[0029] In a further embodiment, gene therapy is achieved using stable transformation of the organism with an inducible expression system. Suitable inducible expression systems are known in the art and include the CRE-LOX recombinase-based system, suitable for use in mice, and the tetracycline-regulated system, which can be used to treat human subjects.

[0030] In one embodiment, the gene therapy vector is a ribonucleic acid (RNA) vector that comprises or consists of the coding sequence for telomerase reverse transcriptase (TERT) as defined above. In some embodiments, the RNA nucleic acid vector is delivered in 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 in the structure of the RNA, such as those made at the 2' position, the phosphate linkage, and / or the nucleic acid base. Other modifications include encapsulation of the RNA nucleic acid vector in nanoparticles, polymers, endosomes, lipids and lipid-like particles, or other delivery vectors such as viruses. Nanoparticle encapsulation of RNA can physically protect the nucleic acid from degradation and, depending on the particular chemistry, aid in cellular uptake and endosomal escape. Polymers include naturally occurring polymers such as poly(β-amino esters), poly-L-lysine, polyamidoamines and polyethyleneimines, as well as chitosan, that have all been applied to RNA delivery. A chemically well-defined means of delivery is the direct conjugation of bioactive ligands, such as N-acetylgalactosamine, cholesterol, vitamin E, antibodies, peptides, etc., to RNA allowing it to enter the cells of interest.

[0031] Preferably, the RNA nucleic acid vector is a single-stranded RNA molecule, more preferably a messenger RNA (mRNA) vector. Delivery of therapeutic mRNA vectors has been facilitated by maximizing the translation and stability of the mRNA vector, preventing its immune stimulatory activity and the development of in vivo delivery techniques. In some embodiments, the mRNA vector is modified to increase in vivo delivery efficacy. In certain embodiments, the mRNA vector is modified to include incorporation of a 5' cap and / or a 3' poly(A) tail for efficient translation and half-life extension of the mature mRNA. Cap analogues such as 120-150 bp ARCA (anti-reverse cap analogues) and poly(A) tails can also be used. New types of cap analogues that are resistant 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), including sequences involved in the recruitment of RNA-binding proteins (RBPs), can increase the levels of protein products. N1-methyl-pseudouridine base modifications are commonly used to mask mRNA immunostimulatory activity.

[0032] In one embodiment, the regulatory sequence operably linked to the TERT coding sequence is a cytomegalovirus promoter (CMV), although other suitable regulatory sequences are known to those skilled in the art. In another embodiment, the regulatory sequence operably linked 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 the RNA nucleic acid vector is operably linked to a CMV or kidney-specific promoter, and the RNA nucleic acid vector further comprises a poly(A) sequence located 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. Vectors include integrating and non-integrating vectors, such as those based on retroviruses, adenoviruses (AdV), adeno-associated viruses (AAV), antiviruses, poxviruses, alphaviruses and herpes viruses. It appears to be particularly advantageous to use non-integrating viral vectors, such as AAV. In one aspect, this is because non-integrating vectors do not cause any permanent genetic alterations. Secondly, the vector targets adult tissues, avoiding subjecting the subject to the influence of constitutive telomerase expression from an early stage of development. Furthermore, non-integrating vectors effectively incorporate a safety mechanism to avoid overgrowth of TERT-expressing cells. When cells start to proliferate rapidly, they lose the vector (and, as a result, telomerase expression).

[0034] Particular examples of suitable non-integrating vectors include those based on adenovirus (AdV), particularly gutless adenovirus, adeno-associated virus (AAV), integrase-deficient lentivirus, poxvirus, alphavirus and herpesvirus.Preferably, the non-integrating vector used in the present invention is an adeno-associated virus-based non-integrating vector similar to natural adeno-associated virus particle.

[0035] Vectors derived from adeno-associated viruses (AAV) have emerged as one of the vectors of choice for many gene transfer applications due to many desirable properties, including the ability to transduce a wide range of tissues with high efficiency, low immunogenicity and an excellent safety profile, with toxicity absent in many preclinical models. AAV vectors are able to transduce postmitotic 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 transfer has been studied extensively in humans, with promising results in liver, muscle, CNS, and retina.

[0036] AAV preferentially targets postmitotic tissues, which are considered more resistant to cancer than highly proliferative tissues. Examples of adeno-associated virus-based non-integrating vectors include vectors based on any AAV serotype, i.e., 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 alter their tropism range is likely to be important for their use in therapeutics.

[0037] AAV2 is the most well-characterized serotype for gene transfer research in both humans and experimental models.AAV2 shows natural tropism for skeletal muscle, neurons, vascular smooth muscle cells and hepatocytes.Therefore, AAV2 is a good choice of vector for targeting these tissues, especially when using the method or vector of the present invention to treat a condition related to one of these tissues.For example, the treatment of neuromuscular degeneration can be targeted to skeletal muscle and / or neurons in this way.

[0038] Newly isolated serotypes such as AAV7, AAV8, and AAV9 have been successfully employed in preclinical studies, allowing 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 to overcome these immunological responses in subjects, and clinical trials have just begun (ClinicalTrials.gov Identifier: NCT00979238).

[0039] Overall, these encouraging data suggest that AAV vectors are useful tools for treating human diseases with a high safety and efficiency profile.

[0040] The choice of broad tropism adeno-associated viruses, such as those derived from serotype 9 adeno-associated viruses (AAV9), is particularly advantageous when treating conditions associated with short telomere length. AAV9 viruses have shown efficient transduction in a wide range of tissues, with high tropism for the liver, heart and skeletal muscle, thus allowing the beneficial effects of gene therapy to be achieved in more tissues. Furthermore, AAV9 vectors have the unique ability to cross the blood-brain barrier and target the brain upon intravenous injection in adult mice and cats (Foust et al Nature biotechnology 2009).

[0041] One aspect of the present invention provides a system in which the capsid of the adeno-associated virus-based vector (the part of the virus that determines the viral tropism) is made of 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 loaded into the capsid is flanked by the internal terminal repeats (ITRs) of an adeno-associated virus, preferably serotype 2, which have been extensively characterized in the art and present a coding sequence located between the ITRs. As described above, the nucleic acid preferably encodes a functional TERT polypeptide. In one embodiment, the regulatory sequence operably linked to the TERT coding sequence is a cytomegalovirus promoter (CMV), although other suitable regulatory sequences are known to those skilled in the art. In another embodiment, the regulatory sequence operably linked 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 of the capsid protein of the gene therapy vector may be based on the desired site of gene therapy. When 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 transfecting muscle than other AAV serotypes.

[0043] Alternatively, other viral vectors can be used in the present invention. Any vector suitable for use in gene therapy can be used in the present invention. Heilbronn & Weger (2010) Handb Exp Pharmacol. 197:143-70 provides a review of viral vectors useful in gene therapy.

[0044] According to all the above discussions, a vector containing a coding sequence of telomerase reverse transcriptase (TERT) suitable for use in gene therapy is a key point for implementing the present invention. Suitable gene therapy vectors include any kind of particle containing a polynucleotide fragment encoding the telomerase reverse transcriptase (TERT) protein, operably linked to a regulatory element such as a promoter that allows 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 includes within its scope naked DNA molecules such as plasmids or minicircles, i.e. circular DNA molecules that do not contain bacterial DNA sequences, as well as more complex systems, such as particles that have the structure of a virus particle (virion), contain at least one capsid and at least one polynucleotide sequence, and have a size that allows the polynucleotide sequence to be packed 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 contain a region into which the TERT coding sequence and its linked regulatory elements can be inserted so that the telomerase reverse transcriptase protein can be expressed from the polynucleotide sequence upon infection of a cell by the viral particle.

[0045] In one embodiment, the gene therapy vector suitable for use in the present invention is a non-integrating vector, such as an adeno-associated virus-based non-integrating vector.For the purpose of the present invention, the selection of non-integrating vectors seems to be particularly advantageous, since they do not cause any permanent genetic modification.In addition, as mentioned above, such vectors incorporate a safety mechanism to avoid the overgrowth of TERT-expressing cells, which would lose the vector if the cells start to grow rapidly.

[0046] Adeno-associated virus-based vectors derived from serotype 9 adeno-associated virus (AAV9) are preferred because they can achieve beneficial effects in more tissues (see above). In a particularly preferred embodiment, the regulatory sequence operably linked to the TERT coding sequence is a cytomegalovirus promoter (CMV). The nucleic acid sequence encoding TERT is operably linked to a regulatory sequence that drives expression of the coding sequence. As used herein, the term "regulatory element" refers to a nucleic acid sequence that acts as a promoter, i.e., regulates the expression of a nucleic acid sequence operably linked to the promoter. Such a "regulatory element" or "promoter" can control the expression of a linked nucleic acid sequence either constitutively or inducibly. The regulatory sequence can 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 initially and then treated again when TERT expression level has decreased by about 50% of the expression level obtained immediately after treatment. Treatment can be repeated as needed, for example, annually, or once every 5 years or once every 10 years, with the same or alternative vectors to maintain the reduction of age-related disorders. When administering the second or subsequent doses, it may be necessary to use a different gene therapy vector, for example, when using an AAV-based vector, the second or subsequent doses can be a vector with a capsid derived from a different serotype than that 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.Thus, in a further aspect, the present invention relates to the use of a gene therapy method or a nucleic acid vector as described above for use in treating or preventing renal fibrosis, preferably renal or fibrosis associated with short telomere length, in a subject in need thereof.

[0049] The efficacy of treating a condition associated with short telomere length can be measured by various methods known in the art. In one embodiment, the efficacy of treatment is measured by an increase in the lifespan of a treated patient suffering from a condition associated with short telomere length compared to the expected lifespan of an untreated patient suffering from the same condition. In certain embodiments, the lifespan is extended by 5%, 10%, 15%, 20% or more relative to the expected lifespan of a patient suffering from the same condition. In one embodiment, the efficacy of treatment is measured by delayed or prevented renal failure of a treated patient suffering from a condition associated with short telomere length compared to the expected onset of renal failure of an untreated patient suffering from the same condition. In certain embodiments, the delayed onset of bone marrow failure of a treated patient suffering from a condition associated with short telomere length is extended by 5%, 10%, 15%, 20% or more relative to the expected onset of renal failure for an untreated patient suffering from the same condition.

[0050] In one embodiment, the efficacy of the treatment is measured by an increase in the overall fitness of the treated patient suffering from a short telomere length-associated condition, preferably renal fibrosis, compared to the overall fitness of an untreated patient suffering from the same condition. The overall fitness can be determined by measuring a physical attribute associated with a particular condition. Thus, the increase in overall fitness can be determined by a decrease in a physical attribute associated with a particular condition exhibited by the treated patient. In one embodiment, the increase in overall fitness is measured by determining an estimated glomerular filtration rate (eGFR) and / or albuminuria (ACR). A glomerular filtration rate (GFR) of 60 or greater is in the normal range. A GFR of less than 60 may indicate renal disease. A GFR of 15 or less may indicate renal failure.

[0051] A standardized albumin / creatinine ratio (ACR) test indicates whether there is albumin in the urine. A normal amount of albumin in urine is less than 30 mg / g. Anything above 30 mg / g can mean the patient has kidney disease, even if the GFR value is greater than 60. In certain embodiments, the glomerular filtration rate of a treated patient is increased by 5%, 10%, 15%, 20% or more relative to the glomerular filtration rate of an untreated patient suffering from the same condition.

[0052] The effectiveness of treatment can also be measured by directly determining telomere length in a sample taken from a patient.Telomere length can be measured, for example, by using standard hybridization techniques such as fluorescent in situ hybridization (FISH), quantitative fluorescent in situ hybridization (Q-FISH), or high-throughput quantitative fluorescent in situ hybridization (HT Q-FISH) in a sample taken from a patient.Suitable samples for telomere analysis include blood, urine, or tissue biopsy, such as kidney biopsy.

[0053] In certain embodiments, samples can be taken from patients undergoing treatment throughout the course of treatment to determine both absolute telomere length and telomere shortening rate over the course of treatment. Samples can be taken daily or at longer intervals during the course of treatment. In one embodiment, samples are 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. Comparison of telomere length 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 when measured by in situ hybridization techniques (e.g., FISH or Q-FISH, etc.). In an embodiment, the percentage of short telomeres is the percentage of telomeres that exhibit an intensity 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40% or more lower than the average intensity of the sample. In one particular embodiment, the percentage of short telomeres is the percentage of telomeres exhibiting an intensity that is 50% or more lower than the mean intensity of the sample.

[0054] In another embodiment, the percentage of short telomeres is the percentage of telomeres of a certain length, e.g., 8 kb, 7 kb, 6 kb, 5 kb or shorter. In one embodiment, the percentage of short telomeres is the percentage of telomeres of 8 kb or less. In another embodiment, the percentage of short telomeres is the percentage of telomeres of 7 kb or less. In another embodiment, the percentage of short telomeres is the percentage of telomeres of 6 kb or less. In another embodiment, the percentage of short telomeres is the percentage of telomeres of 5 kb or less. In another embodiment, the percentage of short telomeres is the percentage of telomeres of 4 kb or less. In another embodiment, the percentage of short telomeres is the percentage of telomeres of 3 kb or less.

[0055] In one embodiment, the effectiveness of the treatment is measured by a reduction in the proportion of short telomeres in a sample taken from a treated patient suffering from a condition associated with short telomere length, preferably renal fibrosis, compared to a control sample. In one embodiment, the proportion of short telomeres in a sample taken from a treated patient 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 at an early stage of treatment. In another embodiment, the control sample is a sample taken from a patient suffering from the same condition and to which no treatment is provided.

[0056] In a further aspect, the present invention is applied to a subject by administering to the subject a pharmaceutical composition comprising an effective amount of any one of the gene therapy vectors compatible with the present invention described above.

[0057] A "pharmaceutical composition" is intended to include a combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0058] A "composition" is intended to mean a combination of an active agent with another compound or composition, inert (e.g., a detectable agent or label) or active. An "effective amount" is an amount sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications or administrations.

[0059] They usually include components in addition to the active ingredient (such as a gene therapy vector), eg, they typically include one or more pharmaceutical carrier(s) and / or excipient(s).

[0060] The composition is generally administered to the subject in an aqueous form. However, prior to administration, the composition may be in a non-aqueous form. For example, some viral vectors are manufactured in an aqueous form, then filled and dispensed, and also administered in an aqueous form, while other viral vectors are lyophilized during manufacture and reconstituted in an aqueous form at the time of use. Thus, the composition of the present invention may be dried, such as a lyophilized formulation. The composition may include a preservative, such as thiomersal or 2-phenoxyethanol. However, it is preferred that the composition is substantially free (i.e., less than 5 μg / m) of mercury material, such as thiomersal-free.

[0061] It is preferred to include a physiological salt such as a sodium salt to control isotonicity. Sodium chloride (NaCl) is preferred, which may be present at 1-20 mg / ml, e.g., about 10+2 mg / ml NaCl. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dihydrate, magnesium chloride, calcium chloride, etc.

[0062] The compositions will generally have an osmolality of from 200 mOsm / kg to 400 mOsm / kg, preferably from 240 to 360 mOsm / kg, more preferably falling within the range of 290 to 310 mOsm / kg.

[0063] The composition may include one or more buffers. Typical buffers and designations include phosphate buffers; Tris buffers; borate buffers; succinate buffers; histidine buffers (especially with aluminum hydroxide adjuvants); or citrate buffers. Buffers are typically in the range of 5-20 mM.

[0064] The composition may contain material for a single administration or may contain material for multiple administrations (i.e., a "multi-dose" kit). Inclusion of a preservative is preferred in multi-dose configurations. As an alternative (or in addition) to including a preservative in a multi-dose composition, the composition may be housed in a container with a sterile adaptor for removing the material.

[0065] Compositions of the invention for use in humans will typically be administered in a dose of about 0.5ml, although a half dose (ie about 0.25ml) may be administered to children.

[0066] As with the therapeutic methods described herein, the present invention also provides a nucleic acid sequence encoding TERT for use in therapy. The present invention also provides a nucleic acid vector comprising a coding sequence for telomerase reverse transcriptase (TERT) for use in therapy, and a gene therapy vector comprising a coding sequence for telomerase reverse transcriptase (TERT) for use in therapy. In particular, the therapeutic method can be treating or preventing a condition associated with short telomere length. As described for the therapeutic method, the TERT nucleic acid sequence can be the sequence listed in SEQ ID NO: 1 or SEQ ID NO: 3, or a fragment or functional equivalent thereof. The TERT protein can have the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4, or a fragment or functional equivalent thereof.

[0067] The following embodiments also originate from the first aspect and are included herein.

[0068] 1. A method of treating a patient having renal fibrosis associated with short telomere length comprising administering to the patient a nucleic acid vector comprising a coding sequence for telomerase reverse transcriptase (TERT).

[0069] 2. The method of 1, wherein said TERT is encoded by a nucleic acid sequence comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO:1 or SEQ ID NO:3.

[0070] 3. The method according to 1 or 2, wherein the TERT is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO:1 or SEQ ID NO:3.

[0071] 4. The method according to any one of 1 to 3, wherein the TERT is encoded by a nucleic acid sequence consisting solely of the sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

[0072] 5. The method according to any one of 1 to 4, wherein the TERT comprises 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 solely of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0075] 8. A method according to any one of 1 to 7, wherein the nucleic acid sequence encoding TERT is operably linked to a regulatory sequence that drives expression of the coding sequence.

[0076] 9. The method according to any one of 1 to 8, wherein the vector is a non-integrative vector.

[0077] 10. The method according to any one of 1 to 9, wherein the vector is an adeno-associated virus-based non-integrating vector or an 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 of claim 11, wherein the capsid of the adeno-associated virus-based vector is made of capsid proteins of the serotype 9 adeno-associated virus (AAV9), and the nucleic acid sequence contained within the capsid is flanked on both ends by internal terminal repeat sequences corresponding to serotype 2 adeno-associated virus.

[0080] 13. The method of claim 12, wherein the nucleic acid contained in the capsid comprises 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 which is a constitutive promoter.

[0082] 15. The method according to claim 14, wherein the regulatory sequence is a cytomegalovirus (CMV) promoter.

[0083] 16. The method according to any of 1 to 15, wherein said 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 The present invention further provides in a second aspect a non-human knockout animal model characterized in that it exhibits a pathological condition of renal fibrosis, which is obtained or obtainable 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" as used herein refers to a process in 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 sequence of the allele(s) can be by inserting nucleotides into the allele(s) or by partially or completely deleting nucleotides of the allele(s).

[0085] Specifically, the non-human knockout animal model has a knockout mutation in the Tert gene, resulting in knockout of Tert. As used herein, the term "knockout animal" refers to a non-human animal, preferably a mammal, having one or more genetic manipulations that result in the inactivation of the Tert gene, only one allele of the gene, or preferably both alleles. The knockout mutation of one or both alleles of the Tert gene can be present in the germ cells, somatic cells, or both (i.e., the knockout mutation of the Tert gene is present in all animal cells) of the animal. "Germ cells" are referred to herein as germ cells or gametes (eggs and sperm). "Somatic cells" are referred to herein as 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 a rat. The male founder animal is designated G0, and the first, second and third generations are designated G1, G2 and G3, respectively. Preferably, the non-human knockout animal is Tert - / - The non-human animal model is a third generation (G3) or later generation (G4, G5, etc.) of a know-out animal line. Preferably, the non-human animal model is a G3 Tert - / - Mouse. "G3 Tert - / - The term "Tert" as used herein refers to a third generation animal, preferably a mouse, that contains an inactivation in both alleles of the Tert gene, and preferably said inactivation is heritable, i.e., present in the germ cells of the animal. - / - To generate a colony of animals, heterozygous animals Tert + / - and homozygous animals Tert - / - are produced for further phenotyping. Knockout technology is well known in the art.

[0087] Embryonic cells at various developmental stages can be used to introduce genes for the production of knockout animals. Different methods are used depending on the stage of development of the embryonic cells. Such transfected embryonic stem (ES) cells can then colonize the embryo after their introduction into the blastocyst stage embryo and contribute to the germ cells of the resulting chimeric animal. Prior to introducing the transfected ES cells into the blastocyst, the transfected ES cells can be subjected to various selection protocols to enrich for the percentage of ES cells that have incorporated the knockout gene, if the knockout gene provides a means for such selection. Alternatively, PCR can be used to screen for ES cells that have incorporated the knockout.

[0088] Additionally, retroviral infection can also be used to introduce knockout genes into non-human animals. Developing non-human embryos can be cultured in vitro until the blastocyst stage. During this period, the blastomeres can be targeted for retroviral infection. Efficient infection of the blastomeres can be obtained by enzymatic treatment to remove the zona pellucida. The viral vector system used to introduce the knockout is typically a replication-defective retrovirus carrying the knockout. Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells. Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoel. Most of the founders are mosaics for the knockout gene, since integration occurs only in a subset of cells that will form the knockout animal. Additionally, founders can contain various retroviral insertions of the knockout gene at different locations of the genome that generally segregate to the offspring. Additionally, the knockout gene can be introduced into the germline by intrauterine retroviral infection of midgestation embryos. Additional means of using retroviruses or retroviral vectors to generate knockout animals known to those of skill in the art include microinjecting retroviral particles or cells producing retrovirus that have been treated with mitomycin C into the perivitelline space of fertilized eggs or early embryos.

[0089] Once founder animals are produced, they can be bred, inbred, outbred, or crossed to produce a colony of specific animals. Knockout animals are screened and evaluated to select animals with the desired phenotype. Initial screening can be done, for example, using Southern blot analysis or PCR techniques to analyze animal tissues to confirm that the knockout gene has occurred. Know-out animals can be identified by evaluating TERT mRNA expression levels in tissues of knockout animals using techniques including, but not limited to, Northern blot analysis, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR) of tissue samples obtained from the animals. Samples of appropriate tissues can be evaluated immunocytochemically using antibodies specific for the Tert protein or tags such as EGFP. 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 have been inactivated can be screened according to the presence of kidney pathology or fibrosis when a sublethal dose of FA is administered to them.

[0090] As shown in the following examples, G3 Tert - / -The non-human knockout mouse becomes a non-human animal model of renal fibrosis only when administered a sublethal dose of FA. It is therefore noted that the non-human animal model according to the second aspect, characterized by a knockout for 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" refers herein to a dose that does not induce renal fibrosis in wild-type mice. In an embodiment, the sublethal dose is less than 250 mg / kg body weight. Preferably, the dose of FA administered to the non-human animal 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 sublethal doses of FA, and further exhibits increased renal fibrosis as detected by Masson's Trichrome staining and increased renal tubular injury as determined by increased PAS+D staining, as shown in the Examples below.

[0092] In some embodiments, the dose of FA is administered once, preferably at the beginning of the study. In some embodiments, the FA is administered several times. In some other embodiments, the FA is administered once per day for several days (e.g., once a day, once every two days, once a week, once every two weeks, or once a month, etc.). In some embodiments, the dose of FA is administered systemically, preferably intraperitoneally or intravenously.

[0093] The non-human knockout animal to which FA is administered to prepare the non-human animal model of the second embodiment is 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 assessing renal fibrosis associated with the presence of short telomeres, wherein the non-human knockout animal is as defined in the second aspect or any of its embodiments.

[0095] In a fourth aspect, the present invention provides a method for generating a non-human knockout animal model for assessing renal fibrosis associated with the presence of short telomeres, the method comprising: i) a tert-butyl ether derivative, preferably of the third generation (G3 Tert) as defined in the second aspect or any of its embodiments; - / - providing a non-human animal, preferably a rodent, characterized in that it is knocked out for one, preferably both alleles of the gene encoding the telomerase catalytic subunit Tert from said non-human animal, preferably a rodent; ii) administering to the non-human animal of i) at least one sublethal dose of folic acid as also defined in the second aspect or any of its embodiments.

[0096] In a fifth aspect, the present invention provides a non-human knockout animal model for use in assessing 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 of assessing 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 of screening for compounds for ameliorating, treating or preventing renal fibrosis associated with the presence of short telomeres. In a preferred embodiment, the method comprises: i) administering a compound to a non-human animal model of renal fibrosis associated with the presence of short telomeres as defined in the second aspect or any of its embodiments, ii) detecting or assessing a symptom of renal fibrosis; and iii) comparing the symptoms of renal fibrosis with those of a group of animals not administered the compound and selecting a compound that improves the symptoms; and / or and iv) comparing the symptoms of renal fibrosis before and after administration of the test compound.

[0099] In an eighth aspect, the present invention provides a method of screening 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 of its embodiments, and any change in life expectancy or symptoms associated with renal fibrosis associated with the presence of short telomeres is measured and evaluated, and a test compound that improves the change is determined to have a therapeutic effect. Particular embodiments of the screening method of the seventh or eighth aspect include contacting a tissue, organ or cell derived from the non-human knockout animal model with a test compound and measuring and evaluating changes in the tissue, organ or cell to determine which test compound causes an improvement and thus has a therapeutic effect.

[0100] In the seventh or eighth aspect, the test compound found to have a therapeutic effect on renal fibrosis associated with the presence of short telomeres, obtainable by the screening method disclosed herein, can be used for treatment in 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, the case of the non-human animal model of the second aspect or any of its embodiments is compared and evaluated with the case of wild-type mice or the 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, the method comprises: i) administering a compound to a non-human animal model of renal fibrosis associated with the presence of short telomeres as defined in the second aspect or any of its embodiments, ii) detecting or assessing a symptom of renal fibrosis; and iii) comparing the symptoms of renal fibrosis with those of a group of animals not administered the compound and selecting a compound that improves the symptoms; and / or iv) comparing the symptoms of renal fibrosis before and after administration of the test compound, and selecting a compound that improves the symptoms.

[0102] It is noted that the embodiments and definitions contained in the second aspect of the invention also apply to the third, fourth, fifth, sixth, seventh, eighth and ninth aspects.

[0103] Sequence Listing SEQ ID NO: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 அக்க்கியுக்க்கு2 அக்க்கு நுர்க்கு3 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 number 4 அக்குக்க்கு 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 TIFF2025504888000001.tif247170TIFF2025504888000002.tif38170

[0104] The foregoing invention has been described in some detail by way of illustration and example for clarity of understanding, but the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entireties. EXAMPLES

[0105] Example 1: Methods Mice and animal handling Tert heterozygous mice, generated as previously described, were backcrossed to a C57BL / 6 background of >98%. + / -Mice were bred to generate first generation (G1) homozygous Tert - / - We generated knockout mice. - / - The mouse was - / - The mice were generated by continuous breeding of Tert mice on a pure C57BL / 6 background. + / + and G3 Tert - / - Male and female mice were treated with FA (F7876; Sigma-Aldrich). - / - Mice (7, 27 and 47 weeks of age) were euthanized and kidney phenotyped for signs of fibrosis and RNA-seq analysis.

[0106] Trf1 lox / lox Mice64 were crossed with mice expressing CreERT2 recombinase driven by the tamoxifen-inducible ubiquitin C (UBC) promoter65 to express Trf1 lox / lox ;hUBC-CreERT2 or Trf1 + / + hUBC-CreERT2 mice were generated and fed a long-term tamoxifen-containing diet ad libitum starting at 10 weeks of age.

[0107] All mice were generated and maintained under specific pathogen-free conditions with a 12-h light / dark cycle in the animal facility of the Spanish National Cancer Research Centre (CNIO). Mice were housed in plastic cages containing wood shavings or chipped wood bedding with food and water available ad libitum, in accordance with the recommendations of 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 vehicle alone at different doses (50, 100, 125 and 250 mg kg -1 A single intraperitoneal injection of 100 mg / kg of FA was administered to 6- to 8-week-old Tert mice. + / + Mouse and G3 Tert - / - Mice were administered FA. Blood was collected on days 7 and 14 and analyzed by VetScan Comprehensive Diagnostic Profile kit. Mice were euthanized on day 14 after FA injection, and kidneys were collected from FA-treated or vehicle-treated animals and analyzed by immunohistochemistry for renal fibrosis.

[0109] 125mg kg -1 A single intraperitoneal injection of FA by body weight or a low dose of 0.3 M NaHCO3 (200 ul) was administered to 6-8 week-old Tert mice. + / + Mouse and G3 Tert - / - Mice were inoculated. Blood was collected on days 2, 7, and 14 and analyzed by VetScan Comprehensive Diagnostic Profile kit. Mice were euthanized on day 14 and kidneys were perfused with cold PBS and harvested.

[0110] Morphological analysis Kidneys were fixed in 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 area was quantified using the National Institutes of Health (NIH) ImageJ program.

[0111] primary cell culture Primary mouse PTCs were generated at 10–11 week of age using the Tert + / + and G3 Tert - / -mTert-pBabe-puro was a gift from 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 the packaging cell line 293T. After 48 h, the viral supernatant was collected, centrifuged to remove cellular debris, filtered through a 0.45 μm filter (Millipore), and used to express Tert + / + and G3 Tert - / - Isolated PTCs from mice were infected with 2 μg ml -1 Stable cell lines were selected using puromycin. Cells were harvested for RNA extraction and immunofluorescence.

[0112] Immunohistochemical and immunofluorescent staining Renal tissues were fixed in 4% formaldehyde and embedded in paraffin. Paraffin-embedded renal tissue sections with a thickness of 5–7 μm were subjected to immunohistochemical staining. Primary antibodies (and their dilutions) were: rat monoclonal against p21 (HUGO-291H / B5; CNIO Histopathology Core; 1:400), rat monoclonal against p53 (POE316A / E9; CNIO Histopathology Core; 1:400), rat monoclonal against p21 (HUGO-291H / B5; CNIO Histopathology Core; 1:400), rat monoclonal against CD8a (AM-OTO94A; CNIO Histopathology Core), mouse monoclonal against phospho-histone H2AX (Ser139; 05-636; Millipore; 1:400), mouse monoclonal against E-cadherin (610182; BD The antibodies were: rat monoclonal against F4 / 80 (MCA497; AbD serotype; 1:400), rabbit polyclonal against activated caspase-3 (9661; Cell Signaling Technology; 1:400), rabbit monoclonal against CD3e (99940; Cell Signaling Technology; 1:400), rabbit monoclonal against CD4 (25229; Cell Signaling Technology; 1:400), rabbit polyclonal against type VI collagen (ab6588; Abcam; 1:400), rabbit polyclonal against fibronectin (ab2413; Abcam; 1:400), and rabbit polyclonal against Sox9 (AB5535; Millipore; 1:400).

[0113] For immunofluorescence, 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) were used. Images were obtained using a confocal ultraspectromicroscope (Leica TCS-SP5). The percentage of positive staining area 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 tissues 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 calculated by 2. ΔCT 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 (sequences (5'-3')) TIFF2025504888000003.tif248170TIFF2025504888000004.tif189170

[0116] For RNA-seq experiments, total RNA samples (300 ng) were used. When assayed on a PerkinElmer LabChip analyzer, the RNA quality score averaged 6.3 (range 4.7–8.0). Sequencing libraries were prepared using the QuantSeq 3' mRNA-Seq Library Prep Kit for Illumina (FWD) (Lexogen; 015) according to the manufacturer's instructions. Library generation was initiated by reverse transcription with oligo-dT priming, and second strand synthesis was performed from random primers by DNA polymerase. Primers from both steps contained Illumina-compatible sequences. cDNA libraries were purified and 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. Processed reads were analyzed with the Nextpresso pipeline 67 as follows: Sequencing quality was checked with FastQC v0.11.7 (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ). Reads were aligned to the mouse reference genome (GRCm38) by TopHat (v1.0.0) using Bowtie (v 2.0.10) and SAMtools (v0.1.19) (library type fr-secondstrand in TopHat), allowing 3 mismatches and 20 multiple hits. Read counts were obtained with HTSeq-count (v 0.6.1) using mouse gene annotations from GENCODE (GRCm38; vM20 Ensembl 95). GSEA was performed on several gene signatures on a pre-ranked gene list of 1,000 gene set permutations using GSEAPreranked. Only gene sets with significant enrichment levels (FDR q-value <0.25) were considered.

[0117] Telomere analysis Q-FISH determination on paraffin-embedded tissue sections was performed as previously described41. After deparaffinization, tissues were post-fixed in 4% formaldehyde for 5 min, washed three times for 5 min each in PBS, and incubated in pepsin solution (0.1% porcine pepsin, Sigma; 0.01 M HCl, Merck) for 15 min at 37 °C. After another round of washing and fixation as above, slides were dehydrated in an ethanol series (70%, 90% and 100%; 5 min each). After air drying for 10 min, slides were dehydrated in 30 μl of telomere probe mix (10 mM Tris-Cl (pH 7), 25 mM MgCl2, 9 mM citric acid, 82 mM Na2HPO4, 70% deionized formamide (Sigma), 0.25% blocking reagent (Roche) and 0.5 μg ml -1 A telomere PNA probe (Panagene) was added to each slide. A coverslip was added and the slides were incubated at 85°C for 1 h. The slides were incubated for 3 min at RT in a dark humid chamber for an additional 2 h at room temperature. The slides were washed twice with 10 mM Tris-Cl (pH 7), 70% formamide containing 0.1% BSA for 15 min each, with vigorous shaking, then washed three times with TBS 0.08% containing Tween 20 for 5 min each, and then in a DAPI bath (4 μg ml -1 After incubation in 1000x1000 sucrose (Sigma), samples were mounted in Vectashield medium (Vector). Confocal images were acquired using a Leica SP5-MP confocal microscope as stacks of 1 μm each for a total of 5 μm, and maximum projections were made with 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 sizes are similar to those reported previously4,46. Mice were randomly assigned within groups and investigators were blinded to group allocation. Our sample sizes corresponded to the number of mice used in each experiment, as indicated. Quantitative analysis of immunohistochemical staining was performed on whole scanned kidney sections containing 10–15 areas. For immunofluorescence analysis, 10–20 images were collected from each individual. Data distribution was assumed to be normal, 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. Statistical analysis of immunohistochemical / immunofluorescence quantification as well as qPCR and Q-FISH analysis was performed using one-way ANOVA with post-hoc Tukey's test in Prism (GraphPad). Blood and urinary parameters were analyzed using two-way ANOVA with post-hoc Bonferroni test. Statistical significance was defined as P<0.05.

[0119] Example 2: Results Mouse models of renal fibrosis are associated with short telomeres. To test the involvement of short telomeres in renal fibrosis, we first analyzed kidneys from wild-type mice, as well as mice lacking the telomerase catalytic subunit Tert, which we bred for three generations (G3), and found that they had very short telomeres, i.e., G3 Tert - / - The presence of Tert mice was induced (Figure 9a). + / + ) mice and G3 Tert - / - Both mice showed remarkably normal kidney histology, with no evidence of glomerular or tubular defects, and no fibrosis or accumulation of collagen fibers, as judged by Masson's trichrome and Sirius red staining, respectively (Fig. 9b, c). Similarly, we did not detect increased tubular injury or loss of brush border in the kidney, as measured by periodic acid-Schiff with diastase staining (PAS+D; Fig. 9d). Also, G3 Tert - / -Consistent with the grossly normal kidneys of mice, we found that the presence of activated fibroblasts, as determined by the expression of smooth muscle actin (α-SMA); in apoptosis, as determined by the expression of cleaved caspase-3 (CC3); in senescence, as determined by the expression of the p21 cell cycle inhibitor; or in age-matched Tert + / + No differences were observed in the expression of E-cadherin (a marker of EMT associated with tissue fibrosis) compared to mice (Figure 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 extrinsic damage to the kidney. Therefore, we next investigated the effect of 250 mg kg -1 Tert FA, which has previously been described to induce renal fibrosis at high doses of body weight + / + and G3 Tert - / - FA-induced nephropathy has been widely used to study renal interstitial fibrosis4,27,28. In particular, FA administered intraperitoneally to mice leads to the rapid appearance of FA crystals in renal tubules, followed by severe nephrotoxicity.

[0121] To this end, we first investigated the effects of 8- to 9-week-old Tert + / + and G3 Tert - / - Mice were treated with 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: 50, 100 and 125 mg kg -1 Wild-type mice treated with 250 mg kg appeared well and showed normal kidney appearance (Fig. 1b), normal creatinine and blood urea nitrogen (BUN) in the blood (Fig. 1c, d), and no signs of kidney pathology as determined by the absence of fibrosis as shown by Masson's trichrome staining (Fig. 1e) and the absence of tubular injury by PAS+D staining (Fig. 1f). In contrast, mice treated with 250 mg kg -1The majority of wild-type mice (75%) treated with a FA dose of 125 mg kg body weight died on the second day after treatment due to FA-induced acute renal failure (data not shown). -1 was the maximum tolerated dose of FA that did not cause lethality in wild-type mice with normal telomere length. We found that this dose of FA reduced the Tert + / + We reasoned that although it is not sufficient to induce renal fibrosis in mice, it may synergize with short telomeres to induce renal fibrosis in telomerase-deficient mice.

[0122] In fact, the present inventors have - / - Mice were treated with the same dose of FA, 125 mg kg -1 We observed that even after a dose of 0.1 mg / kg / day, the kidneys exhibited a pale color (Figure 1b), suggestive of renal fibrosis. Indeed, the kidneys of G3 Tert treated with increasing doses of FA showed a pale color (Figure 1b), suggesting renal fibrosis. - / - Mice were dosed at 125 mg kg -1 We showed elevated blood creatinine (Fig. 1c) and BUN (Fig. 1d) levels at FA doses of 125 mg kg−1, indicating renal failure, as well as increased fibrosis as detected by Masson's trichrome staining (Fig. 1e), and increased renal tubular injury as determined by increased PAS+D staining (Fig. 1f). -1 FA doses below G3 Tert - / - did not induce a fibrotic phenotype in mice (Fig. 1e). Therefore, we decided to administer 125 mg kg -1 The dose of FA was selected based on body weight.

[0123] Severe renal dysfunction in telomerase-deficient mice treated with sublethal doses of folic acid To address the role of short telomeres in renal fibrosis, we used 8- to 9-week-old Tert + / + and G3 Tert - / - Mice were treated with 125 mg kg -1The mice were subjected to a dose of FA per kg of 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 kidneys were harvested for further analysis. As expected, FA-treated G3 Tert - / - Kidneys from mice appeared paler and damaged compared to similarly treated wild-type kidneys (Fig. 2b, c). To confirm kidney damage, we analyzed urinary parameters and + / + Mice and untreated Tert + / + Mouse and G3 Tert - / - Compared with mice treated with FA, G3 Tert - / - A significant increase in 24-hour urinary albumin excretion and urinary albumin-to-creatinine ratio was observed in mice (FIG. 2b).

[0124] We also examined BUN and creatinine in all mouse cohorts. It is known that administration of high doses of FA induces a transient elevation of BUN and creatinine levels 48 h after injection, followed by subsequent renal dysfunction accompanied by interstitial fibrosis. However, administration of low doses of FA (125 mg kg -1 Two days after injection of 100 mg / kg body weight, untreated and FA-treated wild-type mice (Tert + / + ) showed normal BUN and creatinine levels, indicating normal renal function (Fig. 10a, b). Consistently, we found that low doses of FA (125 mg kg -1 Wild-type mice (Tert + / + ) did not show any decreased motility or increased mortality. Similarly, untreated G3 Tert - / - Mice showed normal creatinine and BUN levels and normal survival rates on day 2. As expected, FA-treated G3 Tert - / - The mice showed a significant increase in both BUN and creatinine levels on days 2, 7 and 14, with creatinine levels gradually increasing from day 2 onwards, indicating renal dysfunction as early as 2 days after FA administration (Fig. 10a, b). + / + and G3 Tert - / -Further biochemical parameters in the blood of mice indicated renal injury (Table 1). Alkaline phosphatase and amylase levels are generally elevated in patients with CKD and in patients requiring dialysis30 and are indicators of renal damage31. Renal dysfunction can result in hyperamylasemia32. On days 7 and 14, FA-treated G3 Tert - / - Mice were treated with FA + / + Mice and untreated Tert + / + Mouse and G3 Tert - / - The rats showed significantly higher levels of alkaline phosphatase and amylase compared to those in mice (Table 1). Hypercalcemia, hyperphosphatemia, hypernatremia and hyperkalemia are common complications in patients with CKD, especially those with end-stage renal disease. We demonstrated that the FA-treated G3 Tert - / - We observed increases in calcium, phosphorus and sodium levels in mice, whereas potassium levels were significantly increased in mice treated with FA. + / + Mice and untreated Tert + / + Mouse and G3 Tert - / - The levels of blood glucose, globulin, total protein and albumin were increased only on day 14 compared to those of mice (Table 1). As an index of renal tubule 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 increased in the FA-treated G3 Tert mice. - / - In mice, FA treatment + / + Mice and untreated Tert + / + Mouse and G3 Tert - / - was significantly elevated compared with mice, whereas albumin was unchanged (Table 1).

[0125] Table 1: Blood parameters of mice on days 2, 7 and 14 after FA. Data are presented as the mean ± standard deviation of the mean. TIFF2025504888000005.tif150170

[0126] Telomerase-deficient mice exhibit collagen deposition and activated myofibroblasts in the kidney after sublethal doses of folic acid It has previously been shown that FA-induced renal injury coincides with the development of segmental interstitial fibrotic lesions approximately 2 weeks after treatment.37 Consistent with this, at 14 days after treatment, we found that areas of interstitial fibrosis, as indicated by both Masson's trichrome and Sirius red staining, were significantly increased in FA-treated G3 Tert - / - In mouse kidneys, G3 Tert - / - , Tert + / + and FA treatment Tert + / + No fibrosis was detected in the kidneys of mice (Fig. 2c), again indicating that short telomeres sensitize to renal fibrosis after subpathological doses of FA that do not induce fibrosis in wild-type mice. - / - Mice were also treated with FA. + / + Mice and untreated Tert + / + Mouse and G3 Tert - / - Compared with normal kidney histology in mice, they developed significant tubulointerstitial damage as indicated by increased PAS+D staining, including tubular dilatation, atrophy and loss of epithelial differentiation after injury (Fig. 2c). Next, we performed immunohistochemistry for α-SMA, fibronectin and collagen VI as well as double immunofluorescence with α-SMA and vimentin to detect interstitial myofibroblasts (Fig. 3a). Again, we found that FA-treated Tert + / + Mice and untreated Tert + / + Mouse and G3 Tert - / - Compared to undetectable staining in FA-treated G3 Tert mice - / - In mice, we only detected increased staining for α-SMA, fibronectin, collagen VI, and vimentin (Fig. 3a).

[0127] EMT is accompanied by a change from the apical-basal polarity of epithelial cells to the anterior-posterior polarity of mesenchymal cells, and the expression of mesenchymal markers such as fibroblast-specific protein-1, vimentin, N-cadherin, and α-SMA. These changes induce enhanced migration ability, invasiveness, increased resistance to apoptosis, and increased production of ECM components. FA-treated G3 Tert - / - To demonstrate that the proliferative cells observed in the mice were indeed EMT cells and not tubular cells, we performed double immunofluorescence staining for Ki67 and α-SMA. - / - Compared with mice treated with FA, G3 Tert - / - In mice, 36% of proliferating cells were myofibroblasts (α-SMA + Ki67 + ) (Fig. 3b). To determine the mRNA levels of key fibrotic genes, including Acta2 (encoding α-SMA) and Vim (encoding vimentin), Col1a1, Col3a1 and Col4a1 (encoding type I collagen alpha-1, -3 and -4 chains, respectively) and Fn1 (encoding fibronectin 1), quantitative PCR (qPCR) was performed in FA-treated G3 Tert - / - Increased fibrosis was observed in FA-treated G3 Tert mice but not in similarly treated wild-type or untreated mice. - / - In mouse kidneys, FA treatment reduced Tert + / + Mice and untreated Tert + / + Mouse and G3 Tert - / - was significantly upregulated compared with mice (Fig. 3c).

[0128] Increased renal apoptosis and senescence in telomerase-deficient mice following sublethal doses of folic acid We have shown that short telomeres induce a persistent DNA damage response at 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 TGFβ, 37 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, we investigated the effect of FA-treated G3 Tert - / - We investigated whether the increased renal dysfunction and fibrosis in mice was accompanied by increased cellular senescence or apoptosis, two well-known cellular responses to telomere dysfunction associated with aging. We found that FA treatment reduced the renal dysfunction and fibrosis in mice. + / + Mice and untreated Tert + / + Mouse and G3 Tert - / - Compared to undetectable levels in mice, G3 Tert - / - We observed that induced a significant increase in the levels of CC3 (apoptosis marker), p21 and p53 cell cycle inhibitors and γ-H2AX (DNA damage marker) in IFN-γ mice (Fig. 4a-d). Our model indicates that these events increase renal cell turnover, resulting in telomere shortening. In support of this, we observed increased transcription levels of cell cycle regulators encoding genes CCnd1, Ccnd2, Ccnb1 and Ccne1 (Fig. 11a), suggesting that a pronounced profibrotic phenotype may result in G2 / M arrest. Finally, we found that IFN-γ induced a significant increase in G3 Tert β-amyloid markers in IFN-γ mice compared to those in wild-type mice, as measured by telomere quantitative fluorescent in situ hybridization (Q-FISH) directly on kidney sections. - / - 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 injury and immune infiltration in the kidney Next, we decided to study the pathway by which short telomeres induce renal injury. Kidney injury molecule-1 (KIM-1, also known as hepatitis A virus cellular receptor (HAVCR)1; encoded by the gene Havcr1) is a type 1 transmembrane protein that is undetectable in healthy kidneys but is greatly induced after injury42 and is localized to the apical surface of surviving proximal tubule cells (PTCs)43. Inflammation has been proposed as a key promoter 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). Therefore, we used Havcr1 and Lnc2 as biomarkers of CKD resulting from tubulointerstitial injury. We demonstrated that Havcr1 and Lcn2 are associated with increased renal function following FA treatment, whereas CKD is associated with increased renal function following FA treatment, and increased renal function following FA treatment. - / - Mice and controls, untreated Tert + / + Mouse and G3 Tert - / - Compared to basal expression in mice, FA-treated G3 Tert + / + We found that FA-treated G3 Tert mice were upregulated 134-fold and 206-fold, respectively (Figure 5a). Next, we investigated the expression of molecules involved in immune infiltration, which is also associated with tissue fibrosis. We found that FA-treated G3 Tert mice were upregulated 134-fold and 206-fold, respectively (Figure 5b). - / - We observed a 16-fold upregulation of Emr1 mRNA expression (the mouse gene encoding the F4 / 80 antigen) in FA-treated G3 Tert mice (Figure 5a). We also examined the expression of pan-macrophage marker F4 / 80 and T cell markers (CD3e, CD4 and CD8a) by immunohistochemistry. - / - Increase in macrophage colony formation (F4 / 80 positive area) and T cell proliferation in mouse kidneys was observed in FA-treated Tert + / + Mice or untreated Tert + / + and G3 Tert - / - This was observed in comparison with mouse kidneys (Figure 5b).

[0131] Activation of epithelial-mesenchymal transition-related pathways in G3 Tert- / - mice Macrophage recruitment in tissue fibrosis is known to involve TGFβ and is also important for the EMT process45 associated with renal fibrosis46. Therefore, we next sought to determine whether the presence of short telomeres is associated with altered expression of genes involved in EMT. To this end, we performed a randomized controlled study of fibrotic tissue fibrosis in mice treated with untreated or low-dose FA (125 mg kg -1 Ten-week-old Tert mice treated with + / + Mouse and G3 Tert - / - We investigated the expression of EMT and EMT-related pathways, including TGFβ signaling, the major environmental stimulus that induces EMT in adult epithelia, by performing RNA sequencing (RNA-seq) in the kidneys of untreated G3 Tert mice. - / - Untreated Tert mice + / + Mouse gene set enrichment analysis (GSEA) 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 (Fig. 6a, b), suggesting that short telomeres may contribute to a higher basal activation of some EMT genes. Transcriptional upregulation of EMT genes was significantly higher in FA-treated Tert cells compared to untreated animals. + / + Animals and FA treatment G3 Tert - / - These findings were more pronounced in untreated 7-week-old Tert + / + Mouse vs. G3 Tert - / - This was confirmed in 10-, 20-, and 30-year-old mice, as well as in older mice (47 weeks of age; Fig. 12a, b). Thus, short telomeres are associated with altered expression levels of some, but not all, genes involved in EMT in renal epithelial cells and are therefore not sufficient to activate the classical EMT program or to induce fibrosis by itself. Interestingly, treatment with FA significantly reduced the expression levels of both genotypes, including Tert + / + and G3 Tert - / - FA-treated G3 Tert cells induced enrichment of the EMT and TGFβ pathways. - / - In the kidney, FA treatment Tert + / +was higher compared to kidney (NES = 2.42), thus supporting the idea of ​​higher EMT activation in response to FA in telomerase-deficient mice (Fig. 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. TWIST, SNAIL and ZEB1 are transcription factors that control the EMT transcriptional program. Activation of Twist, Snail or Zeb1 is sufficient to induce a mesenchymal phenotype. Therefore, we decided to determine the mRNA expression levels of Tgfb1, Snail1, Snail2, Twist1, Zeb1 and Zeb2 by PCR using reverse transcription (RT-PCR). We found that FA-treated G3 Tert - / - We observed a 32-fold increase in expression of Tgfb1 in FA-treated G3 Tert mice (Fig. 6c). - / - Twist1 was upregulated 7-fold, whereas Zeb1 and Zeb2 were upregulated 6-fold in FA-treated G3 Tert mice (Fig. 6c). - / - 4-fold and 5-fold upregulated in telomerase-deficient mice, respectively (Fig. 6c). Thus, all key EMT players were upregulated in the kidneys of telomerase-deficient mice with short telomeres subjected to a sublethal dose of FA compared to wild-type mice subjected to the same dose, suggesting that short telomeres may contribute to EMT changes after FA treatment. Lysyl oxidase 2 (LOXL2) is necessary and sufficient for hypoxic inhibition of E-cadherin, which mediates cell transformation and induces EMT47. Expression of Loxl2 was upregulated in FA-treated G3 Tert - / -FA-treated G3 Tert mice were upregulated 20-fold (Figure 6c). One of the hallmarks of EMT is the downregulation of E-cadherin isoforms such as Cdh1 and the expression of mesenchymal markers45. Consistent with this, E-cadherin Cdh1 expression was significantly upregulated in FA-treated G3 Tert mice compared to other mouse cohorts, as determined by both mRNA levels and immunohistochemistry. - / - SMAD3 is a key effector in TGFβ signaling. We demonstrated that Smad3 was downregulated two-fold in FA-treated G3 Tert mice (Fig. 6c, d). - / - We found that it was upregulated two-fold in mice (Fig. 6c, d).

[0133] To understand how progenitor 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), paired box 2 (Pax2), sport-like transcription factor 2 (Sall2), activin A receptor type 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 conversion between mesenchymal and epithelial cell states and induce mesenchymal-to-epithelial transformation (MET), playing a key role in the progression of nephrogenesis51. PAX2, a nuclear transcription factor, is associated with MET and is required for renal cell differentiation52. Re-expression of Wt1 and Pax2 in renal tubular epithelial cells plays an important role in promoting EMT, and silencing Pax2 and Wt1 to prevent or reverse renal fibrosis may be of therapeutic value. In this regard, we found that the re-expression of Wt1 and Pax2 in renal tubular epithelial cells was significantly increased in tubular epithelial cells exposed to FA compared to all other groups. - / -We observed a significant increase in the transcript levels of Sox9, Pax2, Wt1 and Acvr2b expression in untreated and treated Tert mice, suggesting that under these conditions, short telomeres sensitized the kidney to undergo EMT (Figure ​(Figure13a). 13a). Sox9 immunostaining was significantly increased in untreated and treated Tert mice. - / - Mouse and G3 Tert + / + Compared with mice treated with FA, G3 Tert - / - Sall2 was significantly increased in FA-treated G3 Tert mice (Fig. 13b). Sall2 is negatively regulated by Wt1 (see 54), and Klotho (Kl) is an anti-aging protein produced mainly in the kidney 55 that regulates telomerase activity 56. Low Kl levels may be a pathological intermediate for the worsening of kidney damage. Interestingly, we found that Sall2 and Kl expression were significantly increased in FA-treated G3 Tert mice (Fig. 13b). - / - We found that the expression of IFN-γ was downregulated in mice (Fig. 13a).

[0134] Sustained expression of the NOTCH signaling pathway in epithelium leads to interstitial fibrosis57. NOTCH is a potent regulator of SNAIL1 and SNAIL2 (see 58). Therefore, we analyzed the expression of Notch receptors (Notch1, Notch2 and Notch3), Notch ligand Jagged 1 (Jag1) and mitochondrial transcription factor A (Tfam) as direct Notch targets59 important for renal function. We found that FA treatment significantly reduced 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 function59. - / - Mice and untreated Tert + / + Mouse and G3 Tert - / - Compared with mice treated with FA, G3 Tert + / + We found a three-fold increase in Notch1 and Notch2 and a four-fold increase in Notch3 and Jag1 in FA-treated Tert mice (Figure 14). - / - Mice and untreated Tert + / + Mouse and G3 Tert - / - Compared with mice treated with FA, G3 Tert + / + We found a five-fold upregulation of Tfam in mice (Figure 14).

[0135] Trf1 deletion-induced telomere dysfunction induces renal fibrosis associated with activation of EMT 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 shelterin telomere protection complex. In particular, we used a mouse model previously described by us in which treatment with tamoxifen led to 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 with Trf1 deletion showed a significantly increased Trf1 expression in the 60 mouse model (Figure 7b). + / + Trf1 mice developed renal fibrosis, as indicated by increased collagen deposition (Masson's trichrome) and fibrotic lesions (Sirius red staining) compared to Trf1 mice (Fig. 7b), as well as by activation of myofibroblasts (SMA staining; Fig. 7b). + / + Compared with mice, Trf1 flox / flox We observed a 3-4-fold increase in mesenchymal markers (Acta2 and Fn1) and a 2-6-fold increase in EMT markers (Tgfb1, Snail1, Snail2, Twist1, Zeb1, Zeb2) in mice (Fig. 7c, d), suggesting that dysfunctional telomeres may also contribute to the transcriptional changes associated with EMT.

[0136] Telomerase overexpression rescues EMT changes associated with short telomeres To further explore the contribution of short telomeres in the activation of the EMT program in the kidney, we investigated whether short telomeres could be rescued by expression of the catalytic subunit of telomerase or TERT61. To this end, we first cultured 10- to 11-week-old Tert + / + Mouse and G3 Tert - / - We isolated kidney epithelial cells, specifically PTCs, 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, wild-type cells and G3 Tert- / - Both G3 Tert cells showed a significant increase in telomere length after 1 week in culture (Figure 8b). - / - The cells exhibited a clear mesenchymal morphology as indicated by their spindle-like shape, which was not observed in wild-type cells, which exhibited a cobblestone morphology (Figure 15a). - / - The cells displayed a fully myofibroblastic phenotype characterized by loss of E-cadherin, enhanced SMA staining, and positivity for Snail1 and TGFβ markers (Fig. 8c and Fig. 15b, c), suggesting that 100% of the cells had undergone 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; Fig. 8c and Fig. S15b, c), and showed some evidence of EMT. Interestingly, mTERT-transduced Tert + / + The cells showed phenotypic reversal, including evidence of MET, restoration of the cobblestone-like phenotype in culture, expression of E-cadherin, and loss of expression of SMA, TGFβ, and Snail1 (Fig. 8c, d and Fig. 15b, c). Importantly, mTERT-transduced G3 Tert - / - The cells also showed a reversal of the mesenchymal phenotype with loss of expression of SMA, TGFβ and Snail1 (Fig. 8c and Fig. 15b, c). Indeed, mTERT-transduced G3 Tert - / - Cells were transduced with G3 Tert - / -Compared to TERT cells, mTERT overexpression and telomere elongation showed reduced expression of many ECM (Acta2, Vim, Col3a1 and Col4a1) and EMT (Tgfb1, Snail1, Snail2 and Zeb1) genes, confirming the restoration of epithelial characteristics upon TERT expression and telomere elongation. Thus, our results show 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 to investigate EMT driven by TERT.

[0137] Example 3: Observations EMT is a cellular plasticity process in which the epithelial layer loses its integrity with concomitant loss of cell polarity and cell-cell interactions mediated by the loss of E-cadherin. The resulting cells express mesenchymal characteristics, including the expression of vimentin and α-SMA. EMT is orchestrated by several transcription factors (EMT-TFs) and plays important roles both during normal development3, as well as in pathological conditions such as cancer and various tissue fibrotic diseases, including renal fibrosis4,62.

[0138] As both cancer and tissue fibrosis are associated with senescence, it is particularly relevant to understand how the known molecular and cellular mechanisms of senescence14 may influence the expression of EMT-TFs and the origin of EMT pathological processes.

[0139] The accumulation of short and dysfunctional telomeres associated with cell division during the lifespan of an organism is considered one of the cardinal features of aging, as it causes persistent DNA damage that is sufficient to impair the regenerative capacity of the adult stem cell compartment.10,13 Indeed, accelerated telomere shortening due to telomerase deficiency in both mice24,25 and humans8 leads to a premature loss of regenerative capacity of tissues, including the development of tissue fibrosis, of which pulmonary fibrosis is one of the most frequent.8,22

[0140] Here, we generated a mouse model of kidney fibrosis associated with short telomeres by challenging telomerase-deficient mice with short telomeres with 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 sublethal doses of FA exhibit all the hallmarks of the human disease, including severe kidney dysfunction, as indicated by elevated blood levels of creatinine and urea. Furthermore, mice with dysfunctional telomeres due to deletion of Trf1, which encodes the shelterin protein, spontaneously develop kidney fibrosis, highlighting the importance of proper telomere function in protecting against fibrotic lesions. Thus, the new mouse model generated here is an excellent tool to understand the role of short dysfunctional telomeres and the resulting DNA damage in the molecular events associated with fibrosis.

[0141] Interestingly, we found here that although short telomeres lead to altered expression of genes involved in EMT, these changes were not sufficient to induce renal fibrosis. These changes were further exacerbated in telomerase-deficient mice treated with FA, which develop renal fibrosis. This is supported by increased expression of Snail1, Snail2, Zeb1, Zeb2 and Twist1 transcription factors, which transform epithelial cells into myofibroblasts. Myofibroblasts deposit ECM-containing collagen, leading to the development of renal fibrosis.

[0142] Corroborating evidence that short telomeres contribute to EMT changes, we show that expression of the mouse telomerase TERT catalytic subunit and subsequent telomere elongation is sufficient to restore the EMT program and restore an epithelial phenotype in kidney cells in culture.

[0143] References 1.Luyckx,V.A.,Tonelli,M.&Stanifer,J.W.The global burden of kidney disease and the sustainable development goals.Bull.World Health Organ.96,414-422C(2018). 2.Humphreys,B.D.Mechanisms of renal fibrosis.Annu.Rev.Physiol.80,309-326(2018). 3.Dongre,A.&Weinberg,R.A.New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer.Nat.Rev.Mol.Cell Biol.20,69-84(2019). 4.Grande,M.T.et al.Snail1-induced partial epithelial-to-mesenchymal transition drives renal fibrosis in mice and can be targeted to reverse established disease.Nat.Med.21,989-997(2015). 5.Inoue,T.,Okada,H.,Takenaka,T.,Watanabe,Y.&Suzuki,H.A case report suggesting the occurrence of epithelial-mesenchymal transition in obstructive nephropathy.Clin.Exp.Nephrol.13,385-388(2009). 6.Martinez,P.&Blasco,M.A.Telomere-driven diseases and telomere-targeting therapies.J.Cell Biol.216,875-887(2017). 7.de Lange,T.Shelterin-mediated telomere protection.Annu.Rev.Genet.52,223-247(2018). 8.Armanios,M.&Blackburn,E.H.The telomere syndromes.Nat.Rev.Genet.13,693-704(2012). 9.Blackburn,E.H.Telomere states and cell fates.Nature 408,53-56(2000). 10.Blasco,M.A.Telomeres and human disease:ageing,cancer and beyond.Nat.Rev.Genet.6,611-622(2005). 11.IJpma,A.S.&Greider,C.W.Short telomeres induce a DNA damage response in Saccharomyces cerevisiae.Mol.Biol.Cell 14,987-1001(2003). 12.Blasco,M.A.,Funk,W.,Villeponteau,B.&Greider,C.W.Functional characterization and developmental regulation of mouse telomerase RNA.Science 269,1267-1270(1995). 13.Flores,I.,Cayuela,M.L.&Blasco,M.A.Effects of telomerase and telomere length on epidermal stem cell behavior.Science 309,1253-1256(2005). 14.Lopez-Otin,C.,Blasco,M.A.,Partridge,L.,Serrano,M.&Kroemer,G.The hallmarks of aging.Cell 153,1194-1217(2013). 15.Armanios,M.Y.et al.Telomerase mutations in families with idiopathic pulmonary fibrosis.N.Engl.J.Med.356,1317-1326(2007). 16.Armanios,M.Telomerase mutations and the pulmonary fibrosis-bone marrow failure syndrome complex.N.Engl.J.Med.367,384(2012). 17.Mitchell,J.R.,Wood,E.&Collins,K.A telomerase component is defective in the human disease dyskeratosis congenita.Nature 402,551-555(1999). 18.Tsakiri,K.D.et al.Adult-onset pulmonary fibrosis caused by mutations in telomerase.Proc.Natl Acad.Sci.USA 104,7552-7557(2007). 19.Coresh,J.,Astor,B.C.,Greene,T.,Eknoyan,G.&Levey,A.S.Prevalence of chronic kidney disease and decreased kidney function in the adult US population:Third National Health and Nutrition Examination Survey.Am.J.Kidney Dis.41,1-12(2003). 20.Beier,F.,Foronda,M.,Martinez,P.&Blasco,M.A.Conditional TRF1 knockout in the hematopoietic compartment leads to bone marrow failure and recapitulates clinical features of dyskeratosis congenita.Blood 120,2990-3000(2012). 21.Bar,C.et al.Telomerase gene therapy rescues telomere length,bone marrow aplasia and survival in mice with aplastic anemia.Blood 127,1770-1779(2016). 22.Povedano,J.M.,Martinez,P.,Flores,J.M.,Mulero,F.&Blasco,M.A.Mice with pulmonary fibrosis driven by telomere dysfunction.Cell Rep.12,86-299(2015). 23.Povedano,J.M.et al.Therapeutic effects of telomerase in mice with pulmonary fibrosis induced by damage to the lungs and short telomeres.eLife 7,e31299(2018). 24.Blasco,M.A.et al.Telomere shortening and tumor formation by mouse cells lacking telomerase RNA.Cell 91,25-34(1997). 25.Lee,H.W.et al.Essential role of mouse telomerase in highly proliferative organs.Nature 392,569-574(1998). 26.Herrera,E.et al.Disease states associated with telomerase deficiency appear earlier in mice with short telomeres.EMBO J.18,2950-2960(1999). 27.Bajwa,A.et al.Sphingosine kinase 2 deficiency attenuates kidney fibrosis via IFN-γ.J.Am.Soc.Nephrol.28,1145-1161(2017). 28.Yang,B.et al.PHF14:an innate inhibitor against the progression of renal fibrosis following folic acid-induced kidney injury.Sci.Rep.7,39888(2017). 29.Long,D.A.,Woolf,A.S.,Suda,T.&Yuan,H.T.Increased renal angiopoietin-1 expression in folic acid-induced nephrotoxicity in mice.J.Am.Soc.Nephrol.12,2721-2731(2001). 30.Damera,S.et al.Serum alkaline phosphatase levels associate with elevated serum C-reactive protein in chronic kidney disease.Kidney Int.79,228-233(2011). 31.Leibovitch,I.,Ben-Chaim,J.,Ramon,J.&Goldwasser,B.Increased serum alkaline phosphatase activity:a possible indicator of renal damage.J.Clin.Lab.Anal.5,406-409(1991). 32.Collen,M.J.,Ansher,A.F.,Chapman,A.B.,Mackow,R.C.&Lewis,J.H.Serum amylase in patients with renal insufficiency and renal failure.Am.J.Gastroenterol.85,1377-1380(1990). 33.Moyses-Neto,M.et al.Acute renal failure and hypercalcemia.Ren.Fail.28,153-159(2006). 34.Barreto,F.C.,Barreto,D.V.,Massy,Z.A.&Drueke,T.B.Strategies for phosphate control in patients with CKD.Kidney Int.Rep.4,1043-1056(2019). 35.Cole,N.I.et al.Serum sodium concentration and the progression of established chronic kidney disease.J.Nephrol.32,259-264(2019). 36.Korgaonkar,S.et al.Serum potassium and outcomes in CKD:insights from the RRI-CKD cohort study.Clin.J.Am.Soc.Nephrol.5,762-769(2010). 37.Yang,H.-C.,Zuo,Y.&Fogo,A.B.Models of chronic kidney disease.Drug Discov.Today Dis.Models 7,13-19(2010). 38.Kalluri,R.&Neilson,E.G.Epithelial-mesenchymal transition and its implications for fibrosis.J.Clin.Invest.112,1776-1784(2003). 39.Blasco,M.A.Telomere length,stem cells and aging.Nat.Chem.Biol.3,640-649(2007). 40.d’Adda di Fagagna,F.et al.A DNA damage checkpoint response in telomere-initiated senescence.Nature 426,194-198(2003). 41.Gonzalez-Suarez,E.,Samper,E.,Flores,J.M.&Blasco,M.A.Telomerase-deficient mice with short telomeres are resistant to skin tumorigenesis.Nat.Genet.26,114-117(2000). 42.Han,W.K.,Bailly,V.,Abichandani,R.,Thadhani,R.&Bonventre,J.V.Kidney injury molecule-1(KIM-1):a novel biomarker for human renal proximal tubule injury.Kidney Int.62,237-244(2002). 43.Humphreys,B.D.et al.Chronic epithelial kidney injury molecule-1 expression causes murine kidney fibrosis.J.Clin.Invest.123,4023-4035(2013). 44.Nickolas,T.L.et al.Sensitivity and specificity of a single emergency department measurement of urinary neutrophil gelatinase-associated lipocalin for diagnosing acute kidney injury.Ann.Intern.Med.148,810-819(2008). 45.Stemmler,M.P.,Eccles,R.L.,Brabletz,S.&Brabletz,T.Non-redundant functions of EMT transcription factors.Nat.Cell Biol.21,102-112(2019). 46.Lovisa,S.et al.Epithelial-to-mesenchymal transition induces cell cycle arrest and parenchymal damage in renal fibrosis.Nat.Med.21,998-1009(2015). 47.Schietke,R.et al.The lysyl oxidases LOX and LOXL2 are necessary and sufficient to repress E-cadherin in hypoxia:insights into cellular transformation processes mediated by HIF-1.J.Biol.Chem.285,6658-6669(2010). 48.Reginensi,A.et al.SOX9 controls epithelial branching by activating RET effector genes during kidney development.Hum.Mol.Genet.20,1143-1153(2011). 49.Lindstrom,N.O.et al.Progressive recruitment of mesenchymal progenitors reveals a time-dependent process of cell fate acquisition in mouse and human nephrogenesis.Dev.Cell 45,651-660(2018). 50.Angelotti,M.L.et al.Characterization of renal progenitors committed toward tubular lineage and their regenerative potential in renal tubular injury.Stem Cells 30,1714-1725(2012). 51.Davies,J.A.et al.Development of an siRNA-based method for repressing specific genes in renal organ culture and its use to show that the Wt1 tumour suppressor is required for nephron differentiation.Hum.Mol.Genet.13,235-246(2004). 52.Rothenpieler,U.W.&Dressler,G.R.Pax-2 is required for mesenchyme-to-epithelium conversion during kidney development.Development 119,711-720(1993). 53.Huang,B.et al.Wt1 and Pax2 re-expression is required for epithelial-mesenchymal transition in 5 / 6 nephrectomized rats and cultured kidney tubular epithelial cells.Cells Tissues Organs 195,296-312(2012). 54.Ma,Y.et al.Cloning and characterization of two promoters for the human HSAL2 gene and their transcriptional repression by the Wilms’ tumor suppressor gene product.J.Biol.Chem.276,48223-48230(2001). 55.Xu,Y.&Sun,Z.Molecular basis of Klotho:from gene to function in aging.Endocr.Rev.36,174-193(2015). 56.Vera,E.,Bernardes de Jesus,B.,Foronda,M.,Flores,J.M.&Blasco,M.A.Telomerase reverse transcriptase synergizes with calorie restriction to increase health span and extend mouse longevity.PLoS ONE 8,e53760(2013). 57.Bielesz,B.et al.Epithelial Notch signaling regulates interstitial fibrosis development in the kidneys of mice and humans.J.Clin.Invest.120,4040-4054(2010). 58.Edeling,M.,Ragi,G.,Huang,S.,Pavenstadt,H.&Susztak,K.Developmental signalling pathways in renal fibrosis:the roles of Notch,Wnt and Hedgehog.Nat.Rev.Nephrol.12,426-439(2016). 59.Huang,S.et al.Jagged1 / Notch2 controls kidney fibrosis via Tfam-mediated metabolic reprogramming.PLoS Biol.16,e2005233(2018). 60.Bejarano,L.et al.Safety of whole-body abrogation of the TRF1 shelterin protein in wild-type and cancer-prone mouse models.iScience 19,572-585(2019). 61.Greider,C.W.&Blackburn,E.H.The telomere terminal transferase of etrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity.Cell 51,887-898(1987). 62.Fazilaty,H.et al.A gene regulatory network to control EMT programs in development and disease.Nat.Commun.10,5115(2019). 63.Liu,Y.et al.The telomerase reverse transcriptase is limiting and necessary for telomerase function in vivo.Curr.Biol.10,1459-1462(2000). 64.Martinez,P.et al.Increased telomere fragility and fusions resulting from TRF1 deficiency lead to degenerative pathologies and increased cancer in mice.Genes Dev.23,2060-2075(2009). 65.Ruzankina,Y.et al.Deletion of the developmentally essential gene ATR in adult mice leads to age-related phenotypes and stem cell loss.Cell Stem Cell 1,113-126(2007). 66.Terryn,S.et al.A primary culture of mouse proximal tubular cells,established on collagen-coated membranes.Am.J.Physiol.Renal Physiol.293,F476-F485(2007). 67.Grana,O.,Rubio-Camarillo,M.,Fdez-Riverola,F.,Pisano,D.G.&Glez-Pena,D.Nextpresso:next-generation sequencing expression analysis pipeline.Curr.Bioinform.13,583-591(2017). 68.Trapnell,C.et al.Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.Nat.Protoc.7,562-578(2012). 69.Langmead,B.,Trapnell,C.,Pop,M.&Salzberg,S.L.Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.Genome Biol.10,R25(2009). 70.Li,H.et al.The Sequence Alignment / Map format and SAMtools.Bioinformatics 25,2078-2079(2009). 71.Anders,S.,Pyl,P.T.&Huber,W.HTSeq-a Python framework to work with high-throughput sequencing data.Bioinformatics 31,166-169(2015). 72.Subramanian,A.et al.Gene-set enrichment analysis:a knowledge-based approach for interpreting genome-wide expression profiles.Proc.Natl Acad.Sci.USA 102,15545-15550(2005). 73.Harley et al.,1990.Nature 345,458-460;Blasco,2007.Nat Chem Biol.3,640-649 74.Flores et al,2008.Genes and Dev 22,654-667 75.Foust et al Nature biotechnology 2009; 76.Heilbronn&Weger(2010)Handb Exp Pharmacol.197:143-70 77.Bernardes de Jesus et al.,2012 EMBO Mol Med.2012 Aug;4(8):691-704.doi:10.1002 / emmm.201200245. 78.Duque et al Molecular therapy 2009

Claims

1. 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.

2. 2. The nucleic acid vector for use according to claim 1, wherein TERT is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO:1 or SEQ ID NO:

3.

3. A nucleic acid vector for use according to any one of claims 1 to 2, wherein TERT comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:

4.

4. A nucleic acid vector for use according to any one of claims 1 to 3, wherein the nucleic acid sequence encoding TERT is operably linked to a regulatory sequence which drives expression of the coding sequence.

5. The nucleic acid vector for use according to any one of claims 1 to 4, wherein the vector is a non-integrating vector.

6. The nucleic acid vector for use according to any one of claims 1 to 5, wherein said nucleic acid vector is ribonucleic acid (RNA), preferably messenger RNA.

7. The nucleic acid vector for use according to any one of claims 1 to 5, wherein the vector is an adeno-associated virus-based non-integrating vector.

8. 8. The nucleic acid vector for use according to claim 7, wherein the vector is an adeno-associated virus-based vector derived from serotype 9 adeno-associated virus (AAV9).

9. 9. The nucleic acid vector for use according to claim 8, wherein the capsid of the adeno-associated virus-based vector is made of capsid proteins of the serotype 9 adeno-associated virus (AAV9) and the nucleic acid sequence contained within the capsid is flanked on both ends by internal terminal repeats corresponding to serotype 2 adeno-associated virus.

10. 10. The nucleic acid vector for use according to claim 9, wherein the nucleic acid contained in the capsid comprises a fragment encoding an amino acid sequence encoding TERT.

11. A nucleic acid vector for use according to any of claims 1 to 10, wherein said vector comprises a regulatory sequence which is a constitutive promoter, preferably a cytomegalovirus (CMV) promoter.

12. A non-human animal characterized in that it exhibits a pathological condition of renal fibrosis, which is obtained or obtainable when a sublethal dose of folic acid is administered to the non-human animal whose germ cells contain heritable inactivation of both alleles of the Tert gene.

13. The non-human animal model according to claim 12, wherein the animal is a mammal, preferably a rodent.

14. 14. The non-human animal of claim 12 or 13, wherein the sublethal dose of folic acid is a dose of up to 200 mg / kg body weight, preferably a dose of 125 mg / kg body weight.

15. The non-human animal of any one of claims 12 to 14, wherein the folic acid is administered intraperitoneally.

16. The non-human animal according to any of claims 12 to 15, wherein said folic acid is administered when said animal is between 4 and 10 weeks of age, preferably between 6 and 8 weeks of age.

17. The non-human animal of any one of claims 12 to 16, wherein the folic acid is administered once.

18. The germ cells contain the Tert gene (Tert －／－ The non-human animal comprises a heritable inactivation of both alleles of Tert －／－ A non-human animal according to any one of claims 12 to 17, which is the third generation (G3) of a lineage.

Images