Manipulation of purine nucleotide metabolism as therapies

Abstract

This document provides methods and materials for manipulating telomere length. For example, this document provides methods and materials for using one or more agents that can manipulate (e.g., increase or decrease) telomere length. In some cases, one or more agents that can increase telomere length can be contacted with a cell to increase telomere length in that cell. For example, one or more agents that can increase telomere length can be administered to a mammal having one or more TBDs to increase telomere length in cells within the mammal (e.g., to treat the mammal). In some cases, one or more agents that can decrease telomere length can be contacted with a cell to decrease telomere length in that cell. For example, one or more agents that can decrease telomere length can be administered to a mammal having a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity to decrease telomere length in cells within the mammal (e.g., to treat the mammal).

Description

[0001] MANIPULATION OF PURINE NUCLEOTIDE METABOLISM AS THERAPIES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Patent Application Serial No. 63 / 624,471, filed on January 24, 2024. The disclosure of the prior application is considered part of, and is incorporated by reference in, the disclosure of this application.

[0004] STATEMENT REGARDING FEDERAL FUNDING

[0005] This invention was made with government support under DK135340, GM007753, GM144273, DK107716, and HL154133 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] SEQUENCE LISTING

[0007] This application contains a Sequence Listing that has been submitted electronically as an XML file named “37314-0125WOl_SL.xml”. The XML file, created on January 21, 2025, is 39,076 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0008] TECHNICAL FIELD

[0009] This document relates to methods and materials for manipulating telomere length. In some cases, this document provides methods and materials for using one or more agents that can manipulate (e.g., increase or decrease) telomere length. For example, one or more agents that can increase telomere length can be administered to a mammal having one or more telomere biology disorders (TBDs; e.g., diseases and disorders associated with short telomeres) to increase telomere length in cells within the mammal (e.g., to treat the mammal). For example, one or more agents that can decrease telomere length can be administered to a mammal having one or more diseases and / or disorders associated with increased telomere synthesis and / or increased telomerase activity to decrease telomere length in cells within the mammal (e.g., to treat the mammal). BACKGROUND

[0010] The ends of linear chromosomes are capped by hexameric repeat sequences termed telomeres, which are required for genomic integrity (Fagagna, Nature 426: 194-198 (2003); and Takai, Current Biology 13: 1549-1556 (2003)). During genome replication, a terminal portion of telomeres is lost due to the inability of DNA polymerases to completely replicate linear chromosomes (Olovnikov, Journal of Theoretical Biology 41 : 181-190 (1973); and Watson, Nature New Biology 239: 197-201 (1972)). Loss of telomere sequence to a critically short length causes DNA damage signaling leading to cellular senescence and exit from the cell cycle (Harley, Nature 345:458-460 (1990)). In self-renewing cells, the telomerase reverse transcriptase (TERT) compensates for telomere shortening by synthesizing new 5’- GGTTAG-3’ repeats using the telomerase RNA template (TERC) and dNTP substrates (Greider, Nature 337:331-337 (1989); Greider, Cell 43:405-413 (1985); Feng, Science 269: 1236-1241 (1995); and Morin, Cell 59:521-529 (1989)). In prevailing models of human telomere length regulation, the expression, abundance, and recruitment of telomerase are regarded as the critical factors counter-acting telomere attrition and thus determining cellular self-renewal capacity in health and disease (Hockemeyer, Nat Struct Mol Biol 22:848-852 (2015)).

[0011] In the human population, telomere length normally declines throughout life and has well-defined upper and lower boundaries (Alder, Proc Natl Acad Sci USA 115:E2358- E2365 (2018); Aubert, PLoS Genet 8:el002696 (2012); and Baerlocher, Nature Protocols 1 :2365-2376 (2006)), deviation from which at either extreme is linked to lethal diseases. Shorter telomere length is associated with reduced lifespan by mendelian randomization (Codd, Nat Genet 53: 1425-1433 (2021)), and germline mutations in the telomerase components and related genes which result in short telomeres lead to a spectrum of fatal degenerative diseases termed telomere biology disorders (TBDs) (Armanios, Nature Reviews Genetics 13:693-704 (2012)) characterized by premature stem cell exhaustion. Conversely, long telomeres are associated with increased risks of various malignancies in the general population by mendelian randomization (Codd, Nat Genet 53:1425-1433 (2021); and The Telomeres Mendelian Randomization Collaboration, JAMA Oncol 3:636 (2017)), and rare germline mutations that lead to long telomeres predispose patients to melanoma, chronic lymphoblastic leukemia and other cancers (DeBoy, New England Journal of Medicine 388:2422-2433 (2023); Schmutz, eLife 9:e61235 (2020); and Kim, The EMBO Journal 40:el07346 (2021)). Despite these associations, the cellular mechanisms constraining human telomere length remain incompletely defined.

[0012] SUMMARY

[0013] This document provides methods and materials for manipulating telomere length. For example, this document provides methods and materials for using one or more agents that can manipulate (e.g., increase or decrease) telomere length. In some cases, one or more agents that can increase telomere length can be contacted with a cell to increase telomere length in that cell. For example, one or more agents that can increase telomere length can be administered to a mammal having one or more TBDs to increase telomere length in cells within the mammal (e g., to treat the mammal). In some cases, one or more agents that can decrease telomere length can be contacted with a cell to decrease telomere length in that cell. For example, one or more agents that can decrease telomere length can be administered to a mammal having a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity to decrease telomere length in cells within the mammal (e g., to treat the mammal).

[0014] As demonstrated herein, nucleotide salvage efficiency bidirectionally controls telomerase activity and telomere length. In some cases, one or more agents that can manipulate telomere length can be used to increase telomere length in a cell (e g., in a cell within a mammal such as a human). For example, supplementing a cell with deoxy guanosine (dG) and / or deoxy thymidine (dT) can increase telomere length within that cell. For example, increasing a level of a deoxy cytidine kinase (dCK) polypeptide in a cell can increase telomere length within that cell. For example, increasing a level of a deoxynucleoside kinase (dNK; e.g., a Drosophila melanogaster dNK (Dm-dNK)) in a cell can increase telomere length within that cell. For example, inhibiting a sterile alpha motif (SAM) and histidineaspartate (HD) domain-containing protein 1 (SAMHD1) polypeptide in a cell can increase telomere length within that cell. In some cases, one or more agents that can manipulate telomere length can be used to decrease telomere length in a cell (e.g., in a cell within a mammal such as a human). For example, supplementing a cell with guanine and / or guanosine can decrease telomere length within that cell. For example, increasing a level of a purine nucleoside phosphorylase (PNP) polypeptide in a cell can decrease telomere length within that cell. For example, increasing a level of a hypoxanthine-guanine phosphoribosyltransferase (HGPRT) polypeptide in a cell can decrease telomere length within that cell. Having the ability to manipulate (e.g., increase or decrease) telomere length as described herein (e.g., by administering one or more agents that can manipulate (e.g., increase or decrease) telomere length) provides a unique and unrealized opportunity to treat diseases and / or disorders characterized by telomere length.

[0015] Also as demonstrated herein, telomerase-null cells (e.g., 293T cells with short telomeres) expressing (e.g., designed to express) a telomerase (e.g., an exogenous telomerase) can be used to detect the ability of an agent (e.g., a candidate compound) to manipulate telomerase activity. For example, telomerase-null cells expressing a telomerase can be used to identify agents that can manipulate (e.g., increase or decrease) telomere length.

[0016] In general, one aspect of this document features methods for increasing telomere length. The methods can include, or consist essentially of, administering an agent that can increase telomere length to a cell in need of increased telomere length. The cell can be a mammalian cell. The cell can be a human cell. The agent that can increase telomere length can be deoxythymidine (dT), a dNK polypeptide, a SAMHD1 inhibitor, a purine nucleoside phosphorylase (PNP) inhibitor, or a combination of the PNP inhibitor and deoxyguanosine (dG). The PNP inhibitor can be ulodesine.

[0017] In another aspect, this document features methods for treating a TBD. The methods can include, or consist essentially of, administering an agent that can increase telomere length to a mammal having a TBD, where the agent increases telomere length in cells within the mammal. The mammal can be a human. The TBD can be dyskeratosis congenita, Hoyeraal Hreidarsson syndrome, Revesz syndrome, Coats Plus syndrome, aplastic anemia with short telomeres, myelodysplastic syndrome with short telomeres, interstitial lung disease with short telomeres, liver cirrhosis with short telomeres, or a telomere biology disorder (TBD) caused by one or more mutations in DKC1, TERC, TIN2, TERT, NOP 10, NHP2, WRAP53 / TCAB1, TINF2, CTC1, RTEL1, ACD, PARN, NAF1, STN1, ZCCHC8, POTI, RPA1, DCLRE1B, or TYMS. The agent that can increase telomere length can be dT, a dNK polypeptide, a SAMHD1 inhibitor, a PNP inhibitor, or a combination of the PNP inhibitor and dG. The PNP inhibitor can be ulodesine.

[0018] In another aspect, this document features methods for decreasing telomere length. The methods can include, or consist essentially of, administering an agent that can decrease telomere length to a cell in need of decreased telomere length. The cell can be a mammalian cell. The cell can be a human cell. The agent that can decrease telomere length can be guanine, guanosine, or 2’, 5 ’-dideoxyguanosine (2,5ddG).

[0019] In another aspect, this document features methods for treating a mammal having a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity. The methods can include, or consist essentially of, administering an agent that can decrease telomere length to the mammal. The mammal can be a human. The agent that can decrease telomere length can be guanine, guanosine, or 2,5ddG. The disease or disorder can be a cancer.

[0020] In yet another aspect, this document features methods for detecting telomerase activity in a cell. The methods can include, or consist essentially of, contacting a cell having (1) short telomeres and (2) exogenous overexpression of a telomerase with a candidate compound, and identifying the compound as an agent that can increase telomere length when telomerase-mediated telomere lengthening is detected. The cell can include (a) nucleic acid that encodes a telomerase RNA component (TERC) and (b) nucleic acid that can encode a telomerase reverse transcriptase (TERT). The contacting can be performed in vitro. The telomerase-mediated telomere lengthening can be detected using terminal restriction fragment (TRF) Southern blotting.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0022] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figures 1A-1 J: Metabolite screening by TRACE assay reveals purine nucleoside supplementation inhibits telomerase reverse transcriptase activity in human cells. Figure 1 A) Diagram of TRACE assay. Figure IB) Terminal restriction fragment Southern blot (TRF) from TRACE assay performed as follows: TERC-I- 293T cells were transfected with the indicated expression vectors, cultured for 18 hours, then split and harvested at the indicated timepoints after splitting. Figures 1C-1G) TRFs from TRACE assay where cells were transfected as in Figure IB. 18 hours after transfection, cells were split and supplemented as indicated for 30 hours prior to harvesting. Figure 1H) Quantification of TRACE assays from Figures 1C-1G, and Figures 71- 7K, displayed as the relative telomere synthesis compared to untreated cells. Figure II) Quantification of TRACE assay performed as in Figure 1 C. Cells were treated with the indicated compounds at the following doses: dG, 100 pM; all other compounds, 500 pM. Experiment performed in biological triplicate. P values compared with untreated cells were calculated using RM one-way ANOVA with Geisser-Greenhouse correction and Dunnett’s multiple comparison test. Includes data from Figures 1C-1H and IFigures 7I-7K. Figure 1 J) Quantification of data from Figure IF, performed in biological triplicate. Figures 11- 1 J) Data are presented as means; error bars indicate s.d. rC, cytidine; rU, uridine; dC, deoxycytidine; dA, deoxyadenosine; rA, adenosine; rG, guanosine.

[0025] Figures 2A-2M: Metabolism of guanosine nucleosides by PNP and HGPRT to form guanine ribonucleotides inhibits telomerase reverse transcriptase activity and shortens telomeres. Figure 2) Schematic of metabolic pathways for dG and 2,5ddG. Figure 2B) TRF from TRACE assay performed as follows: TERC-I- 293T cells were transfected with the indicated expression vectors, cultured for 18 hours, then split and cultured in the indicated doses of 2,5ddG for 30 hours. Figures 2C-2D) TRF Southern blot of 293T cells or K562 cells cultured in the presence of the indicated compounds and doses for 21 days. Figure 2E) TRF from TRACE assay performed as in Figure 2B. Doses of compounds: ulodesine, IpM; dG, lOOpM; guanosine, 500pM; guanine, 500pM. Figure 2F) Schematic of guanine nucleoside salvage and catabolism. Figure 2G) Quantification of TRF from TRACE assay performed as in Figure 2B with the indicated doses of guanine, uric acid, and xanthine. See Figure 8B-8D for corresponding representative TRFs. Experiment performed in biological triplicate. P- values calculated for each compound using an RM one-way ANOVA with the Geisser- Greenhouse correction and Dunnett’s multiple comparisons test. Figure 2H) TRF from TRACE assay performed as in b. Doses of compounds: febuxostat, lOOpM; dG, lOOpM; guanosine, 500pM; guanine, 500pM. Figure 21) TRF of indicated cells cultured with or without 1 OOpM supplemented guanine for three weeks. Representative TRF shown from three biological replicates. Figure 2J) TRF from TRACE assay performed on TERC-I- 293T targeted with the indicated sgRNA(s). Assay performed as in Figure 2B. dG, lOOpM; guanosine, 500pM; guanine, 500pM. Representative TRF shown from three biological replicates. Figure 2K) Quantification of Figure 2J. dG, lOOpM; guanosine, 500pM; guanine, 500pM. P-values calculated for each genotype using an RM one-way ANOVA with the Geisser-Greenhouse correction and Dunnett’s multiple comparisons test. Figure 2L) Quantification of TRF from TRACE assay performed using TERC-I- 293T targeted with the indicated sgRNA(s). Assay performed as in Figure 2B. See Figures 8H-8I for corresponding representative TRFs. Experiment performed in biological duplicate. P-values calculated with a one-way ANOVA with Dunnett’s multiple comparisons test. Figure 2M) TRF from TRACE assay performed as in b. Representative TRF shown from three biological replicates. Data in Figure 2G and 2L are presented as means and error bars indicate s.d.

[0026] Figures 3A-3G: Salvage of dG nucleotides via DCK activates telomerase reverse transcription in a manner modulated by the dNTPase SAMHD1. Figure 3) TRF Southern blot of TRACE assay from TERC-I- 293T cells transfected with the indicated expression vectors, cultured for 18 hours, then split and cultured in the indicated doses of dG for 30 hours. A representative blot is shown from three biological replicates. Figure 3B) Schematic of dG salvage pathways. Figure 3C) TRF Southern blot of TRACE assay performed as in a using either TERC-I- 293T cells (left), or TERC-I- 293T cells which had been targeted with sgRNAs targeting DCK (right). Cells were treated with DI-87 at 2pM and with dG as indicated. A representative blot is shown from two biological replicates. Figure 3D) TRF Southern blot of TRACE assay performed as in a where cells were treated with dG and dC as indicated. Figure 3E) TRF Southern blot of TRACE assay performed as in a using TERC- / - 293T cells overexpressing eGFP or DCK. Cells were treated with dG as indicated. Figure 3F) TRF Southern blot of TRACE assay performed as in a using TERC- / - 293T cells targeted with sgRNA(s) against AAVS1 or SAMHD1. Cells were treated with dG as indicated. A representative blot is shown from two biological replicates. Figure 3G) TRF Southern blot of TRACE assay performed as in a using TERC-I- 293 T cells targeted with sgRNAs against SAMHD1. Cells were treated with DI-87, dC, and dG as indicated.

[0027] Figures 4A-4E Expression of an efficient deoxynucleoside kinase from Drosophila melanogaster promotes telomerase activity from dG treatment and lengthens telomeres. Figure 4A) Schematic of Dm-dNK activity. Figure 4B) Immunoblot for V5-tagged Dm-dNK in TERC -I- 293T cells stably expressing Dm-dNK. Figures 4C-4F) TRF Southern blot of TRACE assay from TERC -I- 293T cells overexpressing either eGFP or Dm-dNK which were transfected with the indicated doses of dG, guanine, or guanosine for 30 hours. Figure 4G) TRF Southern blot of 293T cells which were transduced with lentivirus for constitutive overexpression of eGFP or Dm-dNK and cultured for one month. A representative blot is shown from three biological replicates. Figure 4H) TRF Southern blot of K562 cells which were transduced with lentivirus for doxycycline inducible overexpression of eGFP or Dm- dNK and cultured for one month in the presence of 1 pg / mL of doxycycline. A representative blot is shown from two biological replicates. Figure 41) Quantification of Figures 4G and 4H. Data are presented as means and error bars indicate s.d. P-values were calculated using a paired two-sided t test.

[0028] Figures 5A-5F: Manipulation of deoxynucleotide salvage by expressing Dm-dNK or treatment with dG plus ulodesine drives telomere lengthening in fibroblasts from patients with TBDs. Figure 5A) TRF Southern blot from TERT expressing fibroblasts from a healthy donor overexpressing eGFP or Dm-dNK as indicated and cultured for five weeks. Figure 5B) TRF Southern blot from TERT expressing fibroblasts from TBD patients harboring mutations in the indicated genes overexpressing eGFP or Dm-dNK as indicated and cultured for one month. Figure 5C) Quantification of median telomere length from Figure 5B. Error bars represent s.d. P value was calculated using a paired two-sided t test. Figure 5D) TRF Southern blot from TERT transformed fibroblasts from a healthy donor which were treated with IpM ulodesine and dG as indicated for three weeks. Figure 5E), TRF Southern blot from TERT transformed fibroblasts from TBD patients harboring mutations in the indicated genes which were treated with IpM ulodesine and dG as indicated for three weeks. Figure 5F) Quantification of median telomere length from Figure 5E. Statistical significance determined using paired two-sided t-test. Error bars indicate standard deviation.

[0029] Figure 6: An updated model of human telomere length regulation. To date, most data indicate that human telomere length is regulated by (1) the abundance of telomerase holoenzyme and (2) its interplay with telomere binding proteins. Based on the current findings and emerging human genetic data, we propose a revised model encompassing a third critical dimension of telomere length regulation in human cells: control of dNTP substrate levels available for telomere 5’-GGTTAG-3’ repeat synthesis by telomerase. dNTP substrate accumulation is limited by salvage efficiency including (a) deoxy guanosine (dG) metabolism by DCK, and (b) deoxythymidine (dT) conversion to dTTP via TK1. dTTP can enhance dGTP production by allosteric binding to RNR. Overcoming salvage constraints through dG or dT supplementation or by augmenting kinase activity robustly increases telomerase activity and telomere length. Conversely, guanine nucleoside metabolism via PNP and HGPRT to form guanosine ribonucleotides decreases telomere length, potentially by inhibiting RNR and depletion of dNTPs. Mutations that impair dTTP de novo synthesis led to reduced telomerase substrate levels and impaired telomerase activity, as observed in patients with TYMS locus mutations who develop dyskeratosis congenita. The dNTPase SAMHD1 prevents the accumulation of telomerase dNTP substrates and restricts telomerase activity. Our model provides a mechanistic framework for the recent association between genetic variation in dNTP metabolism genes and telomere length in states of health and disease.

[0030] Figures 7A-7M: TRACE assay is sensitive to genetic and small molecule perturbations to telomerase homeostasis. Figures 7A-7B) RT-qPCR of TERC and TERT expression relative to GAPDH in 293T wild type or TERC-I- cells transfected with the indicated overexpression vectors. Data are shown as mean relative expression and error bars represent S.D. n = 3 biological replicates, / ’-values were calculated using a one-way ANOVA. Figure 7C) TRF from Fig. 7B was stripped and re-probed for the C-rich strand. Figure 7D) TRF from TRACE assay performed as follows: TERC-I- 293T cells were transfected with the indicated expression vectors, split and harvested at the indicated timepoints after transfection. Figure 7E) TRF from TRACE assay performed as follows: TERC-I- 293T cells were transfected with the indicated expression vectors, split 18 hrs after transfected and cultured in the indicated doses of BIBRI 532 for 30 hours prior to harvesting. A representative blot from two independent experiments is shown. Figure 7F) Quantification of Figure 7E. Figure 7G) TRF from TRACE assay where TERC-I- 293T cells were transfected with expression vectors harboring the indicated point mutations followed by culture for 90 hours. A representative blot from two independent experiments is shown.

[0031] Figure 7H) Quantification of Figure 7G) Statistical significance was determined using paired two-sided t-test. i, TRF from TRACE assay performed as in Figure 7G. Statistical significance was determined using paired two-sided t-test. Figure 7J) Quantification of Figure 71 as in Figure 7H. Figures 7K-7M) TRF from TRACE assay where cells were transfected as in Figure 7D, then split 18 hours after transfection and cultured in the indicated compounds for 30 hours prior to harvesting. The data in Figures 7F, 7H, and 7J are presented as means and error bars indicate range.

[0032] Figures 8A-8K: Telomerase inhibition from guanine supplementation. Figure 8A) Quantification of Figures 7C and 7D. Figure 8B) TRF Southern blot of TRACE assay from TERC- / - 293T cells transfected with the indicated expression vectors, cultured for 18 hours, then split and cultured in the indicated compounds for 30 hours. Doses used: forodesine, IpM; dG, lOOpM; guanosine, 500pM; guanine, 500pM. Figures 8C-8E) TRF Southern blot of TRACE assay performed as in Figure 8B. Figure 8F) Genomic DNA from TERC -I- 293T cells manipulated with the indicated sgRNA(s) was PCR amplified with primers specific to the HPRT1 locus as indicated, showing the pools of three sgRNAs targeting HPRT1 generated targeted genomic deletions. Figure 8G) Immunoblot from TERC -I- 293T cell lysates manipulated with indicated sgRNA(s) and probed for HGPRT and -actin confirming HGPRT knockout. Figure 8G) Genomic DNA from TERC -I- 293T cells manipulated with indicated sgRNAs was PCR amplified with primers specific to the APRT and GDA loci as indicated. Figure 81) Quantification of Figure 71. Biological replicates per cell line: 293T, n=5; K562, n=3; Fibroblasts + hTERT, n=3. P values calculated using paired two-sided t- test. Data are plotted as means and error bars indicate s.d. Figures 8J-8K) TRF Southern blot from TRACE assay. Performed on TERC-I- 293T targeted with the indicated sgRNA(s). Assay performed as in Figure 8B with cells split into media containing the indicated doses of guanine. A representative set of blots is shown from two biological replicates. Data presented as means, error bars indicate SD.

[0033] Figures 9A-9M: DCK and SAMHD1 contribute to telomerase regulation in response to manipulation of guanine nucleotide metabolism. Figure 9A) Genomic DNA from TERC - / - 293T cells manipulated with the indicated sgRNA(s) was PCR amplified with primers specific to the DCK locus, showing the three pooled sgRNAs targeting DCK generated on- target genomic deletions. Figures 9B-9C) Immunoblots for DCK expression from TERC-I- 293T cell lysates either manipulated with sgRNAs targeting AA VS1 or DCK loci (Figure 9B) or indicated overexpression vectors (Figure 9C). Figures 9D-9F) Representative gating strategy for cell cycle analysis of TERC-I- 293T cells by DAPI staining and flow cytometry. Cells were first gated on SSC-A versus FSC-A (Figure 9D), doublets were excluded by gating FSC-H versus FSC-A (Figure 9E), and populations were subsequently gated for percentage of cells in Gl, S, and G2 / M phases (Figure 9F). Figures 9G-9H) Representative histograms of DAPI staining in TERC-I- 293T cells with indicated overexpression vectors treated with 20 pM dG for 30 hours. Figures 9I-9J) Quantification of Figures 9G and 9H, respectively, with the mean percentage of cells in each phase of the cell cycle shown in stacked bar charts, n = 3 biological replicates. Figure 9K) Immunoblot from TERC-I- 293 T cell lysates with indicated overexpression vectors treated with 20 pM dG for 30 hours and blotted for pCHKl (S345). UV exposure was used as a positive control. Figure 9L) Immunoblot for SAMHD1 expression in TERC-I- 293T cells manipulated with sgRNAs targeting the AAVS1 and SAMHD1 loci. Figure 9M) TRF Southern blot from TRACE assay of TERC-I- 293T cells targeted with the indicated sgRNA(s) which were transfected with the indicated expression vectors, cultured for 18 hours, then split and cultured in the indicated doses of guanine or guanosine for 30 hours. Note DNA was overloaded in the lane which had the SAMHD1 sgRNAs targeted cells treated with 20 pM guanine, which accounts for the high signal intensity.

[0034] Figures 10A-10O: Effects of dG and ulodesine supplementation in patient fibroblast cells with TBD mutations. Figure 10A) Immunoblot of TERT expression from human skin fibroblasts stably expressing a doxycycline-inducible TERT expression construct (TRE- TERT) grown in indicated doses of doxycycline. TRE-Luciferase expressing fibroblasts were used as a control. Figures 10B-10E) Growth curves of patient fibroblasts expressing TERT with indicated TBD mutations either untreated or grown in 100 pM dG and 1 pM ulodesine. Data are shown as mean population doublings. Error bars indicate S.D. n = 3 biological replicates. E- values were calculated with unpaired t-tests with Welch’s correction. Figures 10F-10I) Representative histograms of DAPI staining in indicated patient fibroblast cells treated for four days with 100 pM dG and 1 pM ulodesine. Figures 10J-10M) Cell cycle analysis quantification of Figures 10F-10I, respectively, to obtain percentage of cells in Gl, S, and G2 / M phases. Data are displayed as mean percentage of cells in each phase of the cell cycle in stacked bar charts, n = 3 biological replicates except for DKC1 p.del37 untreated and treated samples, DKC1 p.A353V untreated samples, and TINF2 p.T284P untreated samples (n = 2 biological replicates). Figures 10N-10O) Immunoblots assessing phosphorylation of CHK1 (S345) in indicated patient fibroblasts as treated in Figures 10F-10I. UV exposure was used as a positive control.

[0035] DETAILED DESCRIPTION

[0036] This document provides methods and materials for manipulating (e.g., increasing or decreasing) telomere length. For example, this document provides methods and materials for using one or more agents that can manipulate telomere length. In some cases, one or more agents that can increase telomere length can be contacted with a cell to increase telomere length in that cell. For example, one or more agents that can increase telomere length can be administered to a mammal having one or more TBDs to increase telomere length in cells within the mammal (e g., to treat the mammal). In some cases, one or more agents that can decrease telomere length can be contacted with a cell to decrease telomere length in that cell. For example, one or more agents that can decrease telomere length can be administered to a mammal having a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity to decrease telomere length in cells within the mammal (e.g., to treat the mammal).

[0037] Telomere length normally declines throughout life. Accordingly, a reference or control value as used herein with respect to telomere length can vary based on the age of the mammal being assessed. For example, the telomere length of a mammal that does not have any disease or disorder associated with altered telomere length (e.g., a healthy mammal) can vary based on the age of the mammal. Additionally, telomere length can vary based on the measurement method used and / or tissue source of the cell being assessed. For example, the telomere length of a mammal that does not have any disease or disorder associated with altered telomere length (e.g., a healthy mammal) can vary based on the cell type being assessed. Thus, it will be appreciated that telomere lengths from comparable samples are used when determining whether or not a particular telomere length is increased or decreased. Exemplary telomere lengths of blood cells (e.g., lymphocytes) from healthy humans can be as shown in Table 1.

[0038] Table 1. Exemplary telomere lengths of healthy humans.

[0039] In some cases, a reference length of a telomere can be as described elsewhere (see, e.g., Demanelis, Science, 369(6509):eaaz6876 (2020); Alder, Proc. Natl. Acad. Sci. USA, 115:E2358-E2365 (2018); Aubert, GPLoS Genet., 8:el002696 (2012); and Baerl ocher, Nature Protocols, 1 :2365-2376 (2006)).

[0040] In some cases, the methods and materials provided herein can include an agent that can increase telomere length. An agent that can increase telomere length can be any appropriate agent that can increase telomere length. In some cases, an agent that can increase telomere length can increase telomerase activity. In some cases, an agent that can increase telomere length can increase telomerase expression (e.g., can increase expression of a telomerase RNA component (TERC) and / or can increase expression of a telomerase reverse transcriptase (TERT)). An agent that can increase telomere length can be any appropriate type of molecule. In some cases, an agent that can increase telomere length can be a polypeptide. In some cases, an agent that can increase telomere length can be a small molecule (e.g., nucleosides and nucleotides). Examples of agents that increase telomere length include, without limitation, pyrimidine nucleobases (e.g., deoxythymidine (dT), dNK polypeptides (e.g., Dm-dNK polypeptides), SAMHD1 inhibitors (e.g., SAMHD1 -targeting gRNA / Cas9 complexes and SAMHD1 -targeting shRNAs), and PNP inhibitors (e.g., ulodesine). In cases where an agent that can increase telomere length includes a PNP inhibitor (e.g., ulodesine), the agent also can include deoxyguanosine (dG). For example, an agent that can increase telomere length can include ulodesine and dG.

[0041] In some cases, an agent that can increase telomere length can increase telomere length in a cell (e.g., in a cell within a mammal) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, or more percent. In some cases, an agent that can increase telomere length can increase telomere length in a cell (e.g., in a cell within a mammal) such that the telomere length is increased relative to a reference length as shown in Table 1.

[0042] In some cases, the methods and materials provided herein can include an agent that can decrease telomere length. An agent that can decrease telomere length can be any appropriate agent that can decrease telomere length. In some cases, an agent that can decrease telomere length can inhibit telomerase activity. In some cases, an agent that can decrease telomere length can inhibit telomerase expression (e.g., can inhibit expression of a telomerase RNA component (TERC) and / or can inhibit expression of a telomerase reverse transcriptase (TERT)). An agent that can decrease telomere length can be any appropriate type of molecule. In some cases, an agent that can decrease telomere length can be a polypeptide. In some cases, an agent that can decrease telomere length can be a small molecule (e.g., nucleosides and nucleotides). Examples of agents that decrease telomere length include, without limitation, guanine, guanosine, and 2’, 5 ’-dideoxyguanosine (2,5ddG).

[0043] In some cases, an agent that can decrease telomere length can decrease telomere length in a cell (e.g., in a cell within a mammal) by, for example, 5, 10, 15, 20, or more percent. In some cases, an agent that can decrease telomere length can decrease telomere length in a cell (e.g., in a cell within a mammal) such that the telomere length is decreased relative to a reference length as shown in Table 1.

[0044] One or more agents that can manipulate (e.g., increase or decrease) telomere length described herein can be formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal having one or more diseases and / or disorders characterized by telomere length. For example, a therapeutically effective amount of one or more agents that can manipulate telomere length described herein can be formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. A pharmaceutical composition can be formulated for administration in solid or liquid form including, without limitation, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules.

[0045] A composition (e.g., a pharmaceutically acceptable composition) including one or more agents that can manipulate (e.g., increase or decrease) telomere length described herein can be administered locally or systemically. A composition containing one or more agents that can manipulate (e.g., increase or decrease) telomere length described herein can be designed for oral, parenteral (including subcutaneous, intramuscular, intravenous, and intradermal), or inhaled administration. For example, a composition containing one or more agents that can manipulate (e.g., increase or decrease) telomere length described herein can be administered systemically by an oral administration to or inhalation by a mammal (e.g., a human).

[0046] Any appropriate amount (e.g., any appropriate dose) of one or more agents that can manipulate (e.g., increase or decrease) telomere length can be administered to a mammal (e g., a human) having one or more diseases and / or disorders characterized by telomere length. An effective amount (e.g., a therapeutically effective amount) of a composition containing one or more agents that can manipulate telomere length described herein can be any amount that can treat a mammal having one or more diseases and / or disorders characterized by telomere length as described herein without producing significant toxicity to the mammal. In some cases, an effective amount of one or more agents that can manipulate telomere length can be about 10 ng / mL. For example, an effective amount of a purine nucleobases (e.g., guanosine, dG, 2,5ddG, adenosine, and dA) can be from about 20 pg / kg to about 500 mg / kg (e.g., from about 20 gg / kg to about 250 mg / kg, from about 20 pg / kg to about 100 mg / kg, from about 20 gg / kg to about 50 mg / kg, from about 20 gg / kg to about 1 mg / kg, from about 20 gg / kg to about 0.5 mg / kg, from about 20 gg / kg to about 0.05 mg / kg, from about 50 gg / kg to about 500 mg / kg, from about 500 gg / kg to about 500 mg / kg, from about 1000 gg / kg to about 500 mg / kg, from about 100 mg / kg to about 500 mg / kg, from about 250 mg / kg to about 500 mg / kg, from about 0.5 mg / kg to about 100 mg / kg, or from about 50 mg / kg to about 250 mg / kg).

[0047] This document also provides methods and materials for treating a mammal (e.g., a human) having one or more diseases and / or disorders characterized by telomere length. In some cases, a disease or disorder associated with telomere length can be a TBD. For example, one or more agents that can increase telomere length can be administered to a mammal having a TBD to increase telomere length in cells within the mammal (e.g., to treat the mammal). In some cases, a disease or disorder associated with telomere length can be a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity. For example, one or more agents that can decrease telomere length can be administered to a mammal having a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity to decrease telomere length in cells within the mammal (e.g., to treat the mammal).

[0048] Any type of mammal having one or more diseases and / or disorders characterized by telomere length can be treated as described herein. Examples of mammals that can be treated as described herein include, without limitation, humans, non-human primates (e.g., monkeys), dogs, cats, horses, cows, pigs, sheep, rabbits, mice, and rats. For example, a human having one or more diseases and / or disorders characterized by telomere length can be treated with one or more agents that can manipulate (e.g., increase or decrease) telomere length as described herein.

[0049] In some cases, the methods provided herein can include identifying a mammal (e.g., a human) as having one or more diseases and / or disorders characterized by telomere length. Any appropriate method can be used to identify a mammal as having one or more diseases and / or disorders characterized by telomere length. For example, imaging techniques to visualize telomere length (e.g., flow cytometry and fluorescence in situ hybridization (flow- FISH) and / or genetic testing for mutations associated with one or more diseases and / or disorders characterized by telomere length (e.g., next-generation sequencing (NGS)) can be used to identify a human or other mammal as having one or more diseases and / or disorders characterized by telomere length. In some cases, a mammal can be identified as having one or more diseases and / or disorders characterized by telomere length as described elsewhere (see, e.g., Baerlocher, Nat. Protoc., l(5):2365-76 (2006); Alter, Blood, 110(5): 1439-47 (2007); Alter, Haematol ogica, 97(3):353-9 (2012); Alter, Proc. Natl. Acad. Sci. USA, 115(10):E2358-E2365 (2018); Heiss, Nat. Genet., 19(l):32-8 (1998); Vulliamy, Nature, 413(6854):432-5 (2001); Yamaguchi, N. Engl. J. Med., 352(14): 1413-24 (2005); Walne, Hum. Mol. Genet., 16(13): 1619-29 (2007); Vulliamy, Proc. Natl. Acad. Sci. USA, 105(23):8073-8 (2008); Savage, Am. J. Hum. Genet., 82(2):501-9 (2008); Zhong, Genes Dev., 25(1): 11-6 (2011); Keller, Pediatr. Blood Cancer, 59(2):311-4 (2012); Ballew, Hum. Genet., 132(4):473-80 (2013); Kocak, Genes Dev., 28(19): 2090- 102 (2014); Tummala, J. Clin. Invest., 125(5):2151-60 (2015); Stanley, Sci. Transl. Med., 8(351):351ral07 (2016); Simon, J. Exp. Med., 213(8): 1429-40 (2016); Gable, Genes Dev., 33(19-20): 1381-1396 (2019); and Sharma, Blood, 139(7): 1039-1051 (2022)).

[0050] When treating a mammal (e.g., a human) having a TBD as described herein (e.g., by administering one or more agents that can increase telomere length), the mammal can have any type of TBD. In some cases, a short telomere can be a telomere including fewer base pairs than a reference length as shown in Table 1. Examples of TBDs include, without limitation, dyskeratosis congenita, Hoyeraal Hreidarsson syndrome, Revesz syndrome, Coats Plus syndrome, aplastic anemia with short telomeres, myelodysplastic syndrome with short telomeres, interstitial lung disease with short telomeres, liver cirrhosis with short telomeres, and TBDs caused by one or more mutations in one or more of DKC1 (e.g., a DKC1 del37L mutation and / or a DKCl A353V mutation), TERC (e.g., a TERC+821 base pair deletion), TIN2 (e.g., a TIN2 p.T284P mutation), TERT, NOP10, NHP2, WRAP53 / TCAB1, TINF2, CTC1, RTEL1, ACD, PARN, NAF1, STN1, ZCCHC8, POTI, RPA1, DCLRE1B, and TYMS.

[0051] In some cases, one or more (e.g., one, two, three, four, or more) agents that can increase telomere length can be used to treat a mammal (e.g., a human) having a TBD. For example, one or more agents that can increase telomere length can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having a TBD) to increase telomere length in cells within the mammal. In some cases, the methods and materials described herein can be effective to increase telomere length in cells within a mammal having a TBD by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, the methods and materials described herein can be effective to increase telomere length in cells within a mammal having a TBD to telomeres having a length as shown in Table 1.

[0052] When treating a mammal (e.g., a human) having a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity as described herein (e.g., by administering one or more agents that can decrease telomere length), the mammal can have any type of disease or disorder associated with increased telomere synthesis and / or increased telomerase activity. In some cases, a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity can be associated with long telomeres. In some cases, a long telomere can be a telomere more base pairs than a reference length as shown in Table 1. Examples of diseases and disorders associated with increased telomere synthesis and / or increased telomerase activity include, without limitation, cancers (e.g., melanomas and chronic lymphoblastic leukemias).

[0053] In some cases, one or more (e.g., one, two, three, four, or more) agents that can decrease telomere length can be used to treat a mammal (e.g., a human) having a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity. For example, one or more agents that can decrease telomere length can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity) to decrease telomere length in cells within the mammal. In some cases, the methods and materials described herein can be effective to decrease telomere length in cells within a mammal having a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, the methods and materials described herein can be effective to decrease telomere length in cells within a mammal having a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity to have a length as shown in Table 1. In cases where a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity is cancer, one or more (e.g., one, two, three, four, or more) agents that can decrease telomere length can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having cancer) to reduce the number of cells within the mammal. In some cases, the methods and materials described herein can be effective to reduce the number of in cells within a mammal having cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.

[0054] In some cases, one or more agents that can increase telomere length described herein can be used as the sole active agent(s) to treat a mammal (e.g., a human) having a TBD.

[0055] In some cases, one or more agents that can increase telomere length described herein can be administered to a mammal (e.g., a human) having a TBD together with one or more (e g., one, two, three, four, or more) additional agents / therapies used to treat a TBD. Examples of agents that can be used to treat a TBD include, without limitation, androgens (e.g., synthetic androgens such as danazol), PAPD5 inhibitors, pyrimidine nucleosides, antioxidants, minerals, and any combinations thereof. In cases where one or more agents that can increase telomere length described herein are used in combination with one or more additional agents used to treat a TBD, the one or more additional agents can be administered at the same time (e.g., in a single composition containing one or more agents that can increase telomere length described herein and the one or more additional agents) or independently. For example, one or more agents that can increase telomere length described herein can be administered first, and the one or more additional agents administered second, or vice versa. Examples of therapies that can be used to treat a TBD include, without limitation, gene therapies, cell therapies, organ transplantations, blood product transfusions, genome editing therapies, and genome engineering therapies. In cases where one or more agents that can increase telomere length described herein used in combination with one or more additional therapies used to treat a TBD, the one or more additional therapies can be performed at the same time or independently of the administration of one or more agents that can increase telomere length described herein. For example, one or more agents that can increase telomere length described herein can be administered before, during, or after the one or more additional therapies are performed. In some cases, one or more agents that can decrease telomere length described herein can be used as the sole active agent(s) to treat a mammal (e.g., a human) having a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity.

[0056] In some cases, one or more agents that can decrease telomere length described herein can be administered to a mammal (e.g., a human) having a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity together with one or more (e.g., one, two, three, four, or more) additional agents / therapies used to treat a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity. In some cases, an agent that can be used to treat a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity can be an anti-cancer agent (e.g., chemotherapeutics including immunotherapeutics such as checkpoint inhibitors and biologies such as antibodies). Examples of agents that can be used to treat a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity include, without limitation, gemcitabine, clofarabine, fludarabine, cladribine, 5 -fluorouracil, azathioprine, mycophenolate mofetil, uridine monophosphate, deoxypyrimidines, and any combinations thereof. In cases where one or more agents that can decrease telomere length described herein are used in combination with one or more additional agents used to treat a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity, the one or more additional agents can be administered at the same time (e.g., in a single composition containing one or more agents that can decrease telomere length described herein and the one or more additional agents) or independently. For example, one or more agents that can decrease telomere length described herein can be administered first, and the one or more additional agents administered second, or vice versa. Examples of therapies that can be used to treat a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity include, without limitation, radiation and anticancer cell therapies such as CAR-T cells. In cases where one or more agents that can decrease telomere length described herein used in combination with one or more additional therapies used to treat a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity, the one or more additional therapies can be performed at the same time or independently of the administration of one or more agents that can decrease telomere length described herein. For example, one or more agents that can decrease telomere length described herein can be administered before, during, or after the one or more additional therapies are performed.

[0057] This document also provides methods and materials for identifying agents that can manipulate telomere length. In some cases, telomerase-null cells (e.g., 293T cells with short telomeres) expressing (e.g., designed to overexpress) a telomerase (e.g., an exogenous telomerase) can be used to whether an agent can manipulate telomere length. For example, methods for identifying agents that can manipulate telomere length can include contacting a candidate compound with telomerase-null cells expressing a telomerase. The method can further include detecting telomerase-mediated telomere lengthening. For example, when telomerase-mediated telomere lengthening is detected, the candidate compound can be identified as an agent that may increase telomere length. For example, when telomerase- mediated telomere lengthening is not detected, the candidate compound can be identified as an agent that may decrease telomere length. In some cases, candidate compounds identified using the methods and materials can then be applied in cell culture (e.g., long-term cell culture) to identify compounds that can increase or decrease telomere length. In some cases, methods and materials for identifying agents that can manipulate telomere length can be as described in Example 1.

[0058] Any appropriate method can be used to detect telomerase-mediated telomere lengthening. In some cases, terminal restriction fragment (TRF) Southern blot can be used to detect telomerase-mediated telomere lengthening. For example, TRF Southern blot can be used to detect telomerase-mediated telomere lengthening within 48 hours of contacting a candidate compound with telomerase-null cells expressing a telomerase.

[0059] Any appropriate telomerase-null cells can be used to identify one or more agents that can manipulate telomere length. In some cases, a telomerase-null cell can have short telomeres. In some cases, a telomerase-null cell can be a mammalian cell such as a human cell. Examples of telomerase-null cells that be used to identify one or more agents that can manipulate telomere length include, without limitation, 293T cells.

[0060] Any appropriate method can be used to overexpress a telomerase in a telomerase-null cell. In some cases, a telomerase-null cell can include (e.g., can be engineered to include) nucleic acid (e g., exogenous nucleic acid) that can encode a telomerase RNA component (TERC) and to include nucleic acid (e.g., exogenous nucleic acid) that can encode a telomerase reverse transcriptase (TERT) such that the telomerase-null cell expresses an exogenous telomerase.

[0061] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0062] EXAMPLES

[0063] Example 1: Metabolic constraint of human telomere length by nucleotide salvage efficiency

[0064] Human telomere length is tightly regulated and associated with diseases at either extreme, but how these bounds are established remains incompletely understood.

[0065] This Example describes the development of a rapid telomere synthesis assay and describes the discovery that nucleoside salvage bidirectionally constrains human telomere length.

[0066] Results

[0067] TRACE - a rapid human cell-based telomerase assay

[0068] The absence of rapid, high-throughput assays to study telomerase reverse transcription in its native cellular setting has been a major barrier to assessing the impact of metabolic perturbations and identifying therapeutically useful targets. Current telomerase assays either rely on measuring telomere length changes in cells after continuous culture requiring several weeks or measuring enzymatic telomeric repeat addition in cell lysates using exogenous dNTPs and primers, potentially obscuring critical aspects of telomerase regulation. To overcome these barriers, we established a method to acutely interrogate the effects of cellular perturbations on telomerase activity within the nucleus at the native chromosome end, called Telomerase Rapid Assessment in the Cellular Environment, or TRACE (Fig. 1A). In TRACE, telomerase-null 293T cells with short telomeres are transfected with vectors encoding the core telomerase components, TERC and TERT (Fig. 7A-7B), leading to telomerase overexpression. In this setting of supra-physiologic telomerase activity, telomerase-mediated telomere lengthening is detectable by terminal restriction fragment (TRF) Southern blot in less than 48 hours (Fig. IB, Fig. 7C-7D), yielding a quantifiable signal that can be used to measure the effects of small molecule or genetic manipulations of telomerase. To evaluate how TRACE responds to small molecules, we applied the telomerase inhibitor BIBRI 532 for 30 hours in the assay and found dose dependent decreases in telomere synthesis (Fig. 7E-7F). To study the capability of TRACE to read out genetic defects in telomerase components, we performed the assay using expression vectors encoding TBD-associated mutations in TERT (Snetselaar, PLoS One 12:e0189467 (2017); and Xin, Blood 109:524-532 (2007)) or TERC (Boyraz, Blood 128:2089-2092 (2016)) and found that these pathogenic variants impaired telomere synthesis (Fig. 7G-7J). Collectively, these data demonstrate that the TRACE assay can rapidly read out small molecule and genetic modulation of telomerase activity in human cells.

[0069] Purine nucleosides inhibit telomerase

[0070] We utilized the TRACE assay to evaluate how the manipulation of nucleotide metabolism impacts telomerase reverse transcriptase activity. We supplemented cells with individual deoxynucleosides (dNs) and ribonucleosides (rNs) at a range of concentrations and found that pyrimidines drove telomerase activation in a dose dependent manner (Fig. 1C, Fig. 7K-7M), most robustly thymidine (dT). In contrast, purine nucleoside supplementation caused telomerase inhibition, with the guanine nucleosides guanosine and deoxyguanosine (dG) inhibiting telomerase activity more potently than adenosine and deoxyadenosine (dA) (Fig. 7D-7J). The inhibitory effect from dG supplementation was unanticipated given prior studies demonstrating that increased levels of dGTP lead to strong activation of purified telomerase enzymatic activity (Morin, Cell 59:521-529 (1989); Maine, Biochemistry 38: 15325-15332 (1999); Sun, Biochemistry 38:4037-4044 (1999); and Chen, E O J 37:e97953 (2018)). Interestingly, dG inhibited telomerase activity to a greater degree at 100 pM than at 500 pM (Fig. 1 J), showing a dose dependent, non-linear relationship between telomerase activity and dG supplementation. Collectively, these results demonstrate that purine nucleoside supplementation can acutely inhibit telomerase reverse transcription in human cells. PNP is required for telomerase inhibition by guanosine and deoxyguanosine

[0071] We next used the TRACE assay to ask whether metabolites of guanosine and dG mediate the observed telomerase inhibition. First, because dG might undergo 5’- phosphorylation by deoxycytidine kinase (DCK), we tested if supplementation of 2’, 5’- di deoxy guanosine (2,5ddG), a dG analog which cannot be directly phosphorylated by DCK (Fig. 2A), could also inhibit telomerase activity. Similar to the effects of guanosine and dG, we saw a dose dependent inhibitory effect of 2,5ddG supplementation in the TRACE assay (Fig. 2B). These results suggest that phosphorylation of dG by DCK to form dG nucleotides is not responsible for dG’s inhibitory effects on telomerase.

[0072] To evaluate the effects of nucleoside supplementation on telomere length in cells without telomerase overexpression, we treated 293T cells and K562 cells with either 2,5ddG or guanosine. In both cases we observed dose dependent telomere shortening after 21 days in culture (Fig. 2C-2D, Fig. 8A). These results validate the TRACE assay’s ability to identify non-natural compounds that cause telomere shortening when applied to cells with endogenous levels of telomerase expression.

[0073] Both guanosine and dG nucleosides are catabolized by purine nucleoside phosphorylase (PNP) to yield guanine and the associated sugar. We thus asked whether treatment with PNP inhibitors would rescue telomerase inhibition from guanine nucleoside supplementation in the TRACE assay. When we applied the PNP inhibitor ulodesine, we found that telomerase reverse transcriptase activity was no longer inhibited by guanosine or dG (Fig. 2E). Ulodesine alone had no effect (Fig. 2E). These results pointed to guanine, the common downstream catabolite of guanosine and dG. Surprisingly and consistent with this, we found that supplementation with guanine itself strongly inhibited telomerase activity in the TRACE assay, in a manner that could not be reversed by ulodesine (Fig. 2E). Similar results were obtained using the structurally distinct PNP inhibitor forodesine (Fig. 8B). These data reveal a novel inhibitory effect of guanine, whether by direct supplementation or catabolism of dG or guanosine nucleosides by PNP, on telomerase activity in human cells. Guanine ribonucleotides inhibit telomere lengthening in human cells

[0074] We next asked whether metabolites of guanine caused the strong telomerase inhibition we observed in the TRACE assay. Guanine can either be salvaged as guanosine monophosphate (GMP) by hypoxanthine-guanine phosphoribosyltransferase (HGPRT), or further catabolized into xanthine and uric acid by guanine deaminase (GDA) and xanthine oxidase, respectively (Fig. 2F). To test whether guanine breakdown products caused telomerase inhibition, we compared the effects of directly supplementing cells with guanine, xanthine, or uric acid in the TRACE assay. Whereas guanine supplementation potently inhibited telomerase reverse transcriptase activity, xanthine and uric acid had minimal effects at doses up to 500 pM (Fig. 2G, Fig. 8C-8E). As a complementary approach to evaluate whether the accumulation of guanine catabolic products drives telomerase inhibition, we tested the xanthine oxidase inhibitor febuxostat in the TRACE assay, and found it was unable to rescue telomerase activity from treatment with either dG, guanosine, or guanine (Fig. 2H). Together, these findings indicated that guanine rather than its downstream degradation products is responsible for telomerase inhibition in cells.

[0075] To study these effects under conditions with physiologic telomerase expression, we treated unmanipulated 293T or K562 cells with 100 pM guanine for 21 days and found reproducible telomere shortening (Fig. 21, Fig. 81). To test this in diploid primary cells, we treated normal human fibroblasts in which TERT was stably expressed with guanine for 21 days, and similarly observed inhibition of telomere lengthening (Fig. 21, Fig. 81). Collectively, these results demonstrate that conversion of guanosine and dG into guanine causes inhibition of telomerase reverse transcription and show that guanine supplementation itself can shorten telomeres in human cancer cell lines with endogenous telomerase levels.

[0076] We next asked if guanine supplementation requires its salvage into cellular ribonucleotide pools via HGPRT to cause telomerase inhibition. Using CRISPR / Cas9, we generated HPRT1 deficient TERC-I- 293T cells and verified efficient on-target gene inactivation (Fig. 8F-8G). We similarly used CRISPR / Cas9 to generate TERC-I- 293T cells deficient foe APRT, encoding adenine phosphoribosyltransferase which converts adenine to AMP, and GDA encoding guanine deaminase which catabolizes guanine to xanthine (Fig. 8H) The AA VS1 safe harbor locus was targeted as a control. Using the TRACE assay, we found that, relative to control cells, the inhibitory effects of guanosine, dG and guanine on telomerase activity were abrogated after HPRT1 inactivation (Fig. 2J-2K). In comparison, telomerase inhibition by guanine in GDA- or 4 / 7 / -targeted cells was similar to controls (Fig. 2L, Fig. 8J-8K). Collectively, these data support a model where treatment with guanosine, dG, or guanine leads to inhibition of telomerase reverse transcriptase activity via the salvage of guanine by HGPRT to form guanine ribonucleotides.

[0077] Guanine salvage into ribonucleotide pools has been shown to reduce dNTP levels including dTTP in human cells (Diehl, Nat Cell Biol 24: 1252-1264 (2022)). dTTP levels are important for telomere length control and can be increased by supplementing cells with dT. Therefore, we asked if the inhibitory effects of guanine treatment on telomerase could be overcome by dT supplementation. Using the TRACE assay, we found that dT supplementation fully rescued guanine-mediated telomerase inhibition (Fig. 2M). Taken together, our results support a model wherein guanine supplementation results in GMP accumulation via HGPRT salvage, which in turn depletes dNTPs needed for telomerase reverse transcription, thus shortening telomere length. Furthermore, combined with our observations of guanine-mediated telomere attrition in the setting of endogenous telomerase expression (Fig. 21, Fig. 81), these data indicate that telomerase activity is constrained in human cells by dNTP substrate availability.

[0078] DCK inefficiently salvages dG to promote telomerase activation

[0079] Our data unexpectedly demonstrate that dG nucleoside supplementation inhibits telomerase via formation of guanosine ribonucleotides. However, in our original TRACE metabolite screen (Fig. 1H), we observed that at high doses dG supplementation had a diminished inhibitory effect on telomerase, with 100 pM dG inhibiting telomerase activity to a greater degree compared to 500 pM dG. We repeated this analysis using a broader dG dose range, and again found that while 100 pM dG strongly inhibited telomerase activity, 500 pM had a smaller inhibitory effect (Fig. 3A). Remarkably, 2.5 mM dG increased telomere synthesis above baseline (Fig. 3A), an observation that would not be possible without TRACE due to the cytotoxicity of dG at this dose after a few days in culture. Given that dGTP is a telomerase substrate, we wondered if salvage of excess supplemented dG by DCK into dG nucleotides could underlie the increased telomerase activity observed with high dose dG, potentially counteracting the inhibitory effects on telomerase from the “default” pathway of dG breakdown into guanine (Fig. 3B). To address this question, we used a series small of molecule and genetic approaches to alter DCK function. We first inactivated DCK in TERC- / - 293T cells using CRISPR / Cas9 (Fig. 9A-9B) and asked if inactivation of DCK could block a potential telomerase activating effect of high dose dG. Using the TRACE assay, we found that dG at high doses no longer stimulated telomerase reverse transcriptase activity in the absence of DCK (Fig. 3C). Next, we applied the DCK inhibitor DI-87 to cells treated with increasing doses of dG in the TRACE assay. In comparison to the effects of dG alone, we found reduced telomerase reverse transcriptase activity at 500 pM and 2.5mM dG in the presence of DI-87 (Fig. 3C). Finally, we asked if dC supplementation could prevent telomerase activation from high dose dG, because DCK is known to prefer dC as a substrate compared with dG (Sarup, Biochemistry 26:590-597 (1987)), and dC supplementation has been shown to inhibit dG salvage by DCK (Abt, J Clin Invest 132:el60852 (2022)) (Fig.

[0080] 3B). Using the TRACE assay, we found that while dC itself had a minimal effect on telomerase activity, the addition of dC to 500 pM dG led to shorter telomeres than 500 pM dG alone (Fig. 3D). These findings rigorously demonstrate that dG at high levels can be salvaged by DCK to form deoxyguanosine nucleotides that result in net telomerase activation, on a backdrop where dG is otherwise metabolized via guanine into guanosine ribonucleotides that cause telomerase inhibition.

[0081] Given that DCK is inefficient at phosphorylating dG, we next asked if increasing cellular DCK activity would enable dG-dependent telomerase activation at lower doses. We generated DCK overexpressing TERC-I- 293 T cells (Fig. 3C) for use in the TRACE assay and found that remarkably dG could now promote telomerase activation at doses as low as 20 pM, with strong activation at 80 pM. In contrast, telomerase activity in eGFP-expressing control cells remained inhibited by dG at 40 and 80 pM doses (Fig. 3E). To test whether these telomere length increases could be explained by altered cell cycle progression, we performed cell cycle analysis but found no significant differences in cells treated with 20 pM dG irrespective of DCK overexpression (Fig. 9D-9J). Furthermore, given concerns that high doses of dG might cause a DNA damage response (Diehl, Nat Cell Biol 24: 1252-1264 (2022)), we assessed phosphorylation of CHK1 at residue serine 345 (pCHKl S345) but found its levels to be undetectable after exposure to 20 pM dG in the presence or absence of DCK overexpression (Fig. 9K). Collectively, these results reveal that dG nucleotide salvage capacity is limiting for telomere elongation in a manner amenable to manipulation, and that dG metabolic pathway choice dictates its net effect on telomerase activity in human cells.

[0082] SAMHD1 restricts telomerase activation from salvaged dG nucleotides

[0083] Salvage of dG into nucleotides by DCK could be promoting telomerase activity at the mono-, di-, or triphosphate levels. The dNTPase SAMHD1 restricts cellular deoxyribonucleotide / / ' / phosphate levels including dGTP and is a negative regulator of telomere length (Fig. 3B). To determine if dG salvage acts at the nucleotide triphosphate level, we asked what impact SAMHD1 disruption would have on telomerase activation after dG supplementation in the TRACE assay. In contrast to the strong telomerase inhibition from 100 pM dG seen in control cells, we found that SAMHD1 deficient TERC-I- 293T cells (Fig. 9L) showed robust telomerase activation at the same dose, an effect that increased further using 500 pM dG (Fig. 3F). In contrast, when we applied guanosine or guanine to SAMHD1 deficient cells, we found that telomerase activity was inhibited to a similar degree compared to cells in which SAMHD1 was intact (Fig. 9M). These results suggest that the telomerase activation seen after dG salvage by DCK is not only mediated by dGTP, but potently restricted by SAMHD1. To verify that the dG dependent telomerase activation in the absence of SAMHD1 was occurring via DCK salvage, we tested the effect of inhibiting DCK and found that DI-87 strongly inhibited telomerase activation in SAMHD1 deficient cells after dG treatment (Fig. 3G). Similarly, we found that dC, which competes with dG as a DCK substrate, also restricted the telomerase activation from dG in SAMHD1 deficient cells (Fig. 3G). These results indicate that the dNTPase SAMHD1 restricts telomerase activation from dG nucleotide salvage downstream of DCK and provide further support for a model where net dGTP accumulation promotes telomerase activity.

[0084] Nucleoside salvage efficiency limits telomerase activity and telomere length

[0085] Given the strong activation of telomerase from dG when we overexpressed DCK, which is inefficient at phosphorylating dG, we wondered if reprogramming cellular metabolism to drive more efficient deoxynucleoside salvage could further promote telomerase activity. The Drosophila melanogaster deoxynucleoside kinase (Dm-dNK) has broad substrate specificity and eight-fold more activity on dG than DCK (Fig. 4A). Thus, we generated Dm-dNK expressing TERC-I- 293T cells (Fig. 4B) and applied the TRACE assay in the setting of dG supplementation. Surprisingly, we found Dm-dNK expressing cells showed higher baseline telomerase activity compared to control eGFP expressing cells without added dG. These results indicate that overexpressing Dm-dNK to reprogram cellular nucleoside salvage capacity is sufficient to promote telomerase activation even in the absence of exogenous nucleoside supplementation (Fig. 4C-4D). When we applied dG to Dm-dNK expressing cells we found a further dose dependent enhancement of telomerase activity up to 500 pM dG (Fig. 4D). In contrast to the striking telomerase activation from dG supplementation, treatment with guanosine or guanine continued to show inhibitory effects telomere synthesis, consistent with the specificity of Dm-dNK for dNs (Fig. 4E_4F). These results confirm that dG salvage into deoxyribonucleotides is limiting and stimulates telomerase activity in human cells.

[0086] We next asked whether overexpressing Dm-dNK could promote telomere lengthening in long term culture of human cells without telomerase overexpression. When we infected cells with Dm-dNK constructs and compared them with eGFP expressing control cells, we found Dm-dNK overexpression led to robust telomere lengthening in 293T cells over 25 days of culture (Fig. 4G and 41). Whereas a similar level of Dm-dNK expression (driven by a CMV promoter) was not well tolerated in K562 cells, use of a doxycycline-inducible Dm- dNK expression construct also showed telomere lengthening compared to controls (Fig. 4H- 41). Taken together, these findings indicate that deoxynucleoside salvage capacity actively constrains telomere synthesis in human cells and can be metabolically reprogrammed to increase telomerase activity and drive telomere lengthening.

[0087] Manipulating deoxynucleoside salvage drives telomere lengthening in cells from TBD patients

[0088] Telomere biology disorders (TBDs) are caused by hypomorphic mutations in the core telomerase components and other telomere maintenance genes. Based on our findings showing overexpression of Dm-dNK could promote telomere lengthening, we asked if the expression of Dm-dNK could overcome genetic defects in telomere synthesis and yield telomere lengthening in cells from TBD patients. To address this question, we utilized a panel of fibroblasts from TBD patients carrying pathogenic mutations in TERC, DKC1 which encodes a member of the telomerase holoenzyme, or TINF2 which encodes a component of the telomere shelterin complex. We first determined that ectopic expression of Dm-DNK robustly increased telomere length in normal human skin fibroblasts stably expressing TERT (Fig. 5A, Fig. 10A). Next, we overexpressed Dm-dNK in TBD patient fibroblasts expressing TERT and found striking increases in telomere length by thousands of base pairs after three weeks in culture (Fig. 5B and 5C). These data indicate that deoxynucleoside salvage capacity actively limits telomere length in human diploid cells, and that augmenting dNTP salvage overcomes disease-causing deficiencies in telomere maintenance genes and restores telomere length in cells from patients with TBDs.

[0089] Next, based on our findings showing that the pathway by which dG is metabolized influences telomerase activity, we hypothesized that shifting the balance towards deoxynucleoside salvage rather than catabolism using small molecules would drive telomere elongation in long term culture. To test this hypothesis in primary human cells, we treated normal fibroblasts stably expressing TERT with the PNP inhibitor ulodesine either alone or in combination with supplemented dG. We found that while treatment with ulodesine alone did not lead to detectable changes in telomere length, the combination of ulodesine with 100 pM dG led to robust telomere lengthening after 21 days in culture (Fig. 5D). We next asked if the combination of ulodesine and dG treatment could promote telomere lengthening in cells derived from patients with TBDs. Remarkably, we found that treatment with ulodesine and 100 pM dG for 21 days drove telomere lengthening across all mutant genotypes tested (Fig. 5E and 5F). Under these conditions, we found no difference in cell growth (measured as population doublings) or cell-cycle progression in two of the four patient fibroblasts, but a reduction in growth and prolongation of S-phase for the other two fibroblasts (Fig. 10B- 10M). None of the cultures showed increases in pCHKl (S345) after dG plus ulodesine, indicating a lack of DNA damage response induction (Fig. ION and 100). Together, these data indicate that the observed telomere length increases after dG and ulodesine treatment cannot be explained by changes in cell growth or S-phase progression, and do not trigger DNA damage responses. Collectively, these findings indicate that deoxynucleotide salvage limits telomere lengthening in human cells, and that genetic or small molecule-based reprogramming of deoxynucleoside salvage can promote telomere restoration in the setting of TBD-causing genetic defects.

[0090] Materials and Methods

[0091] Patient Material

[0092] Biological samples were procured. The patient with mutation of the TINF2 gene presented with dyskeratosis congenita and bone marrow failure in early childhood, consistent with the mutation leading to the p.T284P amino acid change in the ‘hot spot’ region of TINF2, a site where mutations are frequently associated with the TBD dyskeratosis congenita.

[0093] Cell culture

[0094] 293T cells (American Type Culture Collection, ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum, MEM Non-Essential Amino Acids (Gibco), L-glutamate (Coming), and penicillin / streptomycin (Corning) and subcultured using trypsin (Gibco). K562 cells (American Type Culture Collection, ATCC) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, MEM Non-Essential Amino Acids (Gibco), L-glutamate (Coming), and penicillin / streptomycin (Corning). Fibroblasts cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 15% fetal bovine serum, MEM Non- Essential Amino Acids (Gibco), L-glutamate (Corning), and penicillin / streptomycin (Corning) and subcultured using trypsin (Gibco). The TBD patient fibroblasts with the DKC1 del37L (GM01774 (Wong, Genes Dev. 20:2848-2858 (2006))) mutation were obtained from the Cori ell Cell Repository. The TBD patient fibroblasts with the DKC1 A353 V mutation were as described elsewhere (Paulsen, Nat Biomed Eng 1 :878-888 (2017)). The TBD patient fibroblasts with the TERC+I' 821 base pair deletion were as described elsewhere (Agarwal, Nature 464:292-296 (2010)). The TBD patient fibroblasts with the TIN2 p.T284P mutation were generated by culturing punch biopsies under glass coverslips until fibroblast outgrowths were apparent. All fibroblast lines were infected with pCW57.1 TERT lentivirus, selected in puromycin, and cultured in the presence of Ipg / ml doxycycline (Sigma- Aldrich) during experimental perturbations. Lentiviral transduction was performed via spinfection at 931 G for 2 hours in media supplemented with protamine sulfate (Sigma- Aldrich) at 10 pg / ml. 24 hours after removal of virus containing media, cells were selected using blasticidin (InvivoGen) at 10 pg / l for 5-10 days or puromycin (Sigma-Aldrich) at 1 pg / ml for 4-7 days.

[0095] Lentivirus production

[0096] 293T cells were transfected with psPAX2 and pMD2.G in addition to the appropriate transfer vector using Lipofectamine 2000 (Invitrogen) per the manufacturer’s instructions. Lentivirus containing media was harvested 48 and 72 hours after transfection, filtered using 0.45 pM filters (VWR International), and stored at -80°C until use.

[0097] TRACE Assay

[0098] On the evening prior to transfection, TERC-I- 293T cells were plated at 0.75* 106cells per ml of media into a six well plate. The following day, cell media was replaced approximately one hour prior to transfection. Transfection was performed as follows. For each well of a six well plate, 8 pl of Lipofectamine 2000 (Invitrogen) was mixed with 150 pl of Opti-MEM serum free media (Gibco) and incubated for 5 minutes. In a separate tube, 416ng pcDNA-3xHA-TERT and 2083 ng of either pBS-U3-TERC or pMAX-eGFP as indicated were mixed into 150 pl of Opti-MEM. These two solutions were then combined, mixed gently, and incubated for 5 minutes followed by dropwise addition to cells. 18 hours after transfection, cells were split into treatment conditions and cultured for 30 hours unless otherwise indicated. Cells were then harvested, and pellets were stored at -80°C until DNA isolation and terminal restriction fragment length analysis (described below).

[0099] Terminal Restriction Fragment Length Analysis

[0100] DNA was isolated from cell pellets using the Pure Link Genomic DNA Miniprep kit

[0101] (Invitrogen). 0.5-3 pg of DNA was digested with Rsal (NEB) and Hint! (NEB) for 2 hours at 37°C. DNA was then separated by electrophoresis on a 0.6% agarose gel followed by Southern blotting onto Hybond-N+ membrane (Amersham). Detection was performed using the TeloTAGGG Telomere Length assay Kit (Roche) using the provided probe at the recommended concentration or a probe complementary to the C-rich strand of the telomere synthesized using the CCCTAA-probe oligonucleotide (See Oligonucleotides, below). Where indicated, Southern blots were stripped using two washes with 0.2M NaOH and 0.1% sodium dodecyl sulfate (SDS) at 37°C for 15 minutes with shaking followed by detection as described above. Telomere length quantification was performed with the WALTER webtool (v2.0). TRACE assay experiments were quantified using ImageJ (Version 2.9.0 / 1.541) as follows: First, calculate the ratio (R) of signal above 5kb (largely newly synthesized telomere) to below 5kb (largely pre-existing telomere signal for TERC- / - 293T cells) for each sample. Then, to calculate background normalized ratios (Rb), for each sample expressing TERC, subtract R for eGFP expressing cells on the same blot (removing background signal contribution) from sample R. Then, to calculate the relative telomere synthesis for a given sample, divide the Rb of experimental samples by the untreated control sample on the same blot (normalizing relative to untreated). Statistical analysis was performed using GraphPad Prism (version 10.0.0).

[0102] RT-qPCR

[0103] Total cellular RNA was harvested from 293T and TERC- / - 293T cells using Trizol (Thermo Fisher) according to the manufacturer’s protocol. RNA was then DNase treated to remove genomic DNA with the Turbo DNase Kit according to the manufacturer’s protocol (Thermo Fisher). cDNA synthesis was achieved using Superscript III Reverse Transcriptase with random hexamer priming (Thermo Fisher). RT-qPCR was then conducted on cDNA using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) according to the manufacturer’s instructions on a Bio-Rad CFX96 real-time PCR machine. Relative gene expression was quantified using standard AACt methodology.

[0104] Cell cycle analysis by DAPI staining and flow cytometry

[0105] Approximately IxlO6cells per treatment condition were fixed in 70% (v / v) ethanol then stained for DNA content with DAPI followed by flow cytometry analysis on an LSR II analyzer (BD Biosciences) using BD FACSDiva version (8.0.2). Twenty thousand TERC-I- 293T cells and 10,000 patient fibroblast cells were analyzed per sample. Cells were gated on forward versus side scatter and cell cycle phases were first gated in a control sample for each comparison and applied to all samples as described in Fig. 9D-9F using FlowJo version 10.9.0.

[0106] Immunoblotting

[0107] Cells were lysed in RIPA buffer (Thermo Fisher) supplemented with complete protease inhibitor cocktail (Roche) and lysates were quantified using the Bio-Rad DC protein quantification assay according to the manufacturer’s protocol. For immunoblots assessing DNA damage markers (Fig. 9K, Fig. 10N-10O), 15 pg protein was mixed with 2X Laemmli buffer (Bio-Rad) and run on 4-20% tris-glycine SDS gel (Bio-Rad), followed by transfer to a poly vinylidene difluoride membrane. For all other immunoblots, 20 pg protein lysate was used. HGPRT was detected using a rabbit primary antibody from Protein Tech (17758-1-AP) at a 1 : 1000 dilution. TERT was detected using a rabbit primary antibody from Rockland (600-401-252) at a 1 : 1000 dilution. DCK was detected using a rabbit primary antibody from GeneTex (GTX102800) at a 1 : 1000 dilution. SAMHD1 was detected using a rabbit primary antibody from Abeam (ab67820) at a 1 :500 dilution. V5-tagged Dm-dNK was detected using an anti-V5 mouse primary antibody from Invitrogen (SV5-Pkl) at a 1 : 1000 dilution. pCHKl (S345) was detected using a rabbit primary antibody from Cell Signaling Technology at a 1 : 1000 dilution. Rabbit primary antibodies were detected using horseradish peroxidase- conjugated goat anti-rabbit IgG secondary antibody from Abeam (ab6721) at a 1 :5000 dilution. Mouse primary antibodies were detected using horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody from Invitrogen (31430) at a 1 :5000 dilution. Blots were imaged by chemiluminescence on a ChemiDoc Imaging system (Bio-Rad).

[0108] CRISPR / Cas9 gene editing

[0109] Gene editing was performed using the Neon Electroporation Kit (Invitrogen) by complexing 37 pmol Alt-R S.p. Cas9 Nuclease V3 (IDT) and 50 pmol chemically modified sgRNA(s) (Synthego) at room temperature for 20 minutes followed by addition of 200,000 cells suspended in 20 pl buffer R (Thermo Fisher Scientific) with Electroporation Enhancer (IDT). The cell mixture was electroporated using a Neon Transfection System (Thermo Fisher Scientific) using the following settings: HEK 293T, 1200V, 30ms, 1 pulse; K562 1150 V, 10ms, 3 pulses. After electroporation, cells were plated into normal media. Editing efficiency was evaluated by isolating genomic DNA from cells at least 3 days after electroporation followed by PCR amplifying the target locus. PCR products were visualized by electrophoresis on a 2% agarose gel.

[0110] Expression Construct Cloning

[0111] Constructs to express variants in TERT associated with TBDs were generated from the pCDNA-3xHA-hTERT vector using the Q5 site directed mutagenesis kit (NEB) and were verified by Sanger sequencing.

[0112] The DCK expression construct was cloned from 293 T cell cDNA into the pLX304 backbone by Gibson assembly using the NEB HiFi DNA assembly mix. The final construct expresses DCK with a C-terminal fusion to a V5 epitope tag which is present in the pLX304 backbone and the sequence was verified by Sanger sequencing. The cDNA was synthesized by isolating RNA from cells using TRIzol Reagent (Invitrogen), then treating RNA with TURBO DNA-free Kit (Invitrogen). Complementary DNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen) with oligo-dT priming (Invitrogen).

[0113] The PLX304-eGFP construct was generated by PCR amplifying the eGFP sequence from the pXPR Ol 1 vector using attB-flanked primers followed by Gateway cloning (Invitrogen) according to the manufacturers protocol into the pLX304 backbone. The final construct expresses eGFP with a C-terminal fusion to the V5 epitope tag which is present in the pLX304 backbone. The sequence was verified by Sanger sequencing

[0114] The CMV driven Dm-dNK expression construct pLX304 Dm-dNK V5 was cloned from the pOpen-dromedNK plasmid into the pLX304 backbone by Gibson assembly using the NEB HiFi DNA assembly mix. The final construct expresses Dm-dNK with a C-terminal fusion to a V5 epitope tag which is present in the pLX304 backbone. The sequence was verified by Sanger sequencing

[0115] The doxycycline inducible Dm-dNK expression construct was generated by isolating the insert from the pLX304 Dm-dNK V5 using SacI (NEB) and Sall (NEB) and ligating it into the pCW57.1 backbone which had been digested with Nhel (NEB) and Sall (NEB) using Quick Ligase (NEB) generating pCW57. 1 Dm-dNK V5. The sequence was verified by Sanger sequencing. The blasticidin resistant version of this vector used in Figure 4 was generated by digesting pCW57.1 Dm-dNK V5 with Notl and Spel and ligating the Dm-dNK containing fragment into the pCW57-MCSl-P2A-MCS2 (Blast) vector which had also been digested with Notl and Spel using Quick Ligase (NEB).

[0116] The doxycycline inducible eGFP expression construct was generated by PCR amplifying the eGFP sequence from the pXPR Ol 1 vector using attB-flanked primers followed by Gateway cloning (Invitrogen) according to the manufacturers protocol into the pCW57.1 backbone. The sequence was verified by Sanger sequencing. The blasticidin resistant version of this vector used in Figure 4 was generated by digesting pCW57.1 eGFP with Notl (NEB) and Spel (NEB) and ligating the Dm-dNK containing fragment into the pCW57-MCS l-P2A-MCS2 (Blast) vector which had also been digested with Notl (NEB) and Spel (NEB) using Quick ligase (NEB).

[0117] All PCR reactions required for molecular cloning were performed using the Q5 polymerase (NEB).

[0118] Plasmid Sources: pXPR_011 (plasmid 59702; Addgene) pLX304 (plasmid 25890; Addgene) psPAX2 (plasmid 12260; Addgene) pMD2.G (plasmid 12259; Addgene) pBS U3-hTR-5OO (plasmid 28170; Addgene). pCDNA-3xHA-hTERT (plasmid 51637; Addgene). pmaxGFP (Lonza) pHIV7 / SF-U3-TER-500 (Agarwal, Nature 464:292-296 (2010)) pHIV7 / SF-U3-TER-500 G319A (Boyraz, Blood 128:2089-2092 (2016)) pcw57.1 TERT Puro pOpen-dromedNK (plasmid 165579; Addgene) pCW57.1 (plasmid 41393; Addgene) pCW57-MCSl-P2A-MCS2 (Blast) (plasmid 80921; Addgene)

[0119] Nucleosides and other Small Molecules Oligonucleotides sgRNAs were purchased from Synthego, all other oligonucleotides were synthesized by IDT.

[0120]

[0121] Note: / 5Phos / represents 5’ phosphorylation. +N represents locked nucleic acid.

[0122] OTHER EMBODIMENTS

[0123] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for increasing telomere length, wherein said method comprises: administering an agent that can increase telomere length to a cell in need of increased telomere length.

2. The method of claim 1, wherein said cell is a mammalian cell.

3. The method of any one of claims 1-2, wherein said cell is a human cell.

4. The method of any one of claims 1-3, wherein said agent that can increase telomere length is selected from the group consisting of deoxy thy mi dine (dT), a dNK polypeptide, a SAMHD1 inhibitor, a purine nucleoside phosphorylase (PNP) inhibitor, and a combination of the PNP inhibitor and deoxyguanosine (dG).

5. The method of claim 4, wherein the PNP inhibitor is ulodesine.

6. A method for treating a telomere biology disorder (TBD), wherein said method comprises: administering an agent that can increase telomere length to a mammal having said TBD, wherein said agent increases telomere length in cells within the mammal.

7. The method of claim 6, wherein said mammal is a human.

8. The method of any one of claims 6-7, wherein said TBD is selected from the group consisting of dyskeratosis congenita, Hoyeraal Hreidarsson syndrome, Revesz syndrome, Coats Plus syndrome, aplastic anemia with short telomeres, myelodysplastic syndrome with short telomeres, interstitial lung disease with short telomeres, liver cirrhosis with short telomeres, and a telomere biology disorder (TBD) caused by a mutation in DKC1, TERC.TIN2, TERT, NOP 10, NHP2, WRAP53 / TCAB1, TINF2, CTC1, RTEL1, ACD, PARN, NAF1, STN1, ZCCHC8, POTI, RPA1, DCLRE1B, or TYMS.

9. The method of any one of claims 6-8, wherein said agent that can increase telomere length is selected from the group consisting of deoxythymidine (dT), a dNK polypeptide, a SAMHD1 inhibitor, a purine nucleoside phosphorylase (PNP) inhibitor, and a combination of the PNP inhibitor and deoxyguanosine (dG).

10. The method of claim 9, wherein the PNP inhibitor is ulodesine.

11. A method for decreasing telomere length, wherein said method comprises: administering an agent that can decrease telomere length to a cell in need of decreased telomere length.

12. The method of claim 11, wherein said cell is a mammalian cell.

13. The method of any one of claims 11-12, wherein said cell is a human cell.

14. The method of any one of claims 11-13, wherein said agent that can decrease telomere length is selected from the group consisting of guanine, guanosine, and 2’,5’- dideoxy guanosine (2,5ddG).

15. A method for treating a mammal having a disease or disorder associated with increased telomere synthesis and / or increased telomerase activity, wherein said method comprises: administering an agent that can decrease telomere length to said mammal.

16. The method of claim 15, wherein said mammal is a human.

17. The method of any one of claims 15-16, wherein said agent that can decrease telomere length is selected from the group consisting of guanine, guanosine, and 2’,5’- di deoxy guanosine (2,5ddG).

18. The method of any one of claims 15-17, wherein said disease or disorder is a cancer.

19. A method for detecting telomerase activity in a cell, said method comprising: contacting a cell comprising (1) short telomeres and (2) exogenous overexpression of a telomerase with a candidate compound, and identifying said compound as an agent that can increase telomere length when telomerase-mediated telomere lengthening is detected.

20. The method of claim 19, wherein said cell comprises (a) nucleic acid that encodes a telomerase RNA component (TERC) and (b) nucleic acid that can encode a telomerase reverse transcriptase (TERT).

21. The method of any one of claims 19-20, wherein said contacting is performed in vitro.

22. The method of any one of claims 19-21, wherein telomerase-mediated telomere lengthening is detected using terminal restriction fragment (TRF) Southern blotting.

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