{"id":1062252,"date":"2026-07-01T17:26:27","date_gmt":"2026-07-01T17:26:27","guid":{"rendered":"https:\/\/www.europesays.com\/uk\/1062252\/"},"modified":"2026-07-01T17:26:27","modified_gmt":"2026-07-01T17:26:27","slug":"therapeutic-inhibition-of-telomeric-dna-damage-response-rescues-hematopoietic-dysfunction-driven-by-telomere-shortening-and-aging","status":"publish","type":"post","link":"https:\/\/www.europesays.com\/uk\/1062252\/","title":{"rendered":"Therapeutic inhibition of telomeric DNA damage response rescues hematopoietic dysfunction driven by telomere shortening and aging"},"content":{"rendered":"

tASO effectively inhibits tDDR in the hematopoietic organs of Terc
\n \u2212\/\u2212 mice<\/p>\n

To determine whether tDDR was activated in the hematopoietic organs of Terc\u2212\/\u2212 mice and could be effectively inhibited by tASO, we treated young (2- to 3-month-old) G3 Terc\u2212\/\u2212 mice with intraperitoneal injections of tASO targeting either G-rich or C-rich telomeric RNA strands (anti-TeloG or anti-TeloC ASO, respectively) or a control ASO against an unrelated sequence, twice per week for 4 weeks. Two months after tASO treatment, BM cells and splenocytes were collected and analyzed (Fig. 1a<\/a>). tASO efficiently reached the hematopoietic compartment, as demonstrated by fluorescence in situ hybridization (FISH) on BM cells from WT mice treated with the same dosing regimen analyzed 4\u2009h postadministration (Extended Data Fig. 1a<\/a>).<\/p>\n

Fig. 1: Telomeric DDR activation is effectively inhibited by tASO treatment in the hematopoietic organs of Terc\u2212\/\u2212 mice.<\/b>\"Fig.<\/p>\n

a<\/b>, Experimental design. Two- to three-month-old G3 Terc\u2212\/\u2212 mice received intraperitoneal injection of the indicated ASO (15\u2009mg\u2009kg\u22121, twice weekly for four weeks). BM and spleen were collected 2 months after treatment, at 5 months of age. b<\/b>,c<\/b>, Percentages of \u03b3H2AXhigh (left), pKAP1high (middle) and \u03b3H2AXhighpKAP1high (right) cells in BM (b<\/b>) and spleen (c<\/b>) from 5-month-old WT, Terc\u2212\/\u2212 control and Terc\u2212\/\u2212 tASO-treated mice, analyzed by flow cytometry 2 months posttreatment. d<\/b>,e<\/b>, TIFs, defined as \u03b3H2AX-positive DNA damage foci colocalizing with telomeric DNA (d<\/b>), and quantification of mean telomeric signal intensity per cell (e<\/b>) in BM cells from the same experimental groups, assessed by immunoFISH 2 months posttreatment. In d<\/b>, at least 50 nuclei per mouse section were scored. In b<\/b>\u2013e<\/b>, Terc\u2212\/\u2212 controls included untreated mice (empty circles) and control ASO-treated mice (filled circles), whereas Terc\u2212\/\u2212 tASO-treated mice included anti-TeloG-treated mice (empty squares) and anti-TeloC-treated mice (filled squares). Sample sizes were as follows: WT, n\u2009=\u200910 (left and right) and n\u2009=\u200911 (middle); Terc\u2212\/\u2212 controls, n\u2009=\u200914; Terc\u2212\/\u2212 tASO-treated, n\u2009=\u200915 (b<\/b>); WT, n\u2009=\u200911; Terc\u2212\/\u2212 controls, n\u2009=\u200918; Terc\u2212\/\u2212 tASO-treated, n\u2009=\u200919 (c<\/b>); WT, n\u2009=\u20093; Terc\u2212\/\u2212 controls, n\u2009=\u20095; Terc\u2212\/\u2212 tASO-treated, n\u2009=\u20095 (d<\/b>); WT, n\u2009=\u20093; Terc\u2212\/\u2212 controls, n\u2009=\u20096; Terc\u2212\/\u2212 tASO-treated, n\u2009=\u20096 (e<\/b>). Each point represents a single mouse. Data are shown as mean\u2009\u00b1\u2009s.e.m. Statistical significance was determined by one-way ANOVA followed by Tukey\u2019s multiple comparisons test (a<\/b>\u2013e<\/b>).i.p., intraperitoneal. Illustrations in a<\/b> created in BioRender; Rossiello, F. https:\/\/biorender.com\/xjfjttr<\/a>, https:\/\/biorender.com\/twz85eu<\/a> and https:\/\/biorender.com\/etc991g<\/a> (2026).<\/p>\n

Source data<\/a><\/p>\n

\u03b3H2AX and pKAP1, two distinct markers of DDR activation, were analyzed by flow cytometry. Cell populations were defined based on the frequency of \u03b3H2AXhigh, pKAP1high and \u03b3H2AXhighpKAP1high double-positive cells, after validation of antibody performance using 4.5-Gy-irradiated mice as positive controls 1\u2009h postirradiation (Extended Data Fig. 1b\u2013d<\/a>). Terc\u2212\/\u2212 control mice exhibited increased DDR activation compared to age-matched WT mice, and such activation was significantly reduced in tASO-treated Terc\u2212\/\u2212 mice, in BM (Fig. 1b<\/a>) and splenocytes (Fig. 1c<\/a>). Here and in subsequent experiments, unless otherwise specified, untreated and control ASO-treated Terc\u2212\/\u2212 mice, as well as anti-TeloG- or anti-TeloC ASO-treated mice, were pooled into the Terc\u2212\/\u2212 controls and tASO-treated groups, respectively, as no significant biological or statistical differences were observed within each pair. Western blot analyses overall confirmed a reduction in \u03b3H2AX levels in both BM (Extended Data Fig. 1e<\/a>) and spleen (Extended Data Fig. 1f<\/a>) extracts from tASO-treated animals compared to control ASO-treated ones. Consistently, quantification of \u03b3H2AX foci detected by immunofluorescence confirmed an elevated DNA damage burden in Terc\u2212\/\u2212 BM cells relative to WT mice, and a significant reduction following tASO treatment (Extended Data Fig. 1g<\/a>). In addition, to investigate ATM and ATR involvement, we assessed pATM and pCHK1 in BM cells (Extended Data Fig. 1g<\/a>) and observed that pATM levels were increased in Terc\u2212\/\u2212 mice and significantly reduced upon tASO treatment, whereas ATR activity appeared to be minimal and unaffected by tASO treatment.<\/p>\n

To determine whether tASO specifically modulated DDR at telomeres, we quantified tDDR using immunoFISH, combining immunofluorescence detection of \u03b3H2AX with FISH with telomeric probes to identify telomere-dysfunction-induced foci (TIFs). Terc\u2212\/\u2212 control mice exhibited a significant increase in TIF frequency compared to WT mice, reflecting elevated tDDR (Fig. 1d<\/a> and Extended Data Fig. 1h<\/a>). Notably, tASO treatment markedly reduced TIF numbers in Terc\u2212\/\u2212 mice, indicating that tASO effectively targeted tDDR. To confirm the specificity of tASO action toward tDDR only, we examined the cellular response to random DNA damage induced by ionizing radiation. TASO-treated mice displayed \u03b3H2AX and pKAP1 levels in hematopoietic tissues that were indistinguishable from those of untreated or control ASO-treated mice (Extended Data Fig. 1i,j<\/a>), consistent with our previous work in cultured cells7<\/a> demonstrating tASO specificity for tDDR only.<\/p>\n

Finally, to determine whether tASO-mediated suppression of tDDR affected telomere length, we quantified telomeric fluorescence intensity by telomere-FISH. As expected, Terc\u2212\/\u2212 mice showed a significant reduction in telomeric signal compared to WT controls, consistent with telomere shortening; no differences in telomeric fluorescence intensity were detected between control and tASO-treated Terc\u2212\/\u2212 mice (Fig. 1e<\/a>), indicating that tASO inhibits tDDR independently of telomere length modulation. Together, these results demonstrate that tASO selectively attenuates tDDR without impairing global DDR activation or affecting telomere length.<\/p>\n

Given the complex cell composition of hematopoietic organs, we tested whether the observed increase in DDR and its inhibition by tASO varied among different cell types. We used multiparametric flow cytometry to analyze composition and quantify DDR activation in different cell subsets in BM and spleen; the flow cytometric strategy is shown in Extended Data Fig. 2a,b<\/a>. WT, control ASO- and tASO-treated Terc\u2212\/\u2212 mice did not differ with respect to percentages of total (B220+), pre- (B220+IgM\u2212IgD\u2212), immature (B220+IgM+IgD\u2212) and recirculating (B220+IgM+IgD+) B cells in the BM (Extended Data Fig. 2c<\/a>), or those of total (B220+CD19+), transitional (B220+CD19+IgM+IgD\u2212) and mature (B220+CD19+IgM+IgD+) B cells in the spleen (Extended Data Fig. 2d<\/a>). Similarly, no abnormalities were observed in the myeloid cell compartment, with comparable percentages among the three groups of myeloid cells (CD11b+), monocytes (CD11b+Ly-6C+) and granulocytes (CD11b+Ly-6G+) in the BM (Extended Data Fig. 2c<\/a>), and of myeloid cells (CD11b+), monocytes (CD11b+Ly-6C+), granulocytes (CD11b+Ly-6G+) and dendritic cells (CD11b+CD11c+) in the spleen (Extended Data Fig. 2d<\/a>). Overall, these results indicate that immune cell population distributions in these settings remained unchanged across conditions. Analysis of DDR in the same cell populations revealed a consistent pattern, with elevated DDR in Terc\u2212\/\u2212 control mice and DDR reduction in tASO-treated Terc\u2212\/\u2212 mice. The most affected populations were pre-B cells, immature B cells, and total myeloid cells and monocytes in the BM (Extended Data Fig. 3a<\/a>), as well as total B, transitional B, mature B and dendritic cells in the spleen (Extended Data Fig. 3b<\/a>).<\/p>\n

These results demonstrate that tDDR is effectively suppressed by tASO treatment in different cell types in both main hematopoietic organs of Terc\u2212\/\u2212 mice.<\/p>\n

tDDR inhibition by tASO treatment leads to reduced senescence burden and diminished inflammation in hematopoietic organs of Terc
\n \u2212\/\u2212 mice<\/p>\n

Persistent tDDR signaling is a key driver of cellular senescence, a process that contributes to aging and functional decline across multiple tissues, including the hematopoietic system4<\/a>,32<\/a>,33<\/a>. p16INK4a, encoded by the Cdkn2A gene, is a cyclin-dependent kinase inhibitor that plays crucial roles in induction and maintenance of cellular senescence by blocking cell cycle progression and is a widely accepted cellular senescence marker. We therefore tested the impact of tDDR inhibition on cellular senescence by assessing the fraction of Cdkn2A\/p16high cells by flow cytometry in the hematopoietic organs of Terc\u2212\/\u2212 mice. A gating strategy was established in young and aged WT mice and showed the expected increase in Cdkn2A\/p16high cells with age (Extended Data Fig. 4a,b<\/a>). We observed a significant increase in the percentage of Cdkn2A\/p16high cells in BM cells (Fig. 2a<\/a>) and splenocytes (Fig. 2b<\/a>) of Terc\u2212\/\u2212 controls compared to their WT counterparts. Importantly, tASO-treated Terc\u2212\/\u2212 mice showed a significant reduction in Cdkn2A\/p16high cells in both tissues compared to Terc\u2212\/\u2212 controls. Analysis of the different cell subsets revealed a general trend across subtypes, with the most significant changes observed in myeloid cells in the BM (Fig. 2c<\/a>) and B cells in the spleen (Fig. 2d<\/a>).<\/p>\n

Fig. 2: tDDR inhibition decreases senescence burden in the hematopoietic organs of Terc\u2212\/\u2212 mice and lessens inflammation.<\/b>\"Fig.<\/p>\n

a<\/b>,b<\/b>, Percentages of Cdkn2A\/p16high cells in BM (a<\/b>) and spleen (b<\/b>) from 5-month-old WT, Terc\u2212\/\u2212 control and Terc\u2212\/\u2212 tASO-treated mice, analyzed by flow cytometry 2 months posttreatment. c<\/b>,d<\/b>, Percentages of Cdkn2A\/p16high cells within the indicated BM (c<\/b>) and spleen (d<\/b>) cell subsets from the same experimental groups, analyzed by flow cytometry 2 months posttreatment. e<\/b>, Concentrations (pg\u2009ml\u22121) of the indicated cytokines in plasma and BM fluid from the same experimental groups, quantified by multiplex immunoassay 2 months posttreatment. In a<\/b>\u2013e<\/b>, Terc\u2212\/\u2212 controls included untreated mice (empty circles), and control ASO-treated mice (filled circles), whereas Terc\u2212\/\u2212 tASO-treated mice included anti-TeloG-treated mice (empty squares) and anti-TeloC-treated mice (filled squares). Sample sizes were as follows: WT, n\u2009=\u200910; Terc\u2212\/\u2212 controls, n\u2009=\u200914; Terc\u2212\/\u2212 tASO-treated, n\u2009=\u200915 (a<\/b>); WT, n\u2009=\u200910; Terc\u2212\/\u2212 controls, n\u2009=\u200918; Terc\u2212\/\u2212 tASO-treated, n\u2009=\u200918 (b<\/b>); BM subsets (left to right): WT, n\u2009=\u200910, 10, 11, 11, 10, 10, 11; Terc\u2212\/\u2212 controls, n\u2009=\u200913, 13, 14, 14, 13, 13, 14; Terc\u2212\/\u2212 tASO-treated, n\u2009=\u200915 for all subsets (c<\/b>); spleen subsets (left to right): WT, n\u2009=\u200911, 11, 11, 11, 11, 10, 11; Terc\u2212\/\u2212 controls, n\u2009=\u200918, 18, 18, 18, 18, 17, 18; Terc\u2212\/\u2212 tASO-treated, n\u2009=\u200919, 19, 19, 18, 18, 18, 19 (d<\/b>); WT, n\u2009=\u20094 (plasma) and n\u2009=\u20095 (BM fluid); Terc\u2212\/\u2212 controls, n\u2009=\u20097 (IL-3 plasma), n\u2009=\u20098 (IL-6, IL-17A, IFN\u03b3, TNF\u03b1 plasma), n\u2009=\u200910 (IL-3, IL-6, IFN\u03b3, TNF\u03b1 BM fluid) and n\u2009=\u20099 (IL-17A BM fluid); Terc\u2212\/\u2212 tASO-treated, n\u2009=\u20096 (IL-3, IL-17A, IFN\u03b3, TNF\u03b1 plasma), n\u2009=\u20097 (IL-6 plasma), n\u2009=\u200910 (IL-3, IL-6, IL-17A, TNF\u03b1 BM fluid) and n\u2009=\u20099 (IFN\u03b3 BM fluid) (e<\/b>). Each point represents a single mouse. Data are shown as mean\u2009\u00b1\u2009s.e.m. Statistical significance was determined by one-way ANOVA followed by Tukey\u2019s multiple comparisons test (a<\/b> and b<\/b>) or two-way ANOVA followed by Tukey\u2019s multiple comparisons test (c<\/b>\u2013e<\/b>).<\/p>\n

Source data<\/a><\/p>\n

As senescent cells secrete proinflammatory factors that contribute to tissue dysfunction, and persistent inflammation can reinforce senescence, creating a detrimental feedback loop34<\/a>, we analyzed cytokine levels in the plasma and BM fluid of WT, Terc\u2212\/\u2212 control and tASO-treated Terc\u2212\/\u2212 mice (Fig. 2e<\/a>). Among the cytokines examined, IL-3, IL-6, IFN\u03b3 and TNF\u03b1 plasma levels were low among the groups tested, with a trend toward an increase in Terc\u2212\/\u2212 controls compared to WT mice and a significant reduction in the BM fluid of tASO-treated Terc\u2212\/\u2212 mice. IL-17A, a proinflammatory cytokine that has been implicated in BM myelopoiesis and age-related inflammation35<\/a>,36<\/a>, was instead significantly elevated in the plasma of Terc\u2212\/\u2212 controls compared to WT mice and significantly reduced upon tASO treatment.<\/p>\n

These results indicate that tDDR inhibition by tASO treatment leads to reduced senescence burden and attenuation of some associated proinflammatory cytokines.<\/p>\n

tDDR inhibition contrasts progressive immune system dysfunction of older Terc
\n \u2212\/\u2212 mice<\/p>\n

Telomerase-deficient mice recapitulate features of hematopoietic aging, including an altered immune cell composition with lymphoid-to-myeloid skewing27<\/a>,28<\/a>,29<\/a>,30<\/a>,31<\/a>. To complement our flow-cytometry-based immunophenotypic analyses, we performed histological analyses of BM and spleen (Extended Data Fig. 5a<\/a>) in young (5-month-old) Terc\u2212\/\u2212 mice and age-matched WT counterparts. In the BM of Terc\u2212\/\u2212 mice (Extended Data Fig. 5b<\/a>), erythroid colonies showed a significant contraction, whereas segmented myeloid cells expanded slightly, with a more noticeable increase in immature myeloid cell expansion. Terc\u2212\/\u2212 mice exhibited marked pleiomorphism in megakaryocytes, showing considerable variability in shape and size, a feature suggestive of perturbed megakaryopoietic homeostasis. We observed no significant increase in megakaryocyte clustering, a sign of megakaryocytic hyperplasia, or in the occurrence of erythrophagocytic figures, a feature of ineffective hematopoiesis, in Terc\u2212\/\u2212 hosts. When these parameters were summed into an overall BM pathology score, a significant difference between WT and Terc\u2212\/\u2212 mice clearly emerged (Extended Data Fig. 5c<\/a>). For the parameters analyzed in the spleen (Extended Data Fig. 5d,e<\/a>), we did not find significant differences between the two groups; this is consistent with the secondary role of the spleen in hematopoiesis relative to the BM and suggests that the observed BM abnormalities may have been early or moderate enough not to require compensatory mechanisms involving the spleen. Taken together, these findings show that histopathological examination can identify aspects of hematopoietic pathology that can be unappreciated by flow-cytometry-based analyses.<\/p>\n

Next, to determine whether the observed pathology would develop over time and whether tDDR inhibition by tASO would alleviate age-associated hematopoietic abnormalities, we extended our studies to older Terc\u2212\/\u2212 mice treated as in Fig. 1a<\/a> and sacrificed at 12 months of age (Fig. 3a<\/a>). Quantitative analyses of hematoxylin and eosin (H&E)-stained BM and spleen sections (Fig. 3b<\/a>) revealed more severe pathological alterations in adult Terc\u2212\/\u2212 mice compared to age-matched WT controls. Specifically, adult Terc\u2212\/\u2212 mice displayed a significant reduction in erythroid colonies in their BM, accompanied by expansions of both segmented and immature myeloid populations, increased clustering and pleiomorphism of megakaryocytes, indicative of altered platelet turnover, as well as the presence of erythro-hemophagocytosis, suggesting an increase in the fraction of ineffective hematopoiesis. All these alterations were significantly ameliorated by tDDR inhibition (Fig. 3c<\/a>). Similarly, the spleen of adult Terc\u2212\/\u2212 mice exhibited structural and functional abnormalities, including alteration of the ratio between white pulp and red pulp, indicating a contraction of lymphoid tissue secondary to increased red pulp hyperplasia, an increase in myeloid and erythroid precursors consistent with extramedullary hematopoiesis increase, megakaryocyte hyperplasia and clustering, and the presence of hemosiderin-laden macrophages, suggestive of increased hemocatheresis (Fig. 3d<\/a>). tDDR inhibition by tASO treatment effectively reversed both white pulp effacement and red pulp hyperplasia, with a trend toward reduced levels of myeloid\/erythroid precursors and hemosiderin-laden macrophages and minimal impact on megakaryocyte alterations. Overall, the total pathological score, calculated as the sum of the above parameters, revealed a clear and pronounced disease phenotype in Terc\u2212\/\u2212 control mice. tDDR inhibition via tASO treatment effectively reduced the severity of pathology in both BM (Fig. 3e<\/a>) and spleen (Fig. 3f<\/a>).<\/p>\n

Fig. 3: tDDR inhibition promotes tissue homeostasis in the BM and spleen of adult Terc\u2212\/\u2212 mice.<\/b>\"Fig.<\/p>\n

a<\/b>, Experimental design. Two- to three-month-old G3 Terc\u2212\/\u2212 mice received intraperitoneal injection of the indicated ASO (15\u2009mg\u2009kg\u22121, twice weekly for 4 weeks). BM and spleen were collected 9 months after treatment, at 12 months of age. b<\/b>, Representative H&E-stained sections of BM and spleen from 12-month-old WT, Terc\u2212\/\u2212 control and Terc\u2212\/\u2212 tASO-treated mice, analyzed 9 months posttreatment. Data are representative of two independent cohorts of mice with similar results. Original magnification: \u00d7200 (BM) and \u00d7100 (spleen). Scale bars: 146\u2009\u00b5m (BM) and 300\u2009\u00b5m (spleen). c<\/b>,d<\/b>, Histopathological parameters quantified from H&E sections in BM (c<\/b>) and spleen (d<\/b>) from the same experimental groups. e<\/b>,f<\/b>, Total pathological scores for BM (e<\/b>) and spleen (f<\/b>), calculated as the sum of the parameters shown in c<\/b> and d<\/b>, respectively. In c<\/b>\u2013f<\/b>, Terc\u2212\/\u2212 controls included untreated mice (empty circles) and control ASO-treated mice (filled circles), whereas Terc\u2212\/\u2212 tASO-treated mice included anti-TeloG-treated mice (empty squares) and anti-TeloC-treated mice (filled squares). Sample sizes for c<\/b>\u2013f<\/b>: WT, n\u2009=\u20095; Terc\u2212\/\u2212 controls, n\u2009=\u200915; Terc\u2212\/\u2212 tASO-treated, n\u2009=\u200912. Each point represents a single mouse. Data are shown as mean\u2009\u00b1\u2009s.e.m. Statistical significance was determined by one-way ANOVA followed by Tukey\u2019s multiple comparisons test (c<\/b>\u2013f<\/b>). G3, third generation. Illustrations in a<\/b> created in BioRender; Rossiello, F. https:\/\/biorender.com\/etc991g<\/a>, https:\/\/biorender.com\/xjfjttr<\/a> and https:\/\/biorender.com\/twz85eu<\/a> (2026).<\/p>\n

Source data<\/a><\/p>\n

To validate our findings and further investigate the abnormalities in BM and spleen of Terc\u2212\/\u2212 mice, we performed immunohistochemical (IHC) staining on the hematopoietic organs of the same mice analyzed by histology. IHC staining for PAX5, a pan-B cell marker and key transcription factor in B cell commitment and development, revealed reduced percentages of B cells in both BM and spleen of Terc\u2212\/\u2212 mice. Notably, tASO treatment restored B cell levels to those observed in WT mice (Fig. 4a<\/a>). Macrophage marker IBA1 showed no significant changes between WT and Terc\u2212\/\u2212 mice BM; however, macrophage levels were elevated in the spleen of Terc\u2212\/\u2212 controls and were reduced by tASO treatment in both BM and spleen (Fig. 4b<\/a>). Similarly, staining for granulocytic myeloid marker MPO demonstrated an increased percentage of granulocytic cells in both BM and spleen of Terc\u2212\/\u2212 mice, which was effectively restored to WT levels upon tASO treatment (Fig. 4c<\/a>). BM and spleen of Terc\u2212\/\u2212 mice also showed a significant increase in CD41+ cells, representing the megakaryocyte and platelet compartment, with tDDR inhibition effectively reducing these cell levels in both organs (Fig. 4d<\/a>), whereas the percentage of erythroid cells (TER119+), which was reduced in the BM and elevated in the spleen of Terc\u2212\/\u2212 mice, consistent with extramedullary hematopoiesis, was restored to normal levels following tDDR inhibition (Fig. 4e<\/a>).<\/p>\n

Fig. 4: tDDR inhibition preserves immune cell composition in adult Terc\u2212\/\u2212 mice and enhances early humoral immune responses.<\/b>\"Fig.<\/p>\n

a<\/b>\u2013e<\/b>, Percentages of PAX5+ (a<\/b>), IBA1+ (b<\/b>), MPO+ (c<\/b>), CD41+ (d<\/b>) and TER119+ (e<\/b>) cells in BM (left) and spleen (right) of 12-month-old WT, Terc\u2212\/\u2212 control and Terc\u2212\/\u2212 tASO-treated mice, quantified by IHC staining 9 months posttreatment. f<\/b>, Experimental design. Two- to three-month-old G3 Terc\u2212\/\u2212 mice received intraperitoneal injections of the indicated ASO (15\u2009mg\u2009kg\u22121, twice weekly for 4 weeks). Two months after treatment, mice were injected intramuscularly with 1\u2009\u00b5g of SARS-CoV-2 mRNA vaccine and sacrificed 3 weeks later. g<\/b>, Serum SARS-CoV-2 spike-specific IgG levels measured 3 weeks after vaccination in Terc\u2212\/\u2212 controls and Terc\u2212\/\u2212 tASO-treated mice, expressed as percentages relative to Terc\u2212\/\u2212 controls. In a<\/b>\u2013e<\/b> and g<\/b> Terc\u2212\/\u2212 controls included untreated mice (empty circles) and control ASO-treated mice (filled circles), whereas Terc\u2212\/\u2212 tASO-treated mice included anti-TeloG-treated mice (empty squares) and anti-TeloC-treated mice (filled squares). Sample sizes were as follows: WT, n\u2009=\u20096 (BM) and n\u2009=\u20095 (spleen); Terc\u2212\/\u2212 controls, n\u2009=\u200917 (BM) and n\u2009=\u200914 (spleen); Terc\u2212\/\u2212 tASO-treated, n\u2009=\u200919 (BM) and n\u2009=\u200912 (spleen) (a<\/b>); WT, n\u2009=\u20096 (BM) and n\u2009=\u20095 (spleen); Terc\u2212\/\u2212 controls, n\u2009=\u200918 (BM) and n\u2009=\u200915 (spleen); Terc\u2212\/\u2212 tASO-treated, n\u2009=\u200918 (BM) and n\u2009=\u200912 (spleen) (b<\/b>); WT, n\u2009=\u20096 (BM) and n\u2009=\u20095 (spleen); Terc\u2212\/\u2212 controls, n\u2009=\u200918 (BM) and n\u2009=\u200915 (spleen); Terc\u2212\/\u2212 tASO-treated, n\u2009=\u200919 (BM) and n\u2009=\u200912 (spleen) (c<\/b> and d<\/b>); WT, n\u2009=\u20096 (BM) and n\u2009=\u20096 (spleen); Terc\u2212\/\u2212 controls, n\u2009=\u200918 (BM) and n\u2009=\u200916 (spleen); Terc\u2212\/\u2212 tASO-treated, n\u2009=\u200919 (BM) and n\u2009=\u200912 (spleen) (e<\/b>); Terc\u2212\/\u2212 controls, n\u2009=\u200917; Terc\u2212\/\u2212 tASO-treated, n\u2009=\u20099 (g<\/b>). Each point represents a single mouse. Data are shown as mean\u2009\u00b1\u2009s.e.m. Statistical significance was determined by one-way ANOVA followed by Tukey\u2019s multiple comparisons test (a<\/b>\u2013e<\/b>) and two-tailed Student\u2019s t-test (g<\/b>).i.m., intramuscular. Illustrations in e<\/b> created in BioRender; Rossiello, F. https:\/\/biorender.com\/etc991g<\/a> and https:\/\/biorender.com\/agq2d27<\/a> (2026).<\/p>\n

Source data<\/a><\/p>\n

Importantly, when we evaluated the safety of long-term systemic tASO treatment in both young and adult cohorts, tASO-treated Terc\u2212\/\u2212 mice displayed significantly improved weight trajectories (Extended Data Fig. 6a<\/a>), indicating no systemic toxicity and overall health improvement. Spleen weight was comparable between control and tASO-treated groups, indicating the absence of splenomegaly or tissue atrophy (Extended Data Fig. 6b<\/a>). Hematological analyses performed at the time of sacrifice showed no evidence of hematopoietic alterations, with peripheral blood parameters including white blood cells, red blood cells (RBCs), hemoglobin and platelets remaining within the same ranges as those of Terc\u2212\/\u2212 controls (Extended Data Fig. 6c<\/a>). In addition, peripheral blood smear analysis revealed an increased proportion of lymphocytes and a reduced proportion of granulocytes in tASO-treated mice compared to Terc\u2212\/\u2212 controls (Extended Data Fig. 6d<\/a>), consistent with the normalization of immune composition observed by histological and IHC analyses. Finally, longitudinal safety was assessed by observing H&E-stained histopathology sections of hematopoietic tissues and performing necroscopy in all treated and control animals at 9 months posttreatment (Extended Data Fig. 6e<\/a>). Across all conditions examined, no visible tumor formation or signs of overt hematological malignancy were detected. Together, these results indicate that systemic tDDR inhibition by tASO is safe in vivo over both intermediate and long-term time frames.<\/p>\n

Next, we examined telomere length and tDDR to determine whether the long-term tissue benefits induced by tASO were maintained independently of telomere length changes. Relative telomere length measured by quantitative PCR (qPCR) in BM cells and splenocytes revealed the expected telomere shortening in Terc\u2212\/\u2212 mice compared to WT controls; this remained unchanged in tASO-treated Terc\u2212\/\u2212 mice (Extended Data Fig. 7a,b<\/a>), indicating sustained biological effects of tASO without telomere length alterations. In parallel, TIFs were quantified in BM cells by telomere-specific immunoFISH at the same late time point, and tASO-treated mice displayed a significantly reduced TIF burden compared to Terc\u2212\/\u2212 controls (Extended Data Fig. 7c,d<\/a>), indicating persistently attenuated tDDR.<\/p>\n

Next, to determine whether these structural and compositional improvements were accompanied by enhanced immune function, we assessed humoral immune responses following antigenic challenge. Young Terc\u2212\/\u2212 mice treated with tASO were vaccinated with a SARS-CoV-2 mRNA vaccine encoding the viral spike protein 2 months after tASO treatment and sacrificed 3 weeks later, at a time point at which tDDR suppression and senescence burden decrease are detectable but before overt age-associated immune remodeling has occurred (Fig. 4f<\/a>). Serum IgG antibodies specific for the SARS-CoV-2 spike protein were quantified as a functional readout of B cell responsiveness. Although young Terc\u2212\/\u2212 control mice mounted measurable vaccine-induced IgG responses, tASO-treated animals exhibited significantly higher anti-spike IgG levels compared to controls, indicating an enhanced humoral response upon antigenic challenge (Fig. 4g<\/a>). These data demonstrate that tDDR inhibition improves immune functional output even at early stages, preceding the restoration of immune cell composition observed in adult mice.<\/p>\n

Together, these results demonstrate that tASO treatment induces durable suppression of tDDR in vivo without altering telomere length, and that this translates into functional benefits across the lifespan, enhancing immune responsiveness and preserving hematopoietic tissue homeostasis and immune cell composition during aging in Terc\u2212\/\u2212 mice.<\/p>\n

tDDR inhibition enhances Terc
\n \u2212\/\u2212 HSPC fitness<\/p>\n

Given the widespread impact of telomerase deficiency and tASO treatment, we tested whether these effects might arise from altered stem cell biology by examining the composition and functionality of the HSPC compartment, which is known to be compromised in telomerase-deficiency settings28<\/a>,29<\/a>.<\/p>\n

Flow cytometric analysis of BM from young mice (using the gating strategy shown in Extended Data Fig. 8a,b<\/a>) revealed no significant differences in the frequencies of the LIN\u2212SCA1+cKIT+ (LSK) HSPC compartment among WT, Terc\u2212\/\u2212 controls and tASO-treated Terc\u2212\/\u2212 mice (Fig. 5a<\/a>). Deeper characterization of stem and progenitor subsets revealed a modest increase in the long-term HSC (LT-HSC) compartment in tASO-treated mice, accompanied by a concomitant reduction in short-term HSCs (ST-HSCs), without significant changes in downstream progenitor populations (multipotent progenitors (MPP), common lymphoid progenitors, common myeloid progenitors, granulocyte\u2013monocyte progenitors and megakaryocyte\u2013erythroid progenitors) (Fig. 5b<\/a> and Extended Data Fig. 9a<\/a>). Terc\u2212\/\u2212 control mice displayed a pronounced increase in DDR activation in the LSK population (Fig. 5c<\/a>); this was significantly reduced by tASO treatment, restoring DDR levels to those observed in WT mice. Notably, LSK cells from Terc\u2212\/\u2212 control mice showed higher percentages of Cdkn2A\/p16high cells (Fig. 5d<\/a>), with reductions to WT levels following tASO treatment, consistent with the impact of the treatment in the bulk of the organ (Fig. 2a<\/a>). Across all HSC and progenitor populations analyzed, tASO treatment consistently reduced DDR activation (Extended Data Fig. 9b<\/a>) and senescence burden (Extended Data Fig. 9c<\/a>), supporting the conclusion that inhibition of tDDR broadly improves the cellular state of the hematopoietic stem and progenitor hierarchy.<\/p>\n

Fig. 5: tDDR inhibition enhances HSPC fitness of young and adult Terc\u2212\/\u2212 mice.<\/b>\"Fig.<\/p>\n

a<\/b>,b<\/b>, Percentages of LSK cells in BM (a<\/b>) and percentages of LT-HSCs (LIN\u2212SCA1+cKIT+CD135\u2212CD34\u2212), ST-HSCs (LIN\u2212SCA1+cKIT+CD135\u2212CD34+) and MPP (LIN\u2212SCA1+cKIT+CD135+CD34+) within the LSK compartment (b<\/b>) from 5-month-old WT, Terc\u2212\/\u2212 control and Terc\u2212\/\u2212 tASO-treated mice, as assessed by flow cytometry 2 months after treatment. c<\/b>,d<\/b>, Percentages of \u03b3H2AXhigh (left), pKAP1high (middle) and \u03b3H2AXhighpKAP1high (right) cells (c<\/b>) and Cdkn2A\/p16high cells (d<\/b>) within the LSK compartment from the same experimental groups, as analyzed by flow cytometry 2 months posttreatment. e<\/b>, Percentages of Ki67high and Ki67low cells in LT-HSCs (left), ST-HSCs (middle) and MPP (right) from the same experimental groups, as analyzed by flow cytometry 2 months posttreatment. f<\/b>, CFU assay with BM cells from the same experimental groups, performed 2 months after treatment. g<\/b>, CFU assay with BM cells of 5-month-old WT and Terc\u2212\/\u2212 mice untreated or treated in vitro with control ASO or tASO (10\u2009\u03bcM). h<\/b>, Percentages of LSK cells in BM of 12-month-old WT, Terc\u2212\/\u2212 control and Terc\u2212\/\u2212 tASO-treated mice, as analyzed by flow cytometry 9 months after treatment. i<\/b>, CFU assay from BM cells of the same experimental groups analyzed in h<\/b>. In a<\/b>\u2013i<\/b>, Terc\u2212\/\u2212 controls included untreated mice (empty circles) and control ASO-treated mice (filled circles), whereas Terc\u2212\/\u2212 tASO-treated mice included anti-TeloG-treated mice (empty squares), and anti-TeloC-treated mice (filled squares). Sample sizes were as follows: WT, n\u2009=\u200910; Terc\u2212\/\u2212 controls, n\u2009=\u200914; Terc\u2212\/\u2212 tASO-treated, n\u2009=\u200915 (a<\/b>); WT, n\u2009=\u200913; Terc\u2212\/\u2212 controls, n\u2009=\u200920; Terc\u2212\/\u2212 tASO-treated, n\u2009=\u200921 (b<\/b>); WT, n\u2009=\u200911 (left and right) and n\u2009=\u200910 (middle); Terc\u2212\/\u2212 controls, n\u2009=\u200914; Terc\u2212\/\u2212 tASO-treated, n\u2009=\u200915 (c<\/b>); WT, n\u2009=\u200910; Terc\u2212\/\u2212 controls, n\u2009=\u200914; Terc\u2212\/\u2212 tASO-treated, n\u2009=\u200915 (d<\/b>); WT, n\u2009=\u20095; Terc\u2212\/\u2212 controls, n\u2009=\u200910; Terc\u2212\/\u2212 tASO-treated, n\u2009=\u20099 (e<\/b>); WT, n\u2009=\u200916; Terc\u2212\/\u2212 controls, n\u2009=\u200921; Terc\u2212\/\u2212 tASO-treated, n\u2009=\u200926 (f<\/b>); WT, n\u2009=\u20093; Terc\u2212\/\u2212 controls, n\u2009=\u20098; Terc\u2212\/\u2212 tASO-treated, n\u2009=\u20098 (g<\/b>); WT, n\u2009=\u200913; Terc\u2212\/\u2212 controls, n\u2009=\u200919; Terc\u2212\/\u2212 tASO-treated, n\u2009=\u200921 (h<\/b>); WT, n\u2009=\u20099; Terc\u2212\/\u2212 controls, n\u2009=\u200915; Terc\u2212\/\u2212 tASO-treated, n\u2009=\u200918 (i<\/b>). Each point represents a single mouse. Data are shown as mean\u2009\u00b1\u2009s.e.m. Statistical significance was determined by one-way ANOVA followed by Tukey\u2019s multiple comparisons test (a<\/b>, c<\/b>, d<\/b> and f<\/b>\u2013i<\/b>) or two-way ANOVA followed by Tukey\u2019s multiple comparisons test (b<\/b> and e<\/b>).<\/p>\n

Source data<\/a><\/p>\n

Disruptions in the balance between quiescence and proliferation can deplete stem cell pools and compromise hematopoiesis37<\/a>. To investigate HSPC proliferative status, we analyzed the expression of Ki67, a marker of cell cycle progression (using the gating strategy shown in Extended Data Fig. 8b<\/a>). Cells with low Ki67 levels are typically considered to be in a quiescent state, which is critical for long-term hematopoietic maintenance, whereas high Ki67 expression indicates active proliferation necessary for rapid response during hematopoietic stress or injury38<\/a>. In Terc\u2212\/\u2212 control mice, we observed a marked increase in the proportion of Ki67high LSK cells, accompanied by a concomitant reduction in the Ki67low fraction, indicating a shift toward excessive proliferation; this is likely to reflect a compensatory activation of telomere-dysfunctional HSPCs attempting to sustain hematopoietic output under chronic damage, at the expense of increased replicative stress and stem cell fitness (Extended Data Fig. 9d<\/a>). Notably, tDDR inhibition by tASO treatment restored the balance between quiescent and proliferating cells across the entire LSK compartment, indicating global normalization of HSPC cell cycle dynamics. We next investigated whether this effect was uniformly distributed across distinct stem and progenitor subsets within the LSK population. Subset-specific analysis (Fig. 5e<\/a>) revealed that tASO treatment restored quiescence to WT levels in LT- and ST-HSCs, whereas no significant changes were observed in the more downstream MPP compartment. This selective effect indicates that tDDR inhibition preferentially stabilizes the most primitive, self-renewing stem cell populations, which are particularly sensitive to chronic tDDR; by contrast, MPP, which are intrinsically more proliferative and less reliant on long-term quiescence, seem largely unaffected. Together, these findings indicate that tASO treatment improves stem cell fitness by reinstating quiescence primarily within the core stem cell pool rather than altering lineage progression.<\/p>\n

Given the marked effects of tASO treatment, we next evaluated the functional capacity of HSPCs by performing colony forming unit (CFU) assays, an established method for evaluating the fitness of these cells in culture, using the same BM samples from young mice analyzed by flow cytometry (Fig. 5a<\/a>). We observed a significant reduction in colony-forming ability in Terc\u2212\/\u2212 mice compared to their WT counterparts, despite plating an equal number of BM cells, reflecting impaired HSPC functionality (Fig. 5f<\/a>). Notably, tDDR inhibition by tASO treatment in vivo enhanced colony formation to levels comparable to those observed in WT mice. As the percentages of HSPCs remained unchanged in both control and tASO-treated Terc\u2212\/\u2212 mice (Fig. 5a<\/a>), these results indicate that the impaired HSPC functionality in Terc\u2212\/\u2212 mice and its improvement upon tASO treatment are driven by changes in the functional capacity of the cells rather than their overall numbers. To assess whether these effects reflected a cell-intrinsic action of tASO on hematopoietic cells, independent of the in vivo microenvironment, we performed additional CFU assays under fully ex vivo conditions. BM cells isolated from Terc\u2212\/\u2212 mice were cultured in methylcellulose and treated directly with tASO in the absence of stromal support, systemic factors or inflammatory cues (Fig. 5g<\/a>). Under these conditions, tASO treatment again significantly improved colony-forming capacity, demonstrating that tASO can exert a direct, cell-intrinsic effect on HSPCs.<\/p>\n

Next, we extended our analyses to older (12-month-old) Terc\u2212\/\u2212 mice to determine whether the in vivo efficacy of tASO on HSPC functionality was sustained with age. In these older mice, the frequencies of LSK cells again did not show significant differences between the groups (Fig. 5h<\/a>); however, CFU assays revealed a decrease in HSPC functionality in aged Terc\u2212\/\u2212 mice, together with a notable improvement following tASO treatment (Fig. 5i<\/a>), consistent with the effects observed in younger mice. These results indicate that the beneficial effects of tASO treatment on HSPC function persist as animals age.<\/p>\n

Collectively, these results reveal that tASO effectively target the HSPC compartment, inhibiting DDR, reducing cellular senescence and restoring the balance between quiescence and proliferation, thereby improving HSPC fitness.<\/p>\n

tDDR inhibition improves in vivo HSPC reconstitution ability of Terc
\n \u2212\/\u2212 mice<\/p>\n

Having observed improvements in HSPC parameters and ex vivo fitness, we next considered whether these cells could compete with healthy stem cells in vivo. Assessment in vivo is critical to determine how HSPCs perform within their native environment, especially under physiological competition. To model this, we used a competitive BM transplant assay to evaluate the functionality of HSPCs based on their ability to repopulate the hematopoietic system in direct competition with WT donor cells. In this assay, two distinct BM cell populations, typically from different genetic backgrounds or treatments, are cotransplanted into a lethally irradiated mouse recipient. The relative contribution of each donor to hematopoiesis over time indicates its fitness, self-renewal capacity and overall performance in the hematopoietic niche39<\/a>. Telomerase-deficient HSPCs have previously been shown to exhibit reduced repopulating capacity in transplant settings28<\/a>,29<\/a>. We therefore tested whether this defect in Terc\u2212\/\u2212 HSPCs could be improved by tDDR inhibition using tASO treatment (Fig. 6a<\/a>). For this analysis, we used two distinct BM donor populations that were distinguishable by their isoforms of CD45, a surface marker used to track hematopoietic cell origin: CD45.2 BM donor cells sourced from either WT or untreated, control ASO-treated, anti-TeloG-treated or anti-TeloC-treated Terc\u2212\/\u2212 mice (the same mice whose HSPC fitness was analyzed in vitro by CFU assay, in Fig. 5f<\/a>); and competitor CD45.1 WT BM donor cells. All donor mice (CD45.2 and CD45.1) were age-matched (5 months of age) at sacrifice. The BM mixed cell populations were injected in a 1:1 ratio intravenously into lethally irradiated CD45.1 and CD45.2 recipient mice for assessment of the reconstitution ability of the transplanted cells. The percentage of reconstitution (percentage of CD45.2+ cells) was monitored in the peripheral blood cells of the recipient mice over 16 weeks, with assessments every 4 weeks posttransplant (using the gating strategy shown in Extended Data Fig. 10a<\/a>), culminating in the analysis of LSK reconstitution in the BM at the endpoint (gating strategy in Extended Data Fig. 10b<\/a>). As expected, we observed a decreased reconstitution ability of BM from control Terc\u2212\/\u2212 mice, with untreated and control ASO-treated Terc\u2212\/\u2212 mice showing comparable reconstitution ability and therefore being treated as a single group, compared to their WT counterpart in both peripheral blood (Fig. 6b<\/a>) and the LSK compartment (Fig. 6c<\/a>) of recipient mice. Notably, BM from anti-TeloG-treated mice showed an improved reconstitution ability, similar to that of WT mice, in both peripheral blood (Fig. 6b<\/a>) and LSK cells (Fig. 6c<\/a>). Consistent with enhanced HSPC fitness, anti-TeloG-treated donor cells contributed robustly to all major hematopoietic lineages following transplantation, including B cells (Fig. 6d<\/a>), T cells (Fig. 6e<\/a>) and myeloid cells (Fig. 6f<\/a>), indicating preserved multilineage differentiation capacity. These results demonstrate that anti-TeloG tASO treatment functionally enhances HSPC competence and competitive repopulating ability in vivo.<\/p>\n

Fig. 6: tDDR inhibition improves reconstitution ability of Terc\u2212\/\u2212 mice.<\/b>\"Fig.<\/p>\n

a<\/b>, Experimental design of BM competitive transplant. BM cells (CD45.2+) from donor mice (5-month-old WT, Terc\u2212\/\u2212 untreated, control ASO-, anti-TeloG- or anti-TeloC-treated; harvested 2 months after tASO treatment) were mixed in a 1:1 ratio with BM cells (CD45.1+) from competitor age-matched WT mice and transplanted into lethally irradiated CD45.1+CD45.2+ WT recipients. Donor contribution was assessed in peripheral blood at 4, 8, 12 and 16 weeks posttransplant and in BM HSPCs at 16 weeks posttransplant. b<\/b>, Percentages of CD45.2+ cells in total peripheral blood at 4, 8, 12 and 16 weeks posttransplant. c<\/b>, Percentages of CD45.2+ cells within the LSK compartment in recipient BM at 16 weeks posttransplant. d<\/b>\u2013f<\/b>, Percentages of CD45.2+ cells in B cells (CD19+) (d<\/b>), T cells (CD3+) (e<\/b>) and myeloid cells (CD11b+) (f<\/b>) in peripheral blood at 4, 8, 12 and 16 weeks posttransplant. In b<\/b>\u2013f<\/b>, Terc\u2212\/\u2212 controls included untreated mice (empty circles) and control ASO-treated mice (filled circles). Sample sizes were as follows: WT, n\u2009=\u20098 (all time points); Terc\u2212\/\u2212 controls, n\u2009=\u200914 (all time points); Terc\u2212\/\u2212 anti-TeloG-treated, n\u2009=\u20096 (4 weeks) and n\u2009=\u20095 (8\u201316 weeks); Terc\u2212\/\u2212 anti-TeloC-treated, n\u2009=\u20097 (all time points) (b<\/b>); WT, n\u2009=\u20096; Terc\u2212\/\u2212 controls, n\u2009=\u20098; Terc\u2212\/\u2212 anti-TeloG-treated, n\u2009=\u20095; Terc\u2212\/\u2212 anti-TeloC-treated, n\u2009=\u20094 (c<\/b>); WT, n\u2009=\u20097 (4 weeks) and n\u2009=\u20098 (8\u201316 weeks); Terc\u2212\/\u2212 controls, n\u2009=\u200913 (4 weeks) and n\u2009=\u200914 (8\u201316 weeks); Terc\u2212\/\u2212 anti-TeloG-treated, n\u2009=\u20096 (4 weeks) and n\u2009=\u20095 (8\u201316 weeks); Terc\u2212\/\u2212 anti-TeloC-treated, n\u2009=\u20097 (all time points) (d<\/b>); WT, n\u2009=\u20097 (4\u20138 weeks) and n\u2009=\u20098 (12\u201316 weeks); Terc\u2212\/\u2212 controls, n\u2009=\u200913 (4 weeks), n\u2009=\u200912 (8 weeks) and n\u2009=\u200914 (12\u201316 weeks); Terc\u2212\/\u2212 anti-TeloG-treated, n\u2009=\u20096 (4 weeks) and n\u2009=\u20095 (8\u201316 weeks); Terc\u2212\/\u2212 anti-TeloC-treated, n\u2009=\u20097 (all time points) (e<\/b>); WT, n\u2009=\u20097 (4 and 12 weeks) and n\u2009=\u20098 (8 and 16 weeks); Terc\u2212\/\u2212 controls, n\u2009=\u200913 (4 weeks), n\u2009=\u200912 (8 weeks) and n\u2009=\u200914 (12\u201316 weeks); Terc\u2212\/\u2212 anti-TeloG-treated, n\u2009=\u20096 (4 weeks) and n\u2009=\u20095 (8\u201316 weeks); Terc\u2212\/\u2212 anti-TeloC-treated, n\u2009=\u20097 (all time points) (f<\/b>). In c<\/b>, each point represents a single mouse. Data are shown as mean\u2009\u00b1\u2009s.e.m. Statistical significance was determined by one-way ANOVA followed by Tukey\u2019s multiple comparisons test (b<\/b>\u2013f<\/b>). Colored labels indicate pairwise comparisons: red, WT versus Terc\u2212\/\u2212 controls; purple, WT versus Terc\u2212\/\u2212 anti-TeloC-treated mice; blue, Terc\u2212\/\u2212 anti-TeloG-treated versus anti-TeloC-treated mice; black, Terc\u2212\/\u2212 control versus anti-TeloG-treated mice; green, WT versus Terc\u2212\/\u2212 anti-TeloG-treated mice. Illustrations in a<\/b> created in BioRender; Rossiello, F. https:\/\/biorender.com\/etc991g<\/a>, https:\/\/biorender.com\/auo2onz<\/a> and https:\/\/biorender.com\/twz85eu<\/a> (2026).<\/p>\n

Source data<\/a><\/p>\n

tDDR inhibition ameliorates age-associated hematopoietic decline in physiologically aged mice<\/p>\n

To determine whether the beneficial effects of tDDR inhibition by tASO extended beyond genetically modified mice and were relevant to normal aging, we tested the impact of tASO administration in aged WT mice. WT mice were treated using the same regimen applied in Terc\u2212\/\u2212 cohorts, with injections administered at 15\u201316 months of age and analyses performed 2 months after the final dose (18 months of age). Young (5-month-old) WT mice were included as a reference (Fig. 7a<\/a>).<\/p>\n

Fig. 7: tDDR inhibition attenuates age-associated DNA damage signaling, senescence and functional decline in hematopoietic cells of aged WT mice.<\/b>\"Fig.<\/p>\n

a<\/b>, Experimental design. Fifteen- to sixteen-month-old C57BL\/6J WT mice received intraperitoneal injection of the indicated ASO (15\u2009mg\u2009kg\u22121, twice weekly for 4 weeks). Two months after treatment, mice were injected intramuscularly with 1\u2009\u00b5g of SARS-CoV-2 mRNA vaccine and sacrificed 3 weeks later. b<\/b>, Percentages of \u03b3H2AXhigh (left), pKAP1high (middle) and \u03b3H2AXhighpKAP1high (right) cells in BM from young mice, aged controls or aged tASO-treated WT mice, as analyzed by flow cytometry. c<\/b>, Relative Cdkn2A\/p16 mRNA expression in BM cells from the same experimental groups, as measured by qPCR with reverse transcription. d<\/b>,e<\/b>, Percentages of LSK cells in BM (d<\/b>) and of \u03b3H2AXhigh (left), pKAP1high (middle) and \u03b3H2AXhighpKAP1high (right) cells within the LSK compartment (e<\/b>) from the same experimental groups, as assessed by flow cytometry. f<\/b>, CFU assay of BM cells from the same experimental groups. g<\/b>, Serum SARS-CoV-2 spike-specific IgG levels measured 3 weeks after vaccination in the same experimental groups, expressed as a percentage relative to aged controls. h<\/b>, Numbers of colonies grown from BM-derived HSPCs from aged healthy individuals treated as indicated and plated 8 days after 30\u2009\u03bcM treatment ex vivo (n\u2009=\u20092 independent donors, aged 75 and 60 years, respectively). In b<\/b>\u2013g<\/b>, aged WT controls included PBS-treated mice (empty circles) and control ASO-treated mice (filled circles), whereas aged WT tASO-treated mice included anti-TeloG-treated (empty squares) and anti-TeloC-treated mice (filled squares). In h<\/b>, controls included untreated donors (empty circles) and control ASO-treated donors (filled circles), whereas tASO-treated donors included anti-TeloG-treated (empty squares) and anti-TeloC-treated donors (filled squares). Sample sizes were as follows: n\u2009=\u20098 young and n\u2009=\u200910 aged controls, n\u2009=\u20098 aged tASO-treated (b<\/b>); n\u2009=\u20099 young and n\u2009=\u20099 aged controls, n\u2009=\u20099 aged tASO-treated (c<\/b>); n\u2009=\u20099 young and n\u2009=\u20099 aged controls, n\u2009=\u200910 aged tASO-treated (d<\/b>); n\u2009=\u20098 young and n\u2009=\u200910 aged controls, n\u2009=\u20098 aged tASO-treated (e<\/b>); n\u2009=\u20099 young and n\u2009=\u200910 aged controls, n\u2009=\u200910 aged tASO-treated (f<\/b>); n\u2009=\u200911 young and n\u2009=\u200910 aged controls, n\u2009=\u200910 aged tASO-treated (g<\/b>). Each point in b<\/b>\u2013g<\/b> represents a single mouse. Data are shown as mean\u2009\u00b1\u2009s.e.m. Statistical significance was determined by one-way ANOVA followed by Tukey\u2019s multiple comparisons test (b<\/b>\u2013g<\/b>). Illustrations in a<\/b> created in BioRender; Rossiello, F. https:\/\/biorender.com\/etc991g<\/a>, https:\/\/biorender.com\/agq2d27<\/a> and https:\/\/biorender.com\/twz85eu<\/a> (2026).<\/p>\n

Source data<\/a><\/p>\n

We first assessed DDR in the BM by flow cytometry. Consistent with aging-associated hematopoietic stress, aged WT mice exhibited increased frequency of \u03b3H2AXhigh, pKAP1high and \u03b3H2AXhighpKAP1high cells compared to young controls (Fig. 7b<\/a>). tASO-treatments induced a trend toward decreased frequencies of these populations, similar to that observed in Terc\u2212\/\u2212 mice (Fig. 1b<\/a>). When we examined cellular senescence in total BM by quantifying Cdkn2a\/p16 mRNA expression, aged WT mice displayed a robust increase in p16 expression relative to young animals, and tASO treatment led to a consistent and significant reduction in p16 levels, indicating a marked decrease in senescence burden (Fig. 7c<\/a>). Given the central role of HSPCs in age-related hematopoietic decline, we analyzed DDR activation within this compartment. Although the frequency of LSK cells was comparable across groups (Fig. 7d<\/a>), tASO treatment induced a clear reduction in DDR markers within aged WT LSK cells (Fig. 7e<\/a>), demonstrating effective DDR inhibition in HSPCs by tASO in physiologically aged mice.<\/p>\n

We next evaluated whether this molecular improvement translated into enhanced functional capacity. The same BM samples from young and aged WT mice (Fig. 7b\u2013e<\/a>) were plated at equal cell numbers for CFU assays. As expected, BM from aged WT control mice exhibited a pronounced reduction in colony-forming ability compared to that from young WT controls, reflecting age-associated impairment in HSPC functionality (Fig. 7f<\/a>). Notably, tASO-treated aged WT mice showed a strong and significant improvement in colony formation. As the frequency of LSK cells remained unchanged across aged control and tASO-treated groups (Fig. 7d<\/a>), these results indicate that the age-related decline in progenitor activity and its rescue upon tASO treatment are driven by changes in functional capacity rather than differences in HSPC abundance, consistent with observations in Terc\u2212\/\u2212 mice (Fig. 5a,f<\/a>).<\/p>\n

Finally, to determine whether the impact of tASO treatment extended to immune competence, we immunized aged WT mice with an mRNA-based SARS-CoV-2 vaccine following the protocol used for Terc\u2212\/\u2212 cohorts (Fig. 7a<\/a>). As expected, aged WT control mice exhibited a marked reduction in spike-specific IgG responses compared to young WT mice, confirming age-associated immune dysfunction (Fig. 7g<\/a>). Notably, when aged cohorts were compared, tASO-treated aged mice displayed increased spike-specific IgG levels relative to age-matched untreated controls, indicating improved humoral immune responsiveness upon tASO treatment.<\/p>\n

Finally, we explored the impact of tDDR inhibition by tASO in the context of human aging. We treated purified human CD34+ HSPCs isolated from the BM of two aged healthy male individuals (aged 75 and 60 years) with tASO ex vivo and monitored its impact in CFU assays (Fig. 7h<\/a>). Although it was not possible to perform statistical analysis with only two biological replicates, we observed that tASO treatment increased clonogenic potential, with enhanced formation of both erythroid and myeloid colonies compared to control samples, pointing to improved hematopoietic progenitor function in human settings and in normal aging.<\/p>\n

Together, these findings demonstrate that tDDR activation in aged WT mice affects their hematopoietic system, indicating that tDDR is relevant not only in telomere-defective but also in normal, physiological aging, and suggesting that molecular, cellular and functional features observed in treated Terc\u2212\/\u2212 animals, including reduced senescence, attenuated DDR in HSPC and improved HSPC function, occur in normal aging too. Therefore, the beneficial effects of tASO are not restricted to genetic telomerase deficiency but extend to both murine and human physiological aging, supporting the relevance of tDDR targeting in counteracting age-related hematopoietic decline.<\/p>\n","protected":false},"excerpt":{"rendered":"tASO effectively inhibits tDDR in the hematopoietic organs of Terc \u2212\/\u2212 mice To determine whether tDDR was activated…\n","protected":false},"author":2,"featured_media":1062253,"comment_status":"","ping_status":"","sticky":false,"template":"","format":"standard","meta":{"footnotes":"","_share_on_mastodon":"0"},"categories":[8],"tags":[973,291568,3968,68729,12308,70,16,15],"class_list":["post-1062252","post","type-post","status-publish","format-standard","has-post-thumbnail","category-science","tag-ageing","tag-dna-damage-response","tag-general","tag-haematological-diseases","tag-life-sciences","tag-science","tag-uk","tag-united-kingdom"],"share_on_mastodon":{"url":"https:\/\/pubeurope.com\/@uk\/116845898326051516","error":""},"_links":{"self":[{"href":"https:\/\/www.europesays.com\/uk\/wp-json\/wp\/v2\/posts\/1062252","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.europesays.com\/uk\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.europesays.com\/uk\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.europesays.com\/uk\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/www.europesays.com\/uk\/wp-json\/wp\/v2\/comments?post=1062252"}],"version-history":[{"count":0,"href":"https:\/\/www.europesays.com\/uk\/wp-json\/wp\/v2\/posts\/1062252\/revisions"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.europesays.com\/uk\/wp-json\/wp\/v2\/media\/1062253"}],"wp:attachment":[{"href":"https:\/\/www.europesays.com\/uk\/wp-json\/wp\/v2\/media?parent=1062252"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.europesays.com\/uk\/wp-json\/wp\/v2\/categories?post=1062252"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.europesays.com\/uk\/wp-json\/wp\/v2\/tags?post=1062252"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}