Genomic instability refers to the accumulation of somatic mutations within the genome, which increases in frequency with age. These mutations include point mutations, deletions, chromosomal translocations, and DNA strand breaks. They can arise from endogenous sources, such as reactive oxygen species (ROS) generated during metabolism, or from exogenous sources, such as ultraviolet (UV) radiation. In normal cells, proteins such as DNA polymerases, glycosylases, nucleases, ligases, and protein complexes involved in DNA repair pathways, including base excision repair (BER) and nucleotide excision repair (NER), maintain genomic integrity [12]. As organisms age, the efficiency of DNA repair mechanisms declines due to downregulation of genes encoding these proteins, leading to the accumulation of unrepaired DNA damage in the form of mutations [6, 13]. Over time, when genetic damage reaches harmful levels, the cell cycle is halted, and apoptosis or cellular senescence is triggered [14].

Telomeres are protein–DNA structures located at the ends of linear chromosomes that act as protective caps, preventing chromosomal degradation and preserving genomic stability [15]. These repetitive non-coding sequences protect essential DNA from erosion during each round of replication, thereby addressing the end-replication problem [16]. Telomere attrition refers to the progressive shortening of telomeres over time due to both cell division and damage. The telomeric repeat sequence ‘TTAGGG’ is guanine-rich and particularly vulnerable to oxidative stress, especially from ROS [16, 17]. Oxidative damage accelerates telomere shortening beyond that caused by replication alone, and with age, cells exhibit a reduced capacity to counteract oxidative damage [18]. When telomere length reaches a critical threshold, cells undergo cell cycle arrest and are driven into cellular senescence or apoptosis [19].

Epigenetic alterations are heritable changes in gene activity that occur without modification of the underlying DNA sequence [6]. With age, four major types of epigenetic changes are typically observed: alterations in DNA methylation, histone protein dysregulation, non-coding RNA dysregulation, and changes in chromatin remodelling [20]. Ageing is associated with global hypomethylation, especially in repetitive regions, which can inappropriately expose normally silenced DNA to transcriptional activity and lead to aberrant gene expression. Conversely, hypermethylation may occur at promoters of protective genes, such as tumour suppressor genes, silencing their expression and reducing the cell’s capacity to prevent malignant transformation, thereby compromising genomic integrity [6].

Histone proteins, which regulate chromatin compaction and thereby control access to DNA, also undergo age-associated changes in abundance and structure. These changes can inappropriately activate normally silenced genes, such as oncogenes, or repress genes essential for genome maintenance and repair. Histone marks are crucial for recruiting DNA repair factors, and their dysregulation leads to impaired DNA repair [21]. Similarly, age-related dysregulation of non-coding RNAs, including microRNAs and long non-coding RNAs, disrupts normal post-transcriptional and transcriptional gene regulation, contributing to loss of homeostasis [22]. In addition, ageing is characterized by loss of constitutive heterochromatin and disruption of higher-order chromatin organization, due in part to altered chromatin-remodelling complexes, reduced sirtuin activity, and damage to the nuclear lamina [6]. These epigenetic and structural alterations, combined with genomic instability and inefficient repair, promote cellular stress and lead to apoptosis or cellular senescence, thereby contributing to ageing.

Proteostasis refers to the dynamic balance of protein synthesis, folding, quality control, and degradation necessary to maintain a functional proteome. With ageing, this balance becomes disrupted, leading to an accumulation of misfolded and aggregated proteins [23, 24]. Impaired protein degradation and reduced chaperone activity result in fewer correctly folded, functional proteins, while defects in protein trafficking further compromise cellular function. Aggregated proteins exert proteotoxic stress, interfering with normal cell processes and triggering cellular stress responses that may lead to apoptosis or cellular senescence. The accumulation of senescent cells and cell death associated with loss of proteostasis has been implicated in several age-related neurodegenerative disorders [25].

A decline in mitochondrial function and number is a common feature of ageing [26]. Mitochondria primarily generate adenosine triphosphate (ATP) through oxidative phosphorylation, which depends on the electron transport chain (ETC), comprising respiratory complexes I–IV and ATP synthase [27]. Ageing is associated with reduced activity of complexes I and IV, whereas changes in complexes II and III are generally less pronounced [28]. Reduced ETC efficiency promotes electron leakage, increasing ROS production and further damaging cellular components [29].

Mitochondrial DNA (mtDNA) accumulates mutations with age, similar to nuclear DNA, which further impairs mitochondrial function and exacerbates ROS generation. mtDNA is particularly vulnerable because it lacks protective histones and relies mainly on BER, with limited capacity for other repair pathways such as NER and mismatch repair (MMR) [29]. As a result, mtDNA damage accumulates, worsening mitochondrial dysfunction and contributing to cellular stress. In healthy cells, mitochondria undergo continuous fission and fusion to maintain quality control and enable the segregation or removal of damaged components through mitophagy [30]. With age, the expression of fission and fusion proteins becomes dysregulated, leading to either excessively fragmented or hyperfused mitochondria, both of which are functionally compromised [27, 30].

Mitochondrial dysfunction increases ROS production, amplifying macromolecular damage, while reduced oxidative phosphorylation leads to diminished ATP levels and an energy deficit that undermines cellular maintenance and repair, thereby heightening cellular stress [27]. The mitochondrial unfolded protein response (mtUPR), a stress pathway that maintains mitochondrial protein homeostasis, also declines with age. Under proteotoxic stress, the mtUPR activates transcription factors that translocate to the nucleus and upregulate genes encoding mitochondrial chaperones, co-chaperones, and proteases, promoting clearance of misfolded proteins and restoration of proteostasis [27, 31]. With ageing, reduced mtUPR efficiency impairs the removal of damaged proteins, destabilizes the ETC, increases ROS production, and promotes further mtDNA damage, ultimately driving mitochondrial dysfunction and, downstream, cellular senescence or apoptosis [31].

The mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that regulates cellular metabolism, growth, survival, autophagy, and immune responses [32]. mTOR functions in two distinct complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) [32, 33]. mTORC1 primarily senses nutrient and growth factor availability to regulate protein synthesis and metabolism, whereas mTORC2 is more closely associated with cell survival, proliferation, and cytoskeletal organization [34]. Together, these complexes help maintain the balance between anabolism and catabolism in response to cellular conditions [33].

With advancing age, mTOR activity is often found to be chronically elevated, promoting anabolic processes while inhibiting catabolic pathways such as autophagy, leading to loss of metabolic homeostasis [34, 35]. Although the precise mechanisms by which mTOR contributes to ageing are still being elucidated, hyperactivation of this pathway is known to increase oxidative and proteotoxic stress, reduce cellular stress resistance, and impair clearance of damaged macromolecules. These changes collectively accelerate functional decline and promote the onset of age-related pathologies. While the exact mechanism of action of this complex is still not fully understood, it is known to increase oxidative and proteotoxic stress within cells, decrease autophagy, cause mitochondrial dysfunction, and dampen the sensitivity and feedback control of nutrient-sensing pathways, thereby impairing the body’s ability to adapt to nutrient scarcity and hastening cellular senescence [34, 36]. The insulin/insulin-like growth factor (IGF)-1 signalling (IIS) pathway also activates mTOR, which can accelerate ageing when excessively stimulated and increases oxidative damage and ROS generation due to impaired mitochondrial activity [6].

Research shows that the body’s ability to respond to insulin deteriorates with age, although the precise pathophysiological mechanisms underlying this change remain unclear [37, 38]. Reduced insulin sensitivity results in hyperinsulinaemia, which has multiple downstream repercussions in cells and may be related to increased body fat and decreased lean mass [39–41]. Additionally, nutrient signalling through the IIS pathway, mediated by pancreatic beta cells during fasting–feeding cycles, is one of the primary pathways influencing ageing and becomes dysregulated with age [39, 42]. Insulin is secreted in response to glucose and promotes anabolic processes, while IGF-1 is a growth factor stimulated by hormones to support cell division and proliferation [43]. Under appropriate regulation, these mechanisms are beneficial, but with age they can become dysregulated and promote excessive anabolism, disrupting metabolic homeostasis and accelerating ageing [43, 44]. Specifically, the IIS pathway can drive ageing by suppressing the transcription factor forkhead box O (FOXO), which is essential for maintaining cellular quality control, particularly under stress and nutrient deprivation [44–46].

Among the nine hallmarks of ageing originally described by López-Otín et al. [6] (2013), which more recent work has expanded to fourteen, this review focuses on seven that most directly align with the therapeutic pathways discussed, to maintain a relevant scope [47]. These molecular indicators of ageing are not independent phenomena but are interconnected through overlapping pathways that exacerbate one another. Progression of dysfunction in one hallmark can precipitate decline in others. For example, epigenetic alterations can promote genomic instability and influence downstream factors such as telomere attrition and loss of proteostasis [6, 48]. Similarly, overactivation of mTOR increases the likelihood of mitochondrial dysfunction and loss of proteostasis, thereby promoting cellular senescence and further accelerating ageing [49, 50].

Cellular senescence usually occurs when damaged or stressed cells permanently stop dividing. Senescent cells secrete a collection of factors termed the senescence-associated secretory phenotype (SASP), which drives chronic inflammation and tissue damage [51]. The SASP includes pro-inflammatory cytokines such as interleukin (IL)-6, IL-8, IL-1β, tumour necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1), growth factors, and matrix metalloproteinases (MMPs), which are persistently secreted, as described by Coppé et al. [52]. These factors recruit monocytes, macrophages, neutrophils, and other immune cells, leading to chronic immune activation, and can also induce senescence in neighbouring cells, amplifying the impact of ageing [52]. MMPs contribute to the degradation of the extracellular matrix, weakening tissue structural integrity [52]. Additionally, senescent cells activate senescent cell anti-apoptotic pathways (SCAPs), enabling them to resist apoptosis and persist, thereby amplifying their detrimental effects [53, 54]. When cells become senescent or undergo apoptosis in response to damage, the pool of functional cells within tissues declines. This loss is particularly critical in muscle, immune, and brain tissues, which already exhibit reduced regenerative potential with age. Senescence and apoptosis also deplete stem cell pools, as stem cells undergo repeated divisions to replace lost cells and eventually experience exhaustion, losing self-renewal and differentiation capacity despite the presence of telomerase [6, 55]. This stem cell exhaustion leads to impaired regeneration and tissue repair [56] (Figure 1).

Metformin is a small molecule widely noted for its potential anti-ageing effects in patients prescribed this drug as a first-line treatment for type II diabetes [57]. Multiple mechanisms of action have been proposed for metformin, many of which converge on improved geroprotection [58]. Primarily, metformin lowers blood glucose and lipid levels by activating AMP-activated protein kinase (AMPK), a central energy sensor. This activation promotes increased glucose uptake by myocytes, downregulates gluconeogenesis in hepatocytes, improves insulin sensitivity in both myocytes and hepatocytes, suppresses the expression of lipogenic enzymes, enhances fatty acid oxidation, and reduces serum triglyceride and low-density lipoprotein (LDL) levels [58–61]. These effects help maintain glycaemic control and confer cardioprotective benefits [62].

On a broader scale, AMPK activation indirectly suppresses mTOR signalling and shifts cellular metabolism away from excessive anabolism towards more catabolic and stress-resistant states, thereby mitigating pro-ageing mTOR activity [63, 64]. Metformin also reduces ROS production by partially inhibiting mitochondrial complex I and promotes mitochondrial biogenesis through the AMPK–peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) axis [65]. In addition, it exerts anti-inflammatory effects by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling and downregulating pro-inflammatory cytokines such as IL-6 and TNF-α, thereby attenuating the SASP [65]. These actions contribute to improved systemic homeostasis, reduced chronic inflammation, and preservation of genomic and cellular integrity. Furthermore, metformin influences epigenetic regulation by modulating DNA methylation and microRNA expression patterns associated with longevity [66]. Collectively, these multifaceted effects position metformin as a promising gerotherapeutic agent [67, 68]. Although metformin is not currently prescribed solely as an anti-ageing therapy, the landmark Targeting Ageing with Metformin (TAME) trial is in the preparatory phase and is designed to test whether metformin can delay the onset of multiple age-related diseases simultaneously [69] (Figure 2).

Dasatinib and quercetin are agents that have received increasing attention in anti-ageing research. Dasatinib was initially developed as an anticancer drug but was later found to be efficacious when combined with quercetin, forming a potentially powerful senolytic regimen for age-related disorders [70–72]. The dasatinib–quercetin (DQ) combination can selectively eliminate senescent cells, which accumulate with age and contribute to chronic inflammation and tissue dysfunction [73]. Dasatinib inhibits multiple tyrosine kinases that are often overexpressed in senescent cells, selectively targeting senescent preadipocytes, haematopoietic progenitors, chondrogenic stem cells, subsets of fibroblasts, and endothelial cells [70, 71, 74, 75]. It also interferes with SCAPs and induces apoptosis in these pathogenic cells [76]. Quercetin, a flavonoid, helps reduce fat accumulation and senescence markers [75].

Several translational studies have shown that DQ treatment significantly reduces senescent cell burden and improves various health parameters, including bone health, metabolic function, inflammation, and cardiac performance [75, 77, 78]. DQ has also shown promise in aesthetic applications, such as skin rejuvenation, by eliminating senescent dermal fibroblasts and enhancing collagen density [79]. Currently, dasatinib is clinically prescribed for certain forms of leukaemia, whereas quercetin is available over the counter mainly for its antioxidant and anti-inflammatory properties. This combination is not yet approved by the U.S. Food and Drug Administration (FDA) for any indication [79, 80]. However, multiple clinical trials are underway to evaluate its potential to mitigate ageing, including studies on reversing osteoporosis in elderly women, improving physical function in individuals with idiopathic pulmonary fibrosis, enhancing gait and cognitive performance in older adults, slowing the progression of Alzheimer’s disease, and assessing its impact on biological ageing [81–83].

Monoclonal antibodies offer high specificity in targeting ageing-associated pro-inflammatory cytokines such as IL-6 and were initially developed for conditions like autoimmune diseases and cancer [84–86]. More recently, they have been explored as potential agents to reduce chronic low-grade inflammation in ageing-associated disorders [87–89]. IL-6, a widely produced cytokine, exerts pleiotropic effects in tissues surrounding senescent cells [90]. It is a major driver of inflammation and participates in a positive feedback loop with mitochondrial dysfunction [86]. Moreover, elevated IL-6 has been implicated in skewing haematopoietic stem cells towards myeloid differentiation, increasing mutational risk, and adversely affecting cardiac tissue [91, 92].

By suppressing IL-6 signalling, these adverse effects may be mitigated and the pace of ageing-related decline potentially reduced [86]. By neutralising specific SASP components, monoclonal antibody therapies aim to restore tissue homeostasis and function. The humanised monoclonal antibody tocilizumab and the fully human antibody sarilumab bind to IL-6 receptors and prevent IL-6 from engaging them, thereby blocking downstream signalling cascades initiated by this cytokine [93, 94]. Inhibiting IL-6-mediated pathways has shown potential to improve tissue integrity and function [86, 91]. Ongoing clinical trials are investigating whether IL-6 inhibition can slow aspects of biological ageing and improve outcomes in patients with inflammatory disorders such as rheumatoid arthritis, polymyalgia rheumatica, and chronic kidney disease [95].

Stem cell-based therapies seek to replenish or rejuvenate aged tissues. Mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) show considerable promise for treating degenerative conditions of the brain, heart, and musculoskeletal system [96–98]. These cells can repair age-related tissue damage, modulate immune responses, and differentiate into multiple lineages, making them attractive candidates for regenerative applications [96–98]. Ageing, however, impairs the regenerative capacity of endogenous stem cells through mechanisms such as accumulated DNA damage, epigenetic alterations, mitochondrial dysfunction, and telomere shortening, underscoring the need for strategies to enhance stem cell efficacy [98].

At present, stem cell therapy is approved for certain indications, including graft-versus-host disease and specific haematological and immune disorders [99, 100]. Numerous clinical trials are ongoing to evaluate its utility in alleviating ageing-related degenerative conditions, such as Parkinson’s disease, osteoporosis, and ischaemic heart disease [74, 101, 102].

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 is a versatile genome-editing platform that enables precise modification of DNA to silence or alter specific genes [103, 104]. Guided by a 20-nucleotide single-guide RNA (sgRNA), Cas9 introduces double-strand breaks that are repaired by cellular DNA repair pathways, typically non-homologous end joining (NHEJ), resulting in gene knockouts, or by homology-directed repair (HDR) when a repair template is provided [103, 104] (Figure 3). This technology has markedly shortened the time required to generate genetically engineered mice, from over a year to a matter of months [105, 106].

In mouse zygote microinjection or electroporation experiments, high-efficiency editing has been reported, with up to 100% NHEJ and 27% HDR in ten-eleven translocation methylcytosine dioxygenase 2 (Tet2)-targeted embryos [107]. These studies demonstrate that CRISPR-Cas9 can efficiently produce knockouts, knock-ins, large deletions, conditional alleles, and multiplexed gene alterations in a single step [105, 108, 109]. Despite concerns regarding large deletions at target sites, mosaicism, and off-target effects, the speed, scalability, and cost-effectiveness of CRISPR-Cas9 have made it indispensable for functional genomics, disease modelling, and preclinical therapeutic ageing research [105, 108]. For example, CRISPR-Cas9 has been used in mouse models to reduce senescence in hepatocytes and attenuate liver ageing by inactivating the KAT7 gene, which codes for a histone acetyltransferase, thereby extending lifespan [110]. In another study, epigenetic reactivation of telomerase reverse transcriptase (TERT) using CRISPR-Cas9 extended the lifespan of T cells in vitro and delayed their senescence [111]. With a growing number of studies applying CRISPR-Cas9 to interrogate molecular drivers of ageing, CRISPR-based rejuvenation strategies are now actively being explored.

Sirtuin 6 (SIRT6), with its first functional characterisation in 2006, has gained increasing attention as a key molecular target in ageing research due to its multifaceted roles, which have been shown to extend lifespan in preclinical models [112, 113]. SIRT6 is a nicotinamide adenine dinucleotide (NAD⁺)-dependent histone deacetylase involved in critical metabolic signalling pathways. Quercetin derivatives have also been shown to modulate SIRT6 activity, with some acting as activators [114]. SIRT6 promotes longevity by enhancing DNA repair, maintaining chromosomal integrity, regulating energy metabolism, and suppressing SASP production [115–119].

SIRT6 protects telomeres by deacetylating histone H3 in telomeric regions, compacting chromatin to shield DNA from damage [115]. It also promotes BER, NHEJ, and homologous recombination (HR) by activating BER enzymes and poly(ADP-ribose) polymerase 1 (PARP1), thereby enhancing cellular resistance to DNA damage and supporting longevity [116, 117]. This protective effect was demonstrated in a study showing SIRT6-mediated repair of UV-induced DNA damage [116]. In metabolism, SIRT6 represses glycolysis through histone deacetylation, redirecting glucose towards efficient mitochondrial respiration [119]. SIRT6-deficient mice exhibit increased glycolytic flux, leading to dysregulated glucose uptake and hypoglycaemia [119]. SIRT6 also inhibits gluconeogenesis, modulates β-cell insulin secretion, and improves insulin sensitivity [119]. In lipid metabolism, SIRT6 suppresses proprotein convertase subtilisin/kexin type 9 (PCSK9) expression via histone acetylation, preserving LDL receptor levels and reducing cardiovascular risk [120, 121].

Furthermore, SIRT6 attenuates chronic inflammation by deacetylating histones at NF-κB gene promoters, suppressing inflammatory cytokine production [122]. It interacts with nuclear factor erythroid 2-related factor 2 (NRF2) to upregulate antioxidant genes, protecting against ROS [123]. Early human clinical trials are now underway to evaluate SIRT6 upregulation using modulators and activators, with initial data indicating promising safety and effective target engagement [48, 82, 124].

A 2023 University College London study identified the coatomer protein complex I (COPI) pathway as a novel target for senescent cell elimination using RNA interference (RNAi) screens [125]. In normal cells, COPI mediates retrograde vesicle transport between the endoplasmic reticulum and Golgi apparatus, involving cargo selection, ADP-ribosylation factor 1 (ARF1) activation, coat assembly, vesicle budding and scission, uncoating, targeting, and fusion [126]. In senescent cells, ARF1 activity is upregulated [127]. ARF1 requires N-terminal myristoylation for activation; N-myristoyltransferase inhibitors (NMTi) suppress this, thereby repressing COPI function [126].

Pharmacological or genetic COPI suppression induces Golgi apparatus dispersal, impaired autophagy, and apoptosis via the unfolded protein response. While NMTi affect both normal and senescent cells, Golgi apparatus disruption and reduced secretion occur selectively in the latter [125]. This inhibits SASP factors trafficking (e.g., IL-6, IL-8), preventing secretion despite ongoing translation [125]. Although target cells remain senescent, this approach reduces inflammation, limits paracrine senescence spread, protects stem cells, and slows tissue degeneration [126]. Preclinical data are promising, and clinical trials may follow soon [128].

RNAi uses short hairpin RNA (shRNA) or small interfering RNA (siRNA) to silence genes by degrading target messenger RNA (mRNA) [129]. Double-stranded RNA is processed by Dicer into siRNA, which loads into the RNA-induced silencing complex (RISC). RISC binds complementary mRNA, cleaving it for degradation [130, 131]. In anti-ageing research, RNAi targets senescent cells or SASP to restore tissue function [132, 133].

Clinical trials are testing RNAi for age-related conditions. For age-related macular degeneration (AMD), targeting myeloid differentiation primary response 88 (MYD88) mRNA reduces inflammation and tissue damage [134, 135]. In amyloidosis, a precursor to atherosclerosis and dementia, transthyretin (TTR) mRNA targeting reduces amyloid deposition [136–138]. For fibrosis, common in the elderly and impairing organ repair, perilipin 2 (PLIN2) targeting alleviates liver fibrosis [139]. These applications highlight RNAi’s potential in age-related diseases.