Systemic Senolysis in Naturally Aged Mice Using a FAST-PLV Gene Therapy Approach

Systemic Senolysis in Naturally Aged Mice Using a FAST-PLV Gene Therapy Approach | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Systemic Senolysis in Naturally Aged Mice Using a FAST-PLV Gene Therapy Approach John Lewis, Henry Garcia, Douglas Brown, Arun Raturi, Jamil Nehme, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5575296/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Approaches to eliminate senescent cells in vivo using transgenic mouse models have demonstrated significant improvements in lifespan, reduction in cancer incidence, and amelioration of age-related degeneration. These approaches require, however, that the organism be genetically engineered from the embryo and/or repeatedly dosed for the organism’s lifespan, making them challenging to implement in humans using current technologies. To overcome these limitations, we developed a clinically viable senolytic gene therapy consisting of a suicide gene, inducible caspase 9 (iCasp9), under control of the early senescence and tumor suppressive p53 promoter or the late senescence p16 Ink 4 a promoter. In vitro , this gene therapy selectively activates in senescent cells and induces caspase-9-dependent apoptosis. When formulated in the FAST-PLV platform and administered systemically to aged mice, the burden of senescent cells was significantly reduced in various tissues, leading to a 123% increase in post-treatment survival for animals given a combination of p16 and p53 targeted senolytic gene therapies. Treated mice showed significantly reduced frailty, increased physical function, and improved heart health. Gross necropsy indicated a 3-fold reduced tumor incidence. In summary, we demonstrate a novel and redosable senolytic genetic medicine approach that improves healthspan by targeting senescent cells based on their transcriptional activity. Biological sciences/Cell biology/Senescence Biological sciences/Drug discovery/Biologics/Gene therapy Biological sciences/Physiology/Ageing Senescence Aging Senolytic Senolysis Gene therapy Nucleic Acid Delivery Proteolipid Vehicle PLV Inducible Caspase 9 iCasp9 Suicide Gene Therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 In Brief We developed a novel gene therapy that eliminates cells with elevated p53 and/or p16 promoter activity in vivo . This approach significantly reduced the burden of senescent cells in aged mice, resulting in increased health- and lifespan. Introduction With significant medical advances in the modern era, life expectancy has steadily increased, contributing to an aging population 1 . With this, substantial challenges are on the horizon; as age progressively increases, so does the incidence of chronic diseases 2 . Cellular senescence, a state of stable and generally irreversible growth arrest associated with a pro-inflammatory secretory phenotype known as Senescence-Associated Secretory Phenotype (SASP), represents one of the most important hallmarks of aging and contributes to chronic diseases 3–7 .Cellular senescence functions primarily to prevent malignant transformation and is induced by a myriad of stresses, such as genotoxic damage, telomere shortening, and oncogene activation. When these stresses occur, the cell responds by activating p53, which in turn facilitates p21 expression 8 . p21 inhibits cell cycle progression by inhibiting the activity of cyclin-dependent kinase 2 (CDK2) 9 . Though most of these changes have been found to affect p53 at the protein level 10 , several research groups report an important role for the transcriptional activity of the p53 promoter in response to genotoxic stress 11,12 . Activation of p53 and the sequential expression of p21 in response to stress results in an immediate cell cycle arrest necessary for initiating senescence. Maintenance of the senescence-associated growth arrest highly depends on another CDK inhibitor, p16 Ink4a 13 . Expression of p16 Ink4a increases with age, therefore connecting p16-mediated cell cycle arrest and senescence to aging 14 . In addition to undergoing permanent cell cycle arrest, SASP factors have local and systemic effects. SASP factors promote immune cell recruitment, proliferation, and angiogenesis, disrupting surrounding tissue architecture leading to increased incidence of chronic conditions, including cancer, and a general decline in organ function characteristic of aging 15,16 . The elimination of cells expressing p16 Ink4a in transgenic mice has been demonstrated to increase lifespan, prevent age-related organ function decline, and reduce multiple chronic diseases in naturally aged and rapidly aging mouse models 17,18 . Similarly, the elimination of p21-high expressing cells from transgenic animals led to improved metabolic and cardiac functions and lifespan extension. These experiments demonstrated a critical role for senescent cells in facilitating organ decline and numerous age-related disorders, ranging from cancer to neurodegenerative disease to frailty, paving the way for the development of therapeutic interventions aimed at their selective removal, known as senolytics 19–21 . A combination treatment with dasatinib and quercetin efficiently eliminates various types of senescent cells in vivo and has been shown to improve physical function, health span, and lifespan in aged mice 22 . In addition, dasatinib and quercetin have demonstrated some clinical success, decreasing circulating SASP levels and several senescent cell markers in patients with diabetic kidney disease 23 , as well as improving physical function in patients with idiopathic pulmonary fibrosis 24 . ABT-737 and ABT-263, which target the B-cell lymphoma 2 (BCL-2) family of proteins that are upregulated in senescent cells and serve to resist apoptotic stimuli 25,26 , were shown to achieve senolysis in various disease models. Currently, newly formulated analogues of ABT-263 are under clinical evaluation for the treatment of macular degeneration. Dasatinib, quercetin, and ABT-737/ABT-263 senolytics are products of drug repurposing and though they have displayed selectivity for senescent cells, at higher concentrations they all impacted normal cells 25,26 . BCL-2 inhibitors have a well-documented history of myelosuppression and have recently been reported to facilitate loss of bone density in aged animals 27–29 . Several alternative senolytic approaches have been shown in the past, including small molecules and peptides that activate p53-mediated apoptosis, inhibitors of Na2+/K+ exchangers, natural compounds such as fisetin, and various polyphenols, CAR T cells, and even vaccines 30–35 . However, most of these approaches remained limited by efficacy, cost, and lack of reproducibility. An important biological obstacle to the application of current senolytic approaches is the heterogeneity of senescent cells, with growing evidence showing that the timing, type of induction, and initial cell type can impact the genetic signature of the senescent cell 36 . To target new markers of senescence, small molecule approaches require extensive development to achieve an appropriate level of specificity. Contrary to small molecule drugs that interact with all cells in the body, genetic approaches can be developed quickly to target specific gene signatures 37 . The efficacy, safety, and beneficial effect of intermittent clearance of senescent cells through the removal of p16– or p21-expressing cells have been shown using transgenic mouse models 21,38–40 . To overcome the limitations surrounding current senolytics, we used these mouse models as a proof-of-concept framework to design a genetic medicine capable of eliminating senescent cells following systemic administration. A plasmid DNA (pDNA) vector was engineered in which the expression of the suicide gene, inducible caspase 9 (iCasp9), is under the control of either the p53 promoter or the p16 Ink4a promoter. Like the mouse models, these senescence-associated promoters restrict suicide gene expression to neoplastic, pre-senescent, or senescent cells. iCasp9 represents an ideal suicide gene to eliminate senescent cells as apoptosis induction is independent of the cell cycle and can bypass upstream abnormalities, such as the upregulation of BCL-2 family proteins 41 . The iCasp9 molecule is non-functional until a Chemical Inducer of Dimerization (CID) is added, which binds to two molecules of iCasp9 and facilitates proximity induced cleavage and activation 42,43 . We chose to utilize a recently developed iCasp9 molecule that is activated by low dose rapamycin, as it is the most cost-effective CID, which increases the accessibility of this therapy 44 . The dose chosen was enough to induce dimerization at a sub-therapeutic level for rapamycin and was dosed once per injection as compared to the standard of care daily therapeutic treatments for organ transplant patients. This level was low even for longevity treatments, where mice are given treatment every other day or daily at an order of magnitude higher. 45 . Here, we describe the development of plasmid DNA-based senolytic gene therapy that relies on selective expression of a late-stage apoptotic protein dependent on transcriptional activation of p53 and/or p16 Ink4a , and we describe its effect for health- and lifespan in aging mice. Results Development of Senolytic Genetic Medicines These two elements, a senescence-associated promoter, and a subsequent temporally controlled apoptotic protein, were combined into pDNA payloads that were delivered using a non-viral system, the Fusogenix FAST-PLV genetic medicine platform which we have recently reported. The Fusogenix PLV platform represents an ideal gene therapy delivery platform, as the use of a FAST protein limits the use of toxic components, making the Fusogenix FAST-PLV platform one of the most well-tolerated non-viral delivery vehicles (Fig. 1 a) 46 . To verify the validity and reliability of our genetic payloads, we compared the expression of endogenous p53 and p16 to the expression of reporters driven by their respective promoter elements. To do so, we transfected BPH1 cells, a prostate cancer cell line, with a p53 promoter-driven fluorescence (GFP) construct of p16 promoter-driven luminescence (fLUC) construct and exposed them to senescence-inducing doses of ionizing radiation 12 , 47 . The endogenous p53 expression reached maximum expression 48 hours after irradiation, which correlated with high fluorescence (Fig. 1 b and Extended Data Fig. 1 a). p16 expression peaked 2 hours after exposure, which also well-correlated with induction of luminescence (Fig. 1 c and Supplementary Fig. 1b ). Though gene therapy gives an opportunity to selectively target cells throughout all stages of senescence induction, there might be limitations in expressing DNA in non-dividing cells due to the nuclear envelope acting as a significant barrier for delivery 48 , 49 . Normal diploid IMR-90 fibroblasts were exposed to 10Gy ionizing radiation to induce senescence. Seven days after exposure, FAST-PLVs encapsulating a pDNA vector in which GFP expression was under the control of the ubiquitous CMV promoter (pDNA-CMV-GFP) were added for 96 hours, following which SA-β-Gal staining and imaging cytometry was conducted. Irradiation resulted in ~ 70% of cells staining for SA-β-Gal ( Extended Data Fig. 1 c), with both the SA-β-Gal + and the SA-β-Gal − populations showing GFP expression ( Extended Fig. 1 d). These results indicate that it is possible to achieve transgene expression from cytosolically delivered pDNA in growth-arrested senescent cells using the Fusogenix FAST-PLV platform. To show the efficacy of cell elimination using a DNA payload, we used a variant of the senolytic payload with a GFP-tagged iCasp9, p53-iCasp9-GFP, and p16-iCasp9-GFP constructs. H1299 cells were incubated with FAST-PLV encapsulating either of the constructs. Cells were then exposed to 100 nM CID (for construct activation) for 24 hours. Cells receiving the p53-iCasp9-GFP construct in combination with CID displayed a significantly reduced GFP + fraction, indicative of the elimination of cells co-expressing iCasp9 and GFP (Fig. 1 d). Cells treated with the p16-iCasp9-GFP construct in combination with CID had a similar decrease in GFP (Fig. 1 e). Taken together, these results indicate that the senescence-associated p53 and p16 promoters can drive expression of the iCasp9 gene and are sufficient to facilitate apoptosis induction in vitro . Senolytic Genetic Medicine Reduces Senescent Cell Burden in vivo To evaluate the senolytic effect of our gene therapy approach i n vivo , we injected 24-month-old C57/B6 mice intravenously with PBS or PLV-encapsulating p53-iCasp9, p16-iCasp9, either individually or in combination. Twenty-four hours after PLV or PBS injection, mice received an intraperitoneal injection of 0.1 mg/kg of CID to induce dimerization and activation of Caspase-9. The treatment cycle was repeated once a week for three weeks, and animals sacrificed one week after the final CID injection (Fig. 2 a). Immunohistochemical staining showed a significant reduction of the senescence marker p19 ARF (mouse homolog to p14 ARF in humans) in the kidney cortex of mice receiving the PLV platform (Fig. 2 b). In accordance, kidneys of mice receiving the combination treatment p53-iCasp9 and p16-iCasp9 vectors had a significantly lower SA-β-Gal + population than control mice receiving PBS (Fig. 2 c-e). Altogether these data validated the senolytic properties of our gene therapy approach in vivo . Senolytic PLV Platform Increase Healthspan of Naturally Aged Mice To study the potential impact of our therapy on the aging trajectory of C57bl6 mice, we utilized a battery of tests and measurements in naturally aged mice. Mice at an average age of 24 months were acclimated to a series of physical frailty tests over 2 months to establish baseline levels, after which they were treated with either PLV containing an inactive mutant of the iCasp9 gene (n = 88) or the combination PLV treatment of p16 and p53-iCasp9 (n = 84). CID was administered IP 24 hours after each dose as a CID. Clinical and physical frailty measurements were conducted monthly (Fig. 3 a). For this study we expanded the SA-β-Gal analysis to 11 tissues (Cerebellum, Cortex, Fat, Gastrocnemius, Heart, Hippocampus, Kidney, Liver, Lung, Quadriceps, and Skin). Most of the tissues showed a decrease in the number of SA-β-Gal + cells and aggregation of the data across all tissues demonstrated a significant reduction in SA-β-Gal + levels (Fig. 3 b,c ). Clinical frailty was assessed using a series of observational parameters each scored with 0 (not observed), 0.5 (mild presentation), and 1 (strong presentation). These 31 parameters were aggregated into an overall clinical frailty score for each mouse. Mice treated with the control PLV platform showed a gradual increase in clinical frailty over time, as expected, whereas mice treated with the senolytic PLV combination showed an attenuation of frailty progression (Fig. 3 d). Physical frailty was assessed using established physical tests that were developed for mice to approximate the Fried Phenotype used in the clinic to assess frailty in elderly patients 50 – 52 . Activity, strength, speed, endurance, and body structure were measured individually and aggregated to create a physical function score ( Extended Data Fig. 2 a-e ) . Mice treated with control PLV showed a gradual decrease in physical function over time, as expected, whereas mice treated with the senolytic PLV combination showed maintained physical function in comparison (Fig. 3 e). We then algorithmically combined clinical frailty with physical function to generate a vitality score, which showed a significant improvement in vitality with the senolytic PLV platform versus control (Fig. 3 f) 53 . Fried et al and other groups adapting Fried’s Frailty to mice also describe frailty in a Boolean fashion, with an organism being frail or not-frail according to how they perform against established benchmarks. Utilizing similar percentage cutoffs to determine the prevalence of frailty, the percentage of the population that is frail is lower in mice treated with the senolytic PLV platform, with a greater than two-fold reduction in the prevalence of frailty one month after cessation of senolytics (Fig. 3 g). Cardiovascular health is one of the leading causes of death, and while not directly measured as a component of frailty, it has broad downstream effects on it. Using an ECG machine designed for mice, various parameters measured for each mouse were scored against an established benchmark, with 0 being within the normal range and 1 being significantly outside the normal range and therefore dysfunctional. The scores were aggregated to generate a hearty frailty score. Aged mice treated with the senolytic PLV platform had significantly lower frailty scores than aged mice treated with control PLV (Fig. 3 h). Overall, these data demonstrate that our senolytic PLV gene therapy can effectively reduce the progression of clinical and physical frailty in aged mice. Treatment of Naturally Aged Mice Improves Lifespan During the course of tracking frailty metrics in the mice, gross necropsy was performed on every mouse that perished to gain insights into the pathologies afflicting them. Evidence of tumor burden, including tumor type (if identifiable), and other determinable metrics such as hepatomegaly and kidney alterations were observed. While it is true that gross necropsy cannot determine the presence of neoplastic lesions, small tumors, or micro-metastasis, it can be used to estimate large tumor burden. Surprisingly, mice that were treated with the combination senolytic PLV platform had a 3-fold lower observable tumor burden, with the most significant sub-type being intestinal tumors (Fig. 4 a). Considering the improved healthspan and lower burden of cancer observed in the cohort receiving the senolytic gene therapy, we decided to study whether systemic treatment with p53 and p16 driven caspase PLV platforms was sufficient to extend lifespan. Mice of approximately 105 weeks of age were injected intravenously with PBS, or PLV encapsulating p53-iCasp9, p16-iCasp9, or a combination. Twenty-four hours after PLV or PBS injection, mice received an intraperitoneal injection of 0.1 mg/kg of CID. This treatment cycle was repeated once a month for one year, or until mice died of natural causes or displayed significant clinical morbidity warranting veterinarian-directed euthanasia. Mice injected with PBS had a median lifespan of 122.5 weeks, which is similar to previous reports of C57/B6 mice at the Jackson Laboratory 54 . Compared to PBS control, mice treated with p53-iCasp9 monotherapy had an 11.8% increase in overall survival [54% post-treatment survival], mice treated with p16-iCasp9 monotherapy had a 17.6% increased overall survival [111% post-treatment survival], and mice treated with the combination of p53-iCasp9 and p16-iCasp9 had a 20.2% increased overall survival [123% post-treatment survival]. (Fig. 4 b,c). Due to the relatively small group sizes in this study (n = 10), the only treatment group with a statistically significant increase in survival was the combination p53-iCasp9 + p16-iCasp9 group. This suggests that these two promoters are synergistic, likely targeting at least two distinct populations of senescent or pre-senescent cells and potentially neoplastic cells. Discussion Here, we describe the development of two senolytic genetic medicines that can be used as monotherapies or in combination to reduce senescent cell burden, attenuate the onset of frailty and potentially tumor burden and eventually leading to extended longevity in mice. As p53 and p16 Ink 4 a are major drivers of cellular senescence, we sought to develop a transcriptionally active gene therapy dependent on one or both factors 13 , 55 . We developed two pDNA vectors where the p16 or p53 promoter regions are upstream of the iCasp9 suicide gene sequence. As demonstrated by previous studies, only senescent cells or pre-senescent cells 13 would achieve sufficient caspase expression following systemic delivery of the genetic senolytic and no adverse events were reported in response to this clearance 21 . Following the administration of the CID, cells expressing the iCasp9 suicide gene undergo selective apoptosis induction 42 – 44 . Our in vitro results indicate that irradiated SA-β-Gal + cells treated with either the p53-iCasp9 or p16-iCasp9 construct undergo selective apoptosis induction following CID administration. Gene therapy holds great promise for treating a myriad of age-related diseases, with more than 2600 clinical trials being initiated worldwide 56 . Despite this, most potential gene therapies reach a bottleneck, as safety and efficacy concerns prevent most treatments from reaching the clinic 57 . The recent success of lipid nanoparticles (LNPs) as delivery vehicles for mRNA-based vaccines has revitalized the gene therapy landscape 58 . However, LNPs present with their own challenges and safety concerns, which limit tolerability following systemic delivery 59 . Given that aging is a complex process that typically results in frailty even in the absence of disease, senolytics must be designed with particular attention being paid to safety. By restricting gene expression to senescent cells, our senescent promoter-based approach eliminates off-target expression resulting in clearance of healthy cells. To prevent toxicity stemming from the nucleic acid delivery vehicle, we utilized the FAST-PLV platform. By combining the beneficial aspects of both viral and non-viral delivery vehicles, the FAST-PLV platform can achieve a high degree of transgene expression without an accompanying increase in toxicity, therefore making it the ideal platform to deliver a genetic senolytic. Furthermore, FAST-PLVs can be administered multiple times without generating humoral responses that impede gene transfer over time, a significant barrier with viral nucleic acid delivery platforms 60 . We found that the kidney cortex of treated mice displayed lower levels of p19 ARF and SA-β-Gal. The kidney represents an interesting target for senolytic treatments as senescent renal cells accumulate with age and have been implicated in renal fibrosis following injury and reduced renal function 61 . In kidneys, ischemic injury was found to induce p19 ARF and p53 expression while having no effect on p16 Ink 4 a expression. Additionally, p19 ARF overexpression in Madin-Darby Canine Kidney (MDCK) cells was found to facilitate cell cycle arrest 62 . Taking these results into consideration, the loss of renal p19 ARF expression in our study was either directly related to treatment and p19 ARF expressing cells are directly eliminated 62 , or the reduced p19 ARF expression was secondary to loss of high p16 Ink 4 a expressing cells as the result of a compensatory feedback mechanism 63 . Regardless, p19 ARF represents a marker of aging and treatment with p53-iCasp9 or p16-iCasp9 resulted in a decreased proportion of p19 ARF expressing cells in the kidney. It is likely that separate senescent cell populations are targeted by each promoter, resulting in a synergistic effect when they are combined. Alcorta et al. 64 found that the p53 target, p21, was elevated as cells neared senescence and once senescent, cells displayed increased p16 Ink 4 a expression. It is likely that the p53-iCasp9 construct targets cells that are in the early stage of senescence or cells that are responding to DNA damage 8 . Thus, in addition to early-senescent cells, neoplastic or cancerous cells may also be targeted by p53-iCasp9, due to the role of the p53 gene in tumor suppression. On the other hand, the p16-iCasp9 construct is likely targeting cells that have entered a growth arrested senescent state and require sustained p16 Ink 4 a expression 13 . Frailty is a multi-factorial pathology that affects a significant percentage of the aging population, and while not always associated with a co-morbidity, potentiates the development of secondary pathologies. In addition, quality of life should not be ignored here: compression of morbidity as a goal of many groups in gerontology can and should include those conditions that do not rank as a clinical pathology but are ubiquitous in the elderly. We showed an attenuation of the onset of frailty and resultant greater healthspan in mice treated with the combination of p16 and p53 senolytic PLV. Other groups have also shown benefits in various frailty metrics, with somewhat indirect mechanisms other than the role of systemic SASP inflammatory markers. More work is needed to elucidate the direct mechanism and/or characterize the various factors at play. A combination of lowered inflammation, improved muscle function, cognitive effects, and more have been shown to have effects on frailty 65 – 67 . Senescent cells play a role in skeletal muscle aging and whether there is a skeletal muscle-specific or general systemic cause, there is an observed skeletomuscular benefit to removing senescent cells 68 . Monthly systemic injections of FAST-PLV encapsulating the p53-iCasp9 and p16-iCasp9 constructs resulted in a 20% increase in survival relative to PBS-injected mice. These results not only demonstrate the senolytic capacity of these DNA constructs but also further validate the tolerability of the FAST-PLV platform. We found that the combination of both the p53-iCasp9 and p16-iCasp9 constructs resulted in better survival than either construct administered as a monotherapy, even at half the dose. In conclusion, we have developed a novel genetic senolytic treatment that relies on the transcriptional activity of senescent cells. By generating a pDNA vector where the p53 or p16 promoter region drives the expression of the suicide gene, iCasp9, we can selectively induce apoptosis in senescent cells with active transcription of either marker. Utilizing FAST-PLV to encapsulate these pDNA vectors enables systemic administration without tolerability concerns that have plagued other non-viral nucleic acid delivery platforms. Treatment with the combination of p53-iCasp9 and p16-iCasp9 resulted in increased median survival. There is growing evidence that the means of induction of senescence, duration of induction, cell type measured, and other subtle factors all play a part in the heterogenous senescent cell population. Thus, we strived to expand past the p16 targeting that has been shown in the past to be effective yet uncomprehensive in senescent cell clearance, with the result being a synergistic effect. The specificity of genetic medicines is seemingly ideal for expanding the breadth of senescent cells that can be targeted without causing undue toxicity, provided the right delivery technology. If ongoing efforts are successful more and more senolytics will enter the clinical path, among them being genetic medicines. Methods Materials Rapamycin was purchased from Fisher Scientific (Cat# BP29631) and dissolved in DMSO (Millipore Sigma, Cat# D8418) to a concentration of 40mg/ml. For in vivo injection, rapamycin was diluted in a solution of 5% PEG-400 (Fisher Scientific, Cat#P167-1) and 5% Tween-80 (Millipore Sigma, Cat# P4780) dissolved in H 2 O. The following lipids were purchased from NOF Co. (Tokyo, Japan): 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dimyristoyl- sn -glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG). 2-dioleoyl- sn -glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were purchased from Avanti Polar Lipids (Alabaster, United States). Anti-mouse CTLA4 (CD152) In Vivo antibody was purchased from BioXCell (Clone 9D9, Cat# BP0164). Anti-p53 antibody (Clone DO-1, Santa Cruz Biotechnology, Cat# sc-126), anti-p16Ink4a antibody (Clone EPR1473, Abcam, Cat# ab108349), rabbit anti-GAPDH (Clone 0411, Santa Cruz Biotechnology, Cat# sc-47724), and mouse anti-β-Tubulin (Clone AA2, Sigma Aldrich, Cat# T8328). All plasmid DNA cloning was done into the p10 plasmid vector produced by Entos Pharmaceuticals. Plasmid DNA preps were expanded and purified by Precision Bio Laboratories (Edmonton, Alberta). Cells and Culturing All cell lines used in this study were purchased from ATCC (Manassas, VA) and cultured in accordance with recommended guidelines. 101 ). Cells were grown in tissue-culture-treated 75 cm2 flasks (VWR 10062-860) until cells reached 80% confluent or nutrients in the media are depleted in a 37°C incubator with humidified atmosphere of 5% CO2 (Nuaire NU-5510). The Trypan Blue assay was used to check for cell viability. FAST-PLV Manufacturing and Validation Lipid formulation composition, manufacturing, and validation was described previously 46 . Briefly, individual lipids were heated in a 37°C water bath for 1 minute, vortexed for 10 seconds each, then combined and vortexed for 10 seconds. The combined lipid mixture was dehydrated in a rotavapor at 60 rpm for 2 hours, under vacuum, then rehydrated with 14 mL 100% ethanol, and sonicated (Branson 2510 Sonicator) at 37°C, set to sonication of 60. The lipid formulation was aliquoted in 500 µL batches and stored at -20°C. The NanoAssemblr Benchtop microfluidics mixing instrument (Precision NanoSystems, Vancouver, BC, NIT0013, and NA-1.5-88, respectively) was used to mix the organic and aqueous solutions and make the PLVs. The organic solution consisted of lipid formulation in EtOH. The aqueous solution consisted of pDNA, 5 nM FAST protein, and 10 mM acetate buffer (pH 4.0). NanoAssemblr was run at a total flow rate of 12 mL/min and a 3:1 aqueous to organic flow rate ratio. PLVs were dialyzed in 8000 MWCO dialysis tubing (BioDesign, D102). The loaded tubing was rinsed with 5 mL of double distilled water and dialyzed in 500 mL of Dialysis Buffer (ENT1844) with gentle stirring (60 rpm) at ambient temperature for 1 hour and was repeated twice with fresh Dialysis Buffer. PLVs were concentrated using a 100 kDa Ultra filter (Amicon, UFC810096) according to the manufacturer’s instructions. PLVs were filter sterilized through 0.2 µm Acrodisc Supor filters (Amicon, UFC910008). Particle size, polydispersity index (PDI), and zeta potential was measured on final samples using the Malvern Zetasizer Range and a Universal 'Dip' Cell Kit (Malvern, ZEN1002) following the manufacturer’s instructions. The nucleic acid encapsulation efficiency and concentration was calculated using a modified Quant-IT PicoGreen dsDNA assay (Thermo Fisher Scientific, Edmonton, Canada). In Vitro Transfection All cells were counted using a hemocytometer prior to plating. All pDNA constructs were prepared with Fusogenix in vitro transfection reagent (formulation 37N as described previously 46 , with slight modifications to the formulation to increase in vitro transfection efficiency, stability, and tolerability). Lipid and pDNA were prepared in separate 1.5 ml tubes. pDNA was added to 10 mM acetate buffer (pH 4.0) to reach a final concentration of 100 ng/uL, and modified lipid formulation 37N was added to 10 mM acetate buffer (pH 4.0) at a 5:1 molar ratio of ionizable lipid:pDNA. The pDNA tube was added dropwise to the lipid tube, gently mixed by inverting, and left at room temperature before adding to cells. For the luciferase assay, 5,000 cells were seeded into 96-well plates (250µL per well) and immediately transfected with the PLV platform. Plates were incubated for 72 hours, following which, a luciferase reporter assay was used to measure expression levels of FLuc produced by p16 promoter. Cell culture media was removed from cells growing in a 96-well plate, and cells washed with 1x PBS. A 50-microliter aliquot of reporter lysis buffer (Promega, Cat# E397A) was added to the cells. The cells were mixed and incubated at room temperature for 15 mins on an orbital shaker. D-luciferin (150µg/mL, GOLDBIO, LUCK-100) was dissolved in 100 mM Tris-HCl (pH 7.8), 5 mM MgCl 2 , 2 mM EDTA, 4 mM DTT, 250 µM acetyl-CoA, and 150 µM ATP. The luciferin substrate (100 µL) of was added via auto-injector to each well immediately before measurement (1–2 second settling time). Luminescence was measured via the FLUOSTAR Omega fluorometer using the MARS data analysis software for analysis. Cells being transfected for cell death assays or Western blotting were seeded at 500,000 cells/well in a 6-well plate and immediately transfected with 10 µg pDNA (p53-iCasp9, p53-iCasp9-GFP, or p53-inactive). Seventy-two hours after transfection with p53-iCasp9-GFP or p16-iCasp9-GFP, H1299 cells have 100 nM RAPA (CID) or vehicle control added for 24 hours before microscopy. Images were analyzed for the number of GFP expressing cells using ImageJ 69 . Western Blot Cells were lysed in ice-cold Pierce RIPA buffer (Thermo Scientific, Cat. No. 89900) 48 hours after transfection with p53-iCasp9. Protein amount was determined using the Pierce BCA protein assay (Thermo Scientific, Cat. No. 23225). Equal amounts of total protein from each lysate were loaded onto Mini-PROTEAN 4–20% Gradient TGX precast gels (BIO-RAD, Cat. No. 456–1095). Separated proteins were transferred to nitrocellulose membranes (BIO-RAD, Cat. No. 1620112). Membranes were blocked with fluorescent Western blocking buffer (Rockland, Cat. No. MB-070) for 1 hour at room temperature. Primary antibodies were diluted 1:1000 in blocking buffer and added to the membranes overnight at 4˚C with shaking. Goat anti-rabbit Alexa Fluor 680 (Thermo Scientific, Cat. No. A27042), or goat anti-mouse Alexa Fluor 750 (Thermo Scientific, Cat. No. A-21084) were diluted 1:10000 in blocking buffer and added for 1 hour at room temperature in the dark. Membranes were visualized on the LI-COR Odyssey. Imaging Flow Cytometry: SA-β-Gal Staining Using the method for SA-β-Gal staining developed by Biran et al. 70 with slight modifications. Cells are lifted, washed, and fixed with 2% paraformaldehyde for 5 minutes. Cells are then washed with 1mM MgCl2/PBS (pH 6 for human cells, pH 5.5 for mouse cells) twice, before resuspending in SA-β-Gal staining buffer: 1mg/ml X-Gal (Sigma Aldrich, Cat# B4252), 5mM K3[Fe(CN)6] (Sigma Aldrich, Cat# 244023), 5mM K4[Fe(CN)6]·3H2O (Sigma Aldrich, Cat# 455989) in 1mM MgCl2/PBS (pH 6 for human cells, pH 5.5 for mouse cells). Cells are incubated for 12 hours in a 37˚C incubator with no CO2 in the dark. Cells are washed twice with flow cytometry buffer and stained with Hoechst 33342 if analysis is to be done immediately. Cells were washed and resuspended in 50 µL of flow cytometry buffer and run on the Amnis ImageStream Mark II imaging flow cytometrer. Cells were gated based on their Area vs Aspect Ratio to identify single cells, then captured based on a positive nuclear stain. SA-β-Gal staining intensity is determined via the mean pixel intensity of the bright field channel (lower values = higher stain intensity), typically a value of -100 to -150 represents the upper cut-off for senescent cells. At least 10,000 events were captured before data was analyzed on the IDEAS imaging software. For SA-β-Gal kidney single cell suspensions, due to a high degree of debris impacting the analysis a mask recapitulating the area occupied by the nuclear stain is generated. This mask is then applied to the bright field channel and expanded 20% to ensure only nucleated cells are included in SA-β-Gal analysis. Cell area is calculated from pixel area with the conversion: 1µm2 = 4 pixels. Mouse Studies All animal studies were carried out according to the guidelines of the Canadian Council on Animal Care (CCAC) and approved by the University of Alberta Animal Care and Use Committee, or the European Community Council Directives of 2010/63/UE and the protocol was approved according to current Italian law (D.Lgs. n. 26/2014) by the Organismo Preposto al Benessere Animale (OPBA, animal care and health committee) of IRCCS INRCA and by the General Direction of Animal Health and Veterinary Drugs of the Italian Ministry of Health with the authorization n° 622/2020-PR, where applicable. Mice were group-housed in IVCs under SPF conditions, with constant temperature and humidity with lighting on a fixed light/dark cycle (12-hours/12-hours) and ad libitum access to food and water. FAST-PLVs were delivered via intravenous injection via the lateral tail vein with a max volume of 200 µL. Rapamycin was dissolved in 5%PEG400/5%Tween-80 (in H 2 O) to give an amount equivalent to 0.1 mg/kg per 100µL and is administered via intraperitoneal injection. Mice are sacrificed via CO2 asphyxiation with cervical dislocation to confirm euthanasia. A total of 149 mice, 51 females and 98 males (mean ± SD age 24.9 ± 1.6 for both sexes) entered the frailty study. After two months of adaptation to manipulations and tests, the mice were sequentially randomized into two age-matched experimental groups: a control group (A group), receiving a monthly i.v. injection of empty PLV, and a treatment group (B group), receiving a monthly i.v. injection of senolytics PLV. All experimenters were blinded to the treatment conditions until the end of the study. Non-invasive measurements of frailty (both clinical frailty index and fried phenotype) were performed once a month in all mice from the inclusion (3rd month, baseline) up to the end of the study. We also recorded the time-to-death data for each mouse and performed necropsies to establish pathologies at death. Mortality occurred when animals died suddenly or were euthanized due to severe illness. Immunohistochemistry Staining Heat-induced antigen retrieval for IHC samples was conducted by immersing rehydrated slides in 10mM sodium citrate (pH 6) and heating until boiling occurred. Slides were blocked in 10% normal rabbit serum (Cat. No. 869019-M, Sigma, Oakville, Canada) with 1% bovine serum albumin (BSA, Cat. No. A9418, Sigma, Oakville, Canada) in TBS with 0.1% Tween-20 for one hour at ambient temperature. Anti-p19ARF antibody (Invitrogen, Cat# PA1-30670) was diluted 1:250 in blocking buffer and incubated on sections overnight. Endogenous peroxidase was blocked with 3% H2O2 in PBS. Goat anti-rabbit HRP (Agilent Dako, Cat# P044801-2) was added to slides for 1 hour. Slides were stained with EnVision FLEX DAB + Chromogen (Agilent Dako, Cat# GV82511-2). Measurement of clinical frailty index We measured both the clinical frailty index (FI) and physical frailty in mice as previously described 71 with slight modifications. We measured the FI in mice based on the validated tool described previously 72–74 . All measurements of frailty were performed within the SPF animal facility of INRCA in a dedicated area. The clinical FI score for each mouse was calculated using the checklist published previously 74 . Clinical assessment included evaluation of the integument, musculoskeletal system, vestibulocochlear and auditory systems, ocular and nasal systems, digestive system, urogenital system, respiratory system, signs of discomfort, as well as body weight and body surface temperature (collected with an infrared thermometer in the ventral zone of the mice). For each parameter, a score of 0 was given if there was no sign of a deficit, a score of 0.5 denoted a mild deficit and a score of 1 indicated a severe deficit. Deficits in body weight and body surface temperature were scored based on their deviation from average reference values obtained from the entire cohort. Values that differed from reference values by less than 1 SD were scored as 0. Values that were ± 1 SD with respect to the reference value were given a frailty value of 0.25; values that differed by ± 2 SD scored 0.5, those that differed by ± 3 SD scored 0.75 and values that were > 3 SD above or below the mean received the maximal frailty value of 1. The sum of the values assigned to the 31 items on the checklist was then divided by 31 to yield a FI score between 0 and 1 for each animal. Measurement of physical frailty The measurement of physical frailty in mice was performed following the same procedure described to translate the physical frailty screening performed in humans 75 to mice ( 52,76,77 ). In order to ensure testing reliability, we performed multiple measurements for each of the five criteria of the frailty assessment (shrinking, weakness, exhaustion, slowness and sedentarity) and the same testers performed all measurements. Each criteria and the respective measurements are listed below: 1) Shrinking: was assessed through a composite score reflecting the body condition of the mice that included current body weight and body length of the mice. The weight was corrected to the previous measurement in the case we detected an increase in weight due to the presence of a tumor or distended abdomen. The body length (nose to base of tail) was measured during the locomotor activity test when the mouse was not in resting state. The locomotor activity area was calibrated with a ruler and the mean of 3 length measurements was calculated using the video analysis software Tracker (v. 5.1.5, https://physlets.org/tracker/). 2) Weakness: This criterion was assessed through a composite score reflecting the forelimb grip strength of the mice that included the measurements from 4 different tests. a) Grip strength meter test (Ugo Basile, Varese, Italy) with a plastic grid 71,78 b) Grip strength meter test (Ugo Basile, Varese, Italy) with an iron bar. c) Home cage lift test. The mouse was gently held by the base of the tail at the top of an empty cage placed above a scale with rapid response and the mean of the two most negative peaks from about 10 attempts was collected. d) Gripping weights lift test with the modification described elsewhere ( 71,79,80 ). 3) Exhaustion: This criterion was assessed through a composite score reflecting the endurance capacity of the mice that included treadmill distance (program: starting at 5 rpm for 2 min and increasing speed from 5 to 50 m/s in 2700 s), mean time to fall at rotarod test (program: starting at 5 m/s for 2 min and increasing speed from 5 to 40 rpm in 300 s) and the score of the gripping weights lift test normalized to body weight. This last measurement was indeed correlated with the other endurance measurements (data not shown) as the test includes an endurance component due to the continuous increase of the weight to be lifted by the mouse 81 . 4) Slowness: This criterion was assessed through a composite score reflecting four different measurements related to the speed of the mice during their normal locomotion. a) We analyzed the distribution of the time spent by the mouse in different speed intervals in an Open Field test (whole test duration 5 min). The speed intervals considered where: I1 (0–1 cm/s), I2 (1–5 cm/s), I3 (5–10 cm/s), I4 (10–15 cm/s), I5 (15–20 cm/s), I6 (20–25 cm/s), I7 (25–30 cm/s), I8 (30–35 cm/s), I9 (35–40 cm/s), I10 (40–90 cm/s). We recorded the highest speed interval that the mouse ran for at least 3 s and assigned as value of the test the mean speed of the interval (e.g. 12.5 for I4 and 37.5 cm/s for I9) as previously described 79,80 . Locomotor activity was conducted by a 5-min open field test on a white wood-chamber (72×72×30 cm) surmounted by a Logitech Brio Ultra HD Webcam 4K 1080 P 60FPS (Logitech Lausanne Switzerland). Tracking and analysis was performed with Biobserve Viewer3 (Biobserve GmbH, Germany) 79,80 . b) An additional measurement for slowness was obtained by recording the maximum speed recorded at rotarod test. c) As previously reported, we also assessed slowness by including the measurement of the mean stride length of the mice with the footprint test 82 as well as d) by measuring the distance between two consecutive hindlimbs paws during a straight walk in the open filed arena (manual tracking of the paws was performed with Tracker v. 5.1.5, https://physlets.org/tracker/). Indeed, there is a strong rationale in support of the relationship between walking speed and stride length, especially in older individuals 83 . 5) Sedentarity (low activity): This criterion was assessed by recording two measurements related to the active behavior of the mice during a locomotor activity test: a) total track length (total distance in cm) and b) the time the mice were not resting (speed above 1 cm/s) during the locomotor activity test. Both measurements were recorded automatically by Biobserve Viewer3 (Biobserve GmbH, Germany). Development of the composite functional scores and detection of the frailty phenotype The results from the multiple measurements related to the same criterion were combined in a unique score following this procedure. All variables were normalized with the MIN-MAX procedure Z= (Xi – Min) / (MAX – MIN) (where Xi is the measurement, Z is the normalized data, MIN and MAX are the minimum and maximum values for X recorded in the population of mice) separately for each sex and the variables assigned to the same criterion were averaged to create a composite score for each criteria. This provided five quantitative composite scores, namely body condition score, strength score, endurance score, speed score and activity score. An overall score representative of physical decline (named Physical Function score) was computed as the mean of the composite scores of the five criteria. To avoid bias due to potential outliers we used the 95th percentile as maximum valuefor all measurements excluding weight. In this last case, we used as MAX the reference weights for mice aged 10–20 months in our colony (28.4 and 33.8 for females and males, respectively). Indeed, the weight of C57BL6/J mice increase until 10 months, then remains relatively stable from 10 to 20 months and only later starts to decline. Hence each measurement range from 1 (or slightly above in some exceptional individuals) to 0. Following the percentiles used by Fried et al. in humans 75 and by others in mice ( 52,76,77,79 ), mice that fell in the bottom 20% of our old cohort for the composite score computed for each criterion were considered positive for frailty for that given criterion. Mice with three or more positive frailty criteria were identified as frail. SA-β-Gal Staining on Frozen Mouse Tissues Tissues were snap frozen in liquid nitrogen and stored in liquid nitrogen until use. Section of 10 µm-thickness were prepared with a cryostat and mounted on slides. Senescence-associated β-galactosidase (SA- β-Gal) staining was performed using a staining kit (Sigma-Aldrich, St.Louis, USA), according to the manufacturer's instructions. Nuclei were counterstained with Nuclear Fast Red (Sigma-Aldrich) and images were acquired using a Zeiss AxioCam HRc mounted on a Leitz Laborlux S light microscope. The percentage of senescent cells was determined by counting of total and SA-β-Gal-positive cells with the positive cell selection tool available in QuPath v. 0.2.3 84 . Statistical analysis A two-tailed Student’s t -test or a one-way analysis of variance (ANOVA) was performed when comparing two groups or more than two groups, respectively. Statistical analysis was performed using Microsoft Excel and Prism 7.0 (GraphPad). Data are expressed as means ± s.d. The difference was considered significant if P < 0.05 (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 unless otherwise indicated). Frailty data were analyzed by generalized linear mixed models (SPSS 26.0, IBM) to account for the longitudinal design of the frailty study in mice. The identifier of each mouse, age group, sex, age of mouse at inclusion, and time were indicated in the model. The linear model was developed assuming normal distribution with the identity link function. The Satterthwaite approximation and robust estimator were used to account for unbalanced data and violation of the assumptions. Differential patterns of survival due to the treatment were estimated by Kaplan-Meier and Cox-regression (SPSS 26.0, IBM) stratified by sex and taking also accounting for possible confounder variables (age at inclusion and frailty index at baseline). Comparisons of SA-β-Gal Staining between control and treatment groups were performed by generalized linear mixed models (SPSS 26.0, IBM) including tissue and treatment as fixed factors, as well as their interaction. The linear model was developed assuming normal distribution with identity link function. The Satterthwaite approximation and robust estimator were used to account for unbalanced data and violations of the assumptions. Declarations Data Availability All processed data are available in the main text or the extended data materials and source data. Acknowledgments This research was supported by an operating grant to John D. Lewis from the Canadian Institutes of Health Research (CIHR), in partnership with the Institute of Aging: Research Nova Scotia, reference number VR1-172710. Dr. Lewis holds the Bird Dogs Chair in Translational Oncology funded by the Alberta Cancer Foundation. Research in the laboratory of Dr. Demaria was supported by a VIDI grant from the Nederlandse organisatie voor gezondheidsonderzoek en zorginnovatie (ZonMw) domain of the Dutch Research Council (NWO; #09150172010029) and by a sponsored research agreement with Oisin Biotechnologies. We thank Katia Carmine-Simmen for her technical support. 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Groningen","correspondingAuthor":false,"prefix":"","firstName":"Jamil","middleName":"","lastName":"Nehme","suffix":""},{"id":389047326,"identity":"b9921539-82d4-43b5-8df9-d1fb981cccbe","order_by":5,"name":"Prakash Bhandari","email":"","orcid":"","institution":"Entos Pharmaceuticals","correspondingAuthor":false,"prefix":"","firstName":"Prakash","middleName":"","lastName":"Bhandari","suffix":""},{"id":389047327,"identity":"db4ec7a7-04c0-422e-84ae-41bb609a5fd4","order_by":6,"name":"Ping Wee","email":"","orcid":"","institution":"Entos Pharmaceuticals","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Wee","suffix":""},{"id":389047328,"identity":"d82bd82e-3854-48de-8f78-59e559accdeb","order_by":7,"name":"Marco Malavolta","email":"","orcid":"https://orcid.org/0000-0002-8442-1763","institution":"INRCA","correspondingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Malavolta","suffix":""},{"id":389047329,"identity":"85e9da1b-0a53-4211-b4a0-619710aaf062","order_by":8,"name":"Liliya Grin","email":"","orcid":"","institution":"Entos Pharmaceuticals","correspondingAuthor":false,"prefix":"","firstName":"Liliya","middleName":"","lastName":"Grin","suffix":""},{"id":389047330,"identity":"96b551f5-5775-4c21-8484-deafd2ec6e4c","order_by":9,"name":"Hector Vega","email":"","orcid":"","institution":"Entos Pharmaceuticals","correspondingAuthor":false,"prefix":"","firstName":"Hector","middleName":"","lastName":"Vega","suffix":""},{"id":389047331,"identity":"f56cf27d-f001-4c7f-ad6f-2ca869fa8889","order_by":10,"name":"Maria Paola Solis Ares","email":"","orcid":"","institution":"Entos Pharmaceuticals","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Paola Solis","lastName":"Ares","suffix":""},{"id":389047332,"identity":"09e62f76-3c3a-4d23-b450-eee06e242549","order_by":11,"name":"Jitendra Kumar","email":"","orcid":"","institution":"Entos Pharmaceuticals","correspondingAuthor":false,"prefix":"","firstName":"Jitendra","middleName":"","lastName":"Kumar","suffix":""},{"id":389047333,"identity":"914b4fa0-a81c-4e23-950f-47fd6d858da6","order_by":12,"name":"Jailal Ablack","email":"","orcid":"","institution":"Entos Pharmaceuticals","correspondingAuthor":false,"prefix":"","firstName":"Jailal","middleName":"","lastName":"Ablack","suffix":""},{"id":389047334,"identity":"5fb72445-0932-49d3-bffd-5499e60dbbcc","order_by":13,"name":"Perrin Beatty","email":"","orcid":"","institution":"Entos Pharmaceuticals","correspondingAuthor":false,"prefix":"","firstName":"Perrin","middleName":"","lastName":"Beatty","suffix":""},{"id":389047335,"identity":"8249551d-687b-40cf-896d-1d8b19ce4cb1","order_by":14,"name":"Gary Hudson","email":"","orcid":"","institution":"Oisin Biotechnologies","correspondingAuthor":false,"prefix":"","firstName":"Gary","middleName":"","lastName":"Hudson","suffix":""},{"id":389047336,"identity":"242264d3-3b18-4502-a491-4bb1b86147b3","order_by":15,"name":"Matthew Sholz","email":"","orcid":"","institution":"Oisin Biotechnologies","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"","lastName":"Sholz","suffix":""},{"id":389047337,"identity":"43facfea-7a9d-4715-aae5-35d02aa0745c","order_by":16,"name":"Roy Duncan","email":"","orcid":"","institution":"Dalhousie University","correspondingAuthor":false,"prefix":"","firstName":"Roy","middleName":"","lastName":"Duncan","suffix":""},{"id":389047338,"identity":"36002d8a-bebb-4a35-a1b4-c5388d392d3b","order_by":17,"name":"Marco Demaria","email":"","orcid":"https://orcid.org/0000-0002-8429-4813","institution":"University Medical Center Groningen","correspondingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Demaria","suffix":""}],"badges":[],"createdAt":"2024-12-03 23:45:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5575296/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5575296/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71222335,"identity":"27ef335a-d031-4a9f-aa1b-7136df79c2e4","added_by":"auto","created_at":"2024-12-12 09:28:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":586154,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDevelopment of a DNA Senolytic. a,\u003c/strong\u003e Schematic showing the mechanism of action of a DNA senolytic. pDNA is encapsulated into the PLV and administered systemically. The senolytic PLV comes into contact with the cell membrane, fuses, and deposits the pDNA directly into the cytoplasm where it is ectopically expressed. Upon activation of senescence-associated promoters such as p16 or p53, the inducible iCasp9 is expressed in target cells. This iCasp9 is inactive until the CID is administered, which then induces apoptosis. \u003cstrong\u003eb,\u003c/strong\u003e BPH1 cells were transfected with a p53-GFP reporter plasmid, irradiated, and fluorescence intensity was measured 48 hours later. Data are represented as the mean ± standard deviation, n = 3. \u003cstrong\u003ec,\u003c/strong\u003e BPH1 cells were transfected with a p16-fLuc reporter plasmid, irradiated, and luminescence was determined. Data are represented as the mean ± standard deviation, n = 3. \u003cstrong\u003ed,e,\u003c/strong\u003e H1299 cells were transfected with a p53-iCasp9r-GFP \u003cstrong\u003ed,\u003c/strong\u003e or p16-iCasp9r-GFP \u003cstrong\u003ee,\u003c/strong\u003eplasmid. At 48 hours post-transfection, cells were treated with either 100nM CID or DMSO vehicle. At 24 hours post-CID, cells were imaged using microscopy and cells per field were counted using ImageJ analysis software. Data are represented as the mean ± standard deviation (n = 3), analyzed using a two-tailed t-test. *p \u0026lt; 0.05, ****p \u0026lt; .0001\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5575296/v1/77f464d30a98969dca13ed8f.png"},{"id":71222810,"identity":"d4927e05-f8b4-47bb-93ce-f1c15dd9aa59","added_by":"auto","created_at":"2024-12-12 09:36:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":848256,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSenolytic Genetic Medicine Reduces Senescent Cell Burden in vivo. a,\u003c/strong\u003e Study timeline schematic. Naturally aged mice were injected with senolytic genetic medicine and 24 hours post injection were intraperitoneally injected with 0.1mg/kg CID. \u003cstrong\u003eb,\u003c/strong\u003e Immunohistochemistry staining of p19\u003csup\u003eARF\u003c/sup\u003e in kidneys from mice injected with senolytic PLV once a week for four weeks. Twenty-four hours after each injection, mice were administered 0.1mg/kg CID. Kidneys were collected 1 week following the last injection. Percent positive staining was assessed using QuPath analysis software. Data are represented as the mean ± standard deviation, n = 3, analyzed using a one-way ANOVA, with Dunnett’s multiple comparisons. (C, D, E) Kidneys from treated mice were dissociated and XGal staining was used to determine senescent cells. \u003cstrong\u003ec,\u003c/strong\u003e Flow cytometry analysis of bright field mean intensity from mice treated with combination PLVs (red) or control (black). \u003cstrong\u003eb,\u003c/strong\u003e Representative images from Imagestream flow cytometry software detecting bright field mean pixel intensity, side scatter to determine size, and DAPI counterstain. \u003cstrong\u003ed,\u003c/strong\u003e Quantification of percent SA-bGal positive cells by XGal staining. Data are represented as the mean ± standard deviation (n = 3), analyzed using a two-tailed t-test. *p \u0026lt; 0.05, **p \u0026lt; .01\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5575296/v1/6e6021bb332eb0470a58e64d.png"},{"id":71222331,"identity":"2af03c2d-d420-4edc-893b-76b7a1fa880c","added_by":"auto","created_at":"2024-12-12 09:28:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":288461,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSenolytic PLV Increase Healthspan of Naturally Aged Mice. a,\u003c/strong\u003e Schematic of the timeline for the study. Mice were adapted to the functional tests for two months, with the second month establishing baseline metrics for the mice. Mice were injected with a combination of p16 and p53 senolytic PLV or inactive mutant control PLV once a month for 4 months. Mice underwent functional testing once a month for a total of 6 times. \u003cstrong\u003eb,\u003c/strong\u003e Relative levels of SA-bGal staining in 11 organs collected from mice treated with combination p16 and p53 senolytic PLVs or an inactive mutant control PLV. Data are represented as ratio to control percentage for each tissue. \u003cstrong\u003ec,\u003c/strong\u003e Quantification of the aggregate percentage of SA-bGal staining across all tissues collected from mice treated with combination p16 and p53 senolytic PLVs or an inactive mutant control PLV. Data are represented as the mean ± standard deviation, n = 8, analyzed using a two-tailed t-test. \u003cstrong\u003ed,\u003c/strong\u003e Using the observational deficit model, each mouse was given a frailty score based on the aggregate of the 31 metrics. Values are reported as the mean estimates obtained by linear mixed model analysis for longitudinal data using cohort, and age (months) as fixed factors (N = 92). \u003cstrong\u003ee,\u003c/strong\u003e Using a battery of functional tests, each mouse was given a functional score for each of the five criteria for frailty (shrinking, weakness, slowness, and sedentarity) Scores were aggregated into one functional score. Values are reported as the mean estimates obtained by linear mixed model analysis for longitudinal data using cohort, and age (months) as fixed factors (N = 92). \u003cstrong\u003ef,\u003c/strong\u003e Each mouse was given a vitality score as an aggregate of their frailty and functional scores. Values are reported as the mean estimates obtained by linear mixed model analysis for longitudinal data using cohort, and age (months) as fixed factors (N = 92). \u003cstrong\u003eg,\u003c/strong\u003eMice were assessed according to whether they had fallen into the bottom 20% of a reference old cohort within each of the five functional criteria. Mice that crossed this threshold in 3 of the 5 are scored as “frail”. Data are represented as the percentage frail. \u003cstrong\u003eh,\u003c/strong\u003e Electrocardiogram measurements were performed, and each mouse was scored according to how well they adhered to a healthy reference population. Data are represented as the mean score ± standard deviation. N = 15. *p \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5575296/v1/6afdabbc0e8b37590ece6b67.png"},{"id":71222809,"identity":"231d2489-d793-4548-a780-01069f80da58","added_by":"auto","created_at":"2024-12-12 09:36:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":215501,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreatment of Naturally Aged Mice Improves Lifespan. a,\u003c/strong\u003e Gross necropsy was performed on each mouse at time of death. Findings of cancer and its location, if determined, were recorded for each mouse. Data are represented as percentage ± standard deviation, N = 12. \u003cstrong\u003eb-e,\u003c/strong\u003e Kaplan-Meier Curve of the survival for p53 senolytic \u003cstrong\u003ec,\u003c/strong\u003e, p16 senolytic \u003cstrong\u003ed,\u003c/strong\u003e and the combination of p53 and p16 senolytic \u003cstrong\u003ee,\u003c/strong\u003e. N = 10. *p \u0026lt; 0.05, *** p \u0026lt; .001\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5575296/v1/b1102ed6995fea1afc72a8d7.png"},{"id":97898268,"identity":"c0436cc0-1a83-4fbd-a0bf-f0386d4afd5e","added_by":"auto","created_at":"2025-12-10 15:38:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2766482,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5575296/v1/c8490add-9436-4c3c-b26f-f56f184e54c4.pdf"},{"id":71222333,"identity":"720b58ee-7d6b-4134-9fcc-acfa59f519bd","added_by":"auto","created_at":"2024-12-12 09:28:51","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":485004,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDataFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-5575296/v1/3820056798c7d0cef6365842.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nD.W.B., P.W., P.B., L.G., H.V., J.A., J.K., M.P.S.A., P.H.B., A.R., R.D., and J.D.L. are employees and/or shareholders of Entos Pharmaceuticals. H.G., G.H., M.S., and J.D.L. are employees and/or shareholders of Oisin Biotechnologies. J.A. and J.D.L. are employees and/or shareholders of OncoSenX. M.S., A.R., R.D., and J.D.L. are authors on a patent related to this work.","formattedTitle":"Systemic Senolysis in Naturally Aged Mice Using a FAST-PLV Gene Therapy Approach","fulltext":[{"header":"In Brief","content":"\u003cp\u003eWe developed a novel gene therapy that eliminates cells with elevated p53 and/or p16 promoter activity \u003cem\u003ein vivo\u003c/em\u003e. This approach significantly reduced the burden of senescent cells in aged mice, resulting in increased health- and lifespan. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eWith significant medical advances in the modern era, life expectancy has steadily increased, contributing to an aging population\u003csup\u003e1\u003c/sup\u003e. With this, substantial challenges are on the horizon; as age progressively increases, so does the incidence of chronic diseases\u003csup\u003e2\u003c/sup\u003e. Cellular senescence, a state of stable and generally irreversible growth arrest associated with a pro-inflammatory secretory phenotype known as Senescence-Associated Secretory Phenotype (SASP), represents one of the most important hallmarks of aging and contributes to chronic diseases\u003csup\u003e3\u0026ndash;7\u003c/sup\u003e.Cellular senescence functions primarily to prevent malignant transformation and is induced by a myriad of stresses, such as genotoxic damage, telomere shortening, and oncogene activation. When these stresses occur, the cell responds by activating p53, which in turn facilitates p21 expression\u003csup\u003e8\u003c/sup\u003e. p21 inhibits cell cycle progression by inhibiting the activity of cyclin-dependent kinase 2 (CDK2)\u003csup\u003e9\u003c/sup\u003e. Though most of these changes have been found to affect p53 at the protein level\u003csup\u003e10\u003c/sup\u003e, several research groups report an important role for the transcriptional activity of the p53 promoter in response to genotoxic stress\u003csup\u003e11,12\u003c/sup\u003e. Activation of p53 and the sequential expression of p21 in response to stress results in an immediate cell cycle arrest necessary for initiating senescence. Maintenance of the senescence-associated growth arrest highly depends on another CDK inhibitor, p16\u003csup\u003eInk4a\u003c/sup\u003e \u003csup\u003e13\u003c/sup\u003e. Expression of p16\u003csup\u003eInk4a\u003c/sup\u003e increases with age, therefore connecting p16-mediated cell cycle arrest and senescence to aging\u003csup\u003e14\u003c/sup\u003e. In addition to undergoing permanent cell cycle arrest, SASP factors have local and systemic effects. SASP factors promote immune cell recruitment, proliferation, and angiogenesis, disrupting surrounding tissue architecture leading to increased incidence of chronic conditions, including cancer, and a general decline in organ function characteristic of aging\u003csup\u003e15,16\u003c/sup\u003e. The elimination of cells expressing p16\u003csup\u003eInk4a\u003c/sup\u003e in transgenic mice has been demonstrated to increase lifespan, prevent age-related organ function decline, and reduce multiple chronic diseases in naturally aged and rapidly aging mouse models\u003csup\u003e17,18\u003c/sup\u003e. Similarly, the elimination of p21-high expressing cells from transgenic animals led to improved metabolic and cardiac functions and lifespan extension. These experiments demonstrated a critical role for senescent cells in facilitating organ decline and numerous age-related disorders, ranging from cancer to neurodegenerative disease to frailty, paving the way for the development of therapeutic interventions aimed at their selective removal, known as senolytics\u003csup\u003e19\u0026ndash;21\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eA combination treatment with dasatinib and quercetin efficiently eliminates various types of senescent cells \u003cem\u003ein vivo\u003c/em\u003e and has been shown to improve physical function, health span, and lifespan in aged mice\u003csup\u003e22\u003c/sup\u003e. In addition, dasatinib and quercetin have demonstrated some clinical success, decreasing circulating SASP levels and several senescent cell markers in patients with diabetic kidney disease\u003csup\u003e23\u003c/sup\u003e, as well as improving physical function in patients with idiopathic pulmonary fibrosis\u003csup\u003e24\u003c/sup\u003e. ABT-737 and ABT-263, which target the B-cell lymphoma 2 (BCL-2) family of proteins that are upregulated in senescent cells and serve to resist apoptotic stimuli\u003csup\u003e25,26\u003c/sup\u003e, were shown to achieve senolysis in various disease models. Currently, newly formulated analogues of ABT-263 are under clinical evaluation for the treatment of macular degeneration.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Dasatinib, quercetin, and ABT-737/ABT-263 senolytics are products of drug repurposing and though they have displayed selectivity for senescent cells, at higher concentrations they all impacted normal cells\u003csup\u003e25,26\u003c/sup\u003e. BCL-2 inhibitors have a well-documented history of myelosuppression and have recently been reported to facilitate loss of bone density in aged animals\u003csup\u003e27\u0026ndash;29\u003c/sup\u003e. Several alternative senolytic approaches have been shown in the past, including small molecules and peptides that activate p53-mediated apoptosis, inhibitors of Na2+/K+ exchangers, natural compounds such as fisetin, and various polyphenols, CAR T cells, and even vaccines\u003csup\u003e30\u0026ndash;35\u003c/sup\u003e. However, most of these approaches remained limited by efficacy, cost, and lack of reproducibility. An important biological obstacle to the application of current senolytic approaches is the heterogeneity of senescent cells, with growing evidence showing that the timing, type of induction, and initial cell type can impact the genetic signature of the senescent cell\u003csup\u003e36\u003c/sup\u003e. To target new markers of senescence, small molecule approaches require extensive development to achieve an appropriate level of specificity. Contrary to small molecule drugs that interact with all cells in the body, genetic approaches can be developed quickly to target specific gene signatures\u003csup\u003e37\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe efficacy, safety, and beneficial effect of intermittent clearance of senescent cells through the removal of p16\u0026ndash; or p21-expressing cells have been shown using transgenic mouse models \u003csup\u003e21,38\u0026ndash;40\u003c/sup\u003e. \u0026nbsp;To overcome the limitations surrounding current senolytics, we used these mouse models as a proof-of-concept framework to design a genetic medicine capable of eliminating senescent cells following systemic administration. \u0026nbsp;A plasmid DNA (pDNA) vector was engineered in which the expression of the suicide gene, inducible caspase 9 (iCasp9), is under the control of either the p53 promoter or the p16\u003csup\u003eInk4a\u003c/sup\u003e promoter. Like the mouse models, these senescence-associated promoters restrict suicide gene expression to neoplastic, pre-senescent, or senescent cells. iCasp9 represents an ideal suicide gene to eliminate senescent cells as apoptosis induction is independent of the cell cycle and can bypass upstream abnormalities, such as the upregulation of BCL-2 family proteins\u003csup\u003e41\u003c/sup\u003e. The iCasp9 molecule is non-functional until a Chemical Inducer of Dimerization (CID) is added, which binds to two molecules of iCasp9 and facilitates proximity induced cleavage and activation\u003csup\u003e42,43\u003c/sup\u003e. We chose to utilize a recently developed iCasp9 molecule that is activated by low dose rapamycin, as it is the most cost-effective CID, which increases the accessibility of this therapy\u003csup\u003e44\u003c/sup\u003e. The dose chosen was enough to induce dimerization at a sub-therapeutic level for rapamycin and was dosed once per injection as compared to the standard of care daily therapeutic treatments for organ transplant patients. This level was low even for longevity treatments, where mice are given treatment every other day or daily at an order of magnitude higher.\u003csup\u003e45\u003c/sup\u003e. Here, we describe the development of plasmid DNA-based senolytic gene therapy that relies on selective expression of a late-stage apoptotic protein dependent on transcriptional activation of p53 and/or p16\u003csup\u003eInk4a\u003c/sup\u003e, and we describe its effect for health- and lifespan in aging mice.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eDevelopment of Senolytic Genetic Medicines\u003c/h2\u003e \u003cp\u003eThese two elements, a senescence-associated promoter, and a subsequent temporally controlled apoptotic protein, were combined into pDNA payloads that were delivered using a non-viral system, the Fusogenix FAST-PLV genetic medicine platform which we have recently reported. The Fusogenix PLV platform represents an ideal gene therapy delivery platform, as the use of a FAST protein limits the use of toxic components, making the Fusogenix FAST-PLV platform one of the most well-tolerated non-viral delivery vehicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea)\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. To verify the validity and reliability of our genetic payloads, we compared the expression of endogenous p53 and p16 to the expression of reporters driven by their respective promoter elements. To do so, we transfected BPH1 cells, a prostate cancer cell line, with a p53 promoter-driven fluorescence (GFP) construct of p16 promoter-driven luminescence (fLUC) construct and exposed them to senescence-inducing doses of ionizing radiation\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. The endogenous p53 expression reached maximum expression 48 hours after irradiation, which correlated with high fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb \u003cb\u003eand Extended Data\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). p16 expression peaked 2 hours after exposure, which also well-correlated with induction of luminescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec \u003cb\u003eand Supplementary Fig.\u0026nbsp;1b\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThough gene therapy gives an opportunity to selectively target cells throughout all stages of senescence induction, there might be limitations in expressing DNA in non-dividing cells due to the nuclear envelope acting as a significant barrier for delivery\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Normal diploid IMR-90 fibroblasts were exposed to 10Gy ionizing radiation to induce senescence. Seven days after exposure, FAST-PLVs encapsulating a pDNA vector in which GFP expression was under the control of the ubiquitous CMV promoter (pDNA-CMV-GFP) were added for 96 hours, following which SA-β-Gal staining and imaging cytometry was conducted. Irradiation resulted in ~\u0026thinsp;70% of cells staining for SA-β-Gal (\u003cb\u003eExtended Data\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), with both the SA-β-Gal\u003csup\u003e+\u003c/sup\u003e and the SA-β-Gal\u003csup\u003e\u0026minus;\u003c/sup\u003e populations showing GFP expression (\u003cb\u003eExtended\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). These results indicate that it is possible to achieve transgene expression from cytosolically delivered pDNA in growth-arrested senescent cells using the Fusogenix FAST-PLV platform.\u003c/p\u003e \u003cp\u003eTo show the efficacy of cell elimination using a DNA payload, we used a variant of the senolytic payload with a GFP-tagged iCasp9, p53-iCasp9-GFP, and p16-iCasp9-GFP constructs. H1299 cells were incubated with FAST-PLV encapsulating either of the constructs. Cells were then exposed to 100 nM CID (for construct activation) for 24 hours. Cells receiving the p53-iCasp9-GFP construct in combination with CID displayed a significantly reduced GFP\u003csup\u003e+\u003c/sup\u003e fraction, indicative of the elimination of cells co-expressing iCasp9 and GFP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Cells treated with the p16-iCasp9-GFP construct in combination with CID had a similar decrease in GFP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Taken together, these results indicate that the senescence-associated p53 and p16 promoters can drive expression of the iCasp9 gene and are sufficient to facilitate apoptosis induction \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSenolytic Genetic Medicine Reduces Senescent Cell Burden in vivo\u003c/h2\u003e \u003cp\u003eTo evaluate the senolytic effect of our gene therapy approach i\u003cem\u003en vivo\u003c/em\u003e, we injected 24-month-old C57/B6 mice intravenously with PBS or PLV-encapsulating p53-iCasp9, p16-iCasp9, either individually or in combination. Twenty-four hours after PLV or PBS injection, mice received an intraperitoneal injection of 0.1 mg/kg of CID to induce dimerization and activation of Caspase-9. The treatment cycle was repeated once a week for three weeks, and animals sacrificed one week after the final CID injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Immunohistochemical staining showed a significant reduction of the senescence marker p19\u003csup\u003eARF\u003c/sup\u003e (mouse homolog to p14\u003csup\u003eARF\u003c/sup\u003e in humans) in the kidney cortex of mice receiving the PLV platform (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In accordance, kidneys of mice receiving the combination treatment p53-iCasp9 and p16-iCasp9 vectors had a significantly lower SA-β-Gal\u0026thinsp;+\u0026thinsp;population than control mice receiving PBS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-e). Altogether these data validated the senolytic properties of our gene therapy approach \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSenolytic PLV Platform Increase Healthspan of Naturally Aged Mice\u003c/h3\u003e\n\u003cp\u003eTo study the potential impact of our therapy on the aging trajectory of C57bl6 mice, we utilized a battery of tests and measurements in naturally aged mice. Mice at an average age of 24 months were acclimated to a series of physical frailty tests over 2 months to establish baseline levels, after which they were treated with either PLV containing an inactive mutant of the iCasp9 gene (n\u0026thinsp;=\u0026thinsp;88) or the combination PLV treatment of p16 and p53-iCasp9 (n\u0026thinsp;=\u0026thinsp;84). CID was administered IP 24 hours after each dose as a CID. Clinical and physical frailty measurements were conducted monthly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). For this study we expanded the SA-β-Gal analysis to 11 tissues (Cerebellum, Cortex, Fat, Gastrocnemius, Heart, Hippocampus, Kidney, Liver, Lung, Quadriceps, and Skin). Most of the tissues showed a decrease in the number of SA-β-Gal\u0026thinsp;+\u0026thinsp;cells and aggregation of the data across all tissues demonstrated a significant reduction in SA-β-Gal\u0026thinsp;+\u0026thinsp;levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb,c\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003eClinical frailty was assessed using a series of observational parameters each scored with 0 (not observed), 0.5 (mild presentation), and 1 (strong presentation). These 31 parameters were aggregated into an overall clinical frailty score for each mouse. Mice treated with the control PLV platform showed a gradual increase in clinical frailty over time, as expected, whereas mice treated with the senolytic PLV combination showed an attenuation of frailty progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Physical frailty was assessed using established physical tests that were developed for mice to approximate the Fried Phenotype used in the clinic to assess frailty in elderly patients\u003csup\u003e\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Activity, strength, speed, endurance, and body structure were measured individually and aggregated to create a physical function score (\u003cb\u003eExtended Data\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-e\u003cb\u003e)\u003c/b\u003e. Mice treated with control PLV showed a gradual decrease in physical function over time, as expected, whereas mice treated with the senolytic PLV combination showed maintained physical function in comparison (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). We then algorithmically combined clinical frailty with physical function to generate a vitality score, which showed a significant improvement in vitality with the senolytic PLV platform versus control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef)\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Fried et al and other groups adapting Fried\u0026rsquo;s Frailty to mice also describe frailty in a Boolean fashion, with an organism being frail or not-frail according to how they perform against established benchmarks. Utilizing similar percentage cutoffs to determine the prevalence of frailty, the percentage of the population that is frail is lower in mice treated with the senolytic PLV platform, with a greater than two-fold reduction in the prevalence of frailty one month after cessation of senolytics (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eCardiovascular health is one of the leading causes of death, and while not directly measured as a component of frailty, it has broad downstream effects on it. Using an ECG machine designed for mice, various parameters measured for each mouse were scored against an established benchmark, with 0 being within the normal range and 1 being significantly outside the normal range and therefore dysfunctional. The scores were aggregated to generate a hearty frailty score. Aged mice treated with the senolytic PLV platform had significantly lower frailty scores than aged mice treated with control PLV (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003eOverall, these data demonstrate that our senolytic PLV gene therapy can effectively reduce the progression of clinical and physical frailty in aged mice.\u003c/p\u003e\n\u003ch3\u003eTreatment of Naturally Aged Mice Improves Lifespan\u003c/h3\u003e\n\u003cp\u003eDuring the course of tracking frailty metrics in the mice, gross necropsy was performed on every mouse that perished to gain insights into the pathologies afflicting them. Evidence of tumor burden, including tumor type (if identifiable), and other determinable metrics such as hepatomegaly and kidney alterations were observed. While it is true that gross necropsy cannot determine the presence of neoplastic lesions, small tumors, or micro-metastasis, it can be used to estimate large tumor burden. Surprisingly, mice that were treated with the combination senolytic PLV platform had a 3-fold lower observable tumor burden, with the most significant sub-type being intestinal tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003eConsidering the improved healthspan and lower burden of cancer observed in the cohort receiving the senolytic gene therapy, we decided to study whether systemic treatment with p53 and p16 driven caspase PLV platforms was sufficient to extend lifespan. Mice of approximately 105 weeks of age were injected intravenously with PBS, or PLV encapsulating p53-iCasp9, p16-iCasp9, or a combination. Twenty-four hours after PLV or PBS injection, mice received an intraperitoneal injection of 0.1 mg/kg of CID. This treatment cycle was repeated once a month for one year, or until mice died of natural causes or displayed significant clinical morbidity warranting veterinarian-directed euthanasia. Mice injected with PBS had a median lifespan of 122.5 weeks, which is similar to previous reports of C57/B6 mice at the Jackson Laboratory\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Compared to PBS control, mice treated with p53-iCasp9 monotherapy had an 11.8% increase in overall survival [54% post-treatment survival], mice treated with p16-iCasp9 monotherapy had a 17.6% increased overall survival [111% post-treatment survival], and mice treated with the combination of p53-iCasp9 and p16-iCasp9 had a 20.2% increased overall survival [123% post-treatment survival]. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb,c). Due to the relatively small group sizes in this study (n\u0026thinsp;=\u0026thinsp;10), the only treatment group with a statistically significant increase in survival was the combination p53-iCasp9\u0026thinsp;+\u0026thinsp;p16-iCasp9 group. This suggests that these two promoters are synergistic, likely targeting at least two distinct populations of senescent or pre-senescent cells and potentially neoplastic cells.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we describe the development of two senolytic genetic medicines that can be used as monotherapies or in combination to reduce senescent cell burden, attenuate the onset of frailty and potentially tumor burden and eventually leading to extended longevity in mice. As p53 and p16\u003csup\u003eInk\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003ea\u003c/sup\u003e are major drivers of cellular senescence, we sought to develop a transcriptionally active gene therapy dependent on one or both factors\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. We developed two pDNA vectors where the p16 or p53 promoter regions are upstream of the iCasp9 suicide gene sequence. As demonstrated by previous studies, only senescent cells or pre-senescent cells\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e would achieve sufficient caspase expression following systemic delivery of the genetic senolytic and no adverse events were reported in response to this clearance\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Following the administration of the CID, cells expressing the iCasp9 suicide gene undergo selective apoptosis induction\u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Our \u003cem\u003ein vitro\u003c/em\u003e results indicate that irradiated SA-β-Gal\u003csup\u003e+\u003c/sup\u003e cells treated with either the p53-iCasp9 or p16-iCasp9 construct undergo selective apoptosis induction following CID administration.\u003c/p\u003e \u003cp\u003eGene therapy holds great promise for treating a myriad of age-related diseases, with more than 2600 clinical trials being initiated worldwide\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Despite this, most potential gene therapies reach a bottleneck, as safety and efficacy concerns prevent most treatments from reaching the clinic\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. The recent success of lipid nanoparticles (LNPs) as delivery vehicles for mRNA-based vaccines has revitalized the gene therapy landscape\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. However, LNPs present with their own challenges and safety concerns, which limit tolerability following systemic delivery\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Given that aging is a complex process that typically results in frailty even in the absence of disease, senolytics must be designed with particular attention being paid to safety. By restricting gene expression to senescent cells, our senescent promoter-based approach eliminates off-target expression resulting in clearance of healthy cells. To prevent toxicity stemming from the nucleic acid delivery vehicle, we utilized the FAST-PLV platform. By combining the beneficial aspects of both viral and non-viral delivery vehicles, the FAST-PLV platform can achieve a high degree of transgene expression without an accompanying increase in toxicity, therefore making it the ideal platform to deliver a genetic senolytic. Furthermore, FAST-PLVs can be administered multiple times without generating humoral responses that impede gene transfer over time, a significant barrier with viral nucleic acid delivery platforms\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe found that the kidney cortex of treated mice displayed lower levels of p19\u003csup\u003eARF\u003c/sup\u003e and SA-β-Gal. The kidney represents an interesting target for senolytic treatments as senescent renal cells accumulate with age and have been implicated in renal fibrosis following injury and reduced renal function\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. In kidneys, ischemic injury was found to induce p19\u003csup\u003eARF\u003c/sup\u003e and p53 expression while having no effect on p16\u003csup\u003eInk\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003ea\u003c/sup\u003e expression. Additionally, p19\u003csup\u003eARF\u003c/sup\u003e overexpression in Madin-Darby Canine Kidney (MDCK) cells was found to facilitate cell cycle arrest\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Taking these results into consideration, the loss of renal p19\u003csup\u003eARF\u003c/sup\u003e expression in our study was either directly related to treatment and p19\u003csup\u003eARF\u003c/sup\u003e expressing cells are directly eliminated\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e, or the reduced p19\u003csup\u003eARF\u003c/sup\u003e expression was secondary to loss of high p16\u003csup\u003eInk\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003ea\u003c/sup\u003e expressing cells as the result of a compensatory feedback mechanism\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Regardless, p19\u003csup\u003eARF\u003c/sup\u003e represents a marker of aging and treatment with p53-iCasp9 or p16-iCasp9 resulted in a decreased proportion of p19\u003csup\u003eARF\u003c/sup\u003e expressing cells in the kidney.\u003c/p\u003e \u003cp\u003eIt is likely that separate senescent cell populations are targeted by each promoter, resulting in a synergistic effect when they are combined. Alcorta \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e found that the p53 target, p21, was elevated as cells neared senescence and once senescent, cells displayed increased p16\u003csup\u003eInk\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003ea\u003c/sup\u003e expression. It is likely that the p53-iCasp9 construct targets cells that are in the early stage of senescence or cells that are responding to DNA damage\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Thus, in addition to early-senescent cells, neoplastic or cancerous cells may also be targeted by p53-iCasp9, due to the role of the p53 gene in tumor suppression. On the other hand, the p16-iCasp9 construct is likely targeting cells that have entered a growth arrested senescent state and require sustained p16\u003csup\u003eInk\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003ea\u003c/sup\u003e expression\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFrailty is a multi-factorial pathology that affects a significant percentage of the aging population, and while not always associated with a co-morbidity, potentiates the development of secondary pathologies. In addition, quality of life should not be ignored here: compression of morbidity as a goal of many groups in gerontology can and should include those conditions that do not rank as a clinical pathology but are ubiquitous in the elderly. We showed an attenuation of the onset of frailty and resultant greater healthspan in mice treated with the combination of p16 and p53 senolytic PLV. Other groups have also shown benefits in various frailty metrics, with somewhat indirect mechanisms other than the role of systemic SASP inflammatory markers. More work is needed to elucidate the direct mechanism and/or characterize the various factors at play. A combination of lowered inflammation, improved muscle function, cognitive effects, and more have been shown to have effects on frailty\u003csup\u003e\u003cspan additionalcitationids=\"CR66\" citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Senescent cells play a role in skeletal muscle aging and whether there is a skeletal muscle-specific or general systemic cause, there is an observed skeletomuscular benefit to removing senescent cells\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. Monthly systemic injections of FAST-PLV encapsulating the p53-iCasp9 and p16-iCasp9 constructs resulted in a 20% increase in survival relative to PBS-injected mice. These results not only demonstrate the senolytic capacity of these DNA constructs but also further validate the tolerability of the FAST-PLV platform. We found that the combination of both the p53-iCasp9 and p16-iCasp9 constructs resulted in better survival than either construct administered as a monotherapy, even at half the dose.\u003c/p\u003e \u003cp\u003eIn conclusion, we have developed a novel genetic senolytic treatment that relies on the transcriptional activity of senescent cells. By generating a pDNA vector where the p53 or p16 promoter region drives the expression of the suicide gene, iCasp9, we can selectively induce apoptosis in senescent cells with active transcription of either marker. Utilizing FAST-PLV to encapsulate these pDNA vectors enables systemic administration without tolerability concerns that have plagued other non-viral nucleic acid delivery platforms. Treatment with the combination of p53-iCasp9 and p16-iCasp9 resulted in increased median survival. There is growing evidence that the means of induction of senescence, duration of induction, cell type measured, and other subtle factors all play a part in the heterogenous senescent cell population. Thus, we strived to expand past the p16 targeting that has been shown in the past to be effective yet uncomprehensive in senescent cell clearance, with the result being a synergistic effect. The specificity of genetic medicines is seemingly ideal for expanding the breadth of senescent cells that can be targeted without causing undue toxicity, provided the right delivery technology. If ongoing efforts are successful more and more senolytics will enter the clinical path, among them being genetic medicines.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv\u003e\n\u003ch2\u003eMaterials\u003c/h2\u003e\n\u003cp\u003eRapamycin was purchased from Fisher Scientific (Cat# BP29631) and dissolved in DMSO (Millipore Sigma, Cat# D8418) to a concentration of 40mg/ml. For in vivo injection, rapamycin was diluted in a solution of 5% PEG-400 (Fisher Scientific, Cat#P167-1) and 5% Tween-80 (Millipore Sigma, Cat# P4780) dissolved in H\u003csub\u003e2\u003c/sub\u003eO. The following lipids were purchased from NOF Co. (Tokyo, Japan): 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dimyristoyl-\u003cem\u003esn\u003c/em\u003e-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG). 2-dioleoyl-\u003cem\u003esn\u003c/em\u003e-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were purchased from Avanti Polar Lipids (Alabaster, United States). Anti-mouse CTLA4 (CD152) In Vivo antibody was purchased from BioXCell (Clone 9D9, Cat# BP0164). Anti-p53 antibody (Clone DO-1, Santa Cruz Biotechnology, Cat# sc-126), anti-p16Ink4a antibody (Clone EPR1473, Abcam, Cat# ab108349), rabbit anti-GAPDH (Clone 0411, Santa Cruz Biotechnology, Cat# sc-47724), and mouse anti-\u0026beta;-Tubulin (Clone AA2, Sigma Aldrich, Cat# T8328). All plasmid DNA cloning was done into the p10 plasmid vector produced by Entos Pharmaceuticals. Plasmid DNA preps were expanded and purified by Precision Bio Laboratories (Edmonton, Alberta).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eCells and Culturing\u003c/h3\u003e\n\u003cp\u003eAll cell lines used in this study were purchased from ATCC (Manassas, VA) and cultured in accordance with recommended guidelines.\u003csup\u003e101\u003c/sup\u003e). Cells were grown in tissue-culture-treated 75 cm2 flasks (VWR 10062-860) until cells reached 80% confluent or nutrients in the media are depleted in a 37\u0026deg;C incubator with humidified atmosphere of 5% CO2 (Nuaire NU-5510). The Trypan Blue assay was used to check for cell viability.\u003c/p\u003e\n\u003ch3\u003eFAST-PLV Manufacturing and Validation\u003c/h3\u003e\n\u003cp\u003eLipid formulation composition, manufacturing, and validation was described previously\u003csup\u003e46\u003c/sup\u003e. Briefly, individual lipids were heated in a 37\u0026deg;C water bath for 1 minute, vortexed for 10 seconds each, then combined and vortexed for 10 seconds. The combined lipid mixture was dehydrated in a rotavapor at 60 rpm for 2 hours, under vacuum, then rehydrated with 14 mL 100% ethanol, and sonicated (Branson 2510 Sonicator) at 37\u0026deg;C, set to sonication of 60. The lipid formulation was aliquoted in 500 \u0026micro;L batches and stored at -20\u0026deg;C. The NanoAssemblr Benchtop microfluidics mixing instrument (Precision NanoSystems, Vancouver, BC, NIT0013, and NA-1.5-88, respectively) was used to mix the organic and aqueous solutions and make the PLVs. The organic solution consisted of lipid formulation in EtOH. The aqueous solution consisted of pDNA, 5 nM FAST protein, and 10 mM acetate buffer (pH 4.0). NanoAssemblr was run at a total flow rate of 12 mL/min and a 3:1 aqueous to organic flow rate ratio. PLVs were dialyzed in 8000 MWCO dialysis tubing (BioDesign, D102). The loaded tubing was rinsed with 5 mL of double distilled water and dialyzed in 500 mL of Dialysis Buffer (ENT1844) with gentle stirring (60 rpm) at ambient temperature for 1 hour and was repeated twice with fresh Dialysis Buffer. PLVs were concentrated using a 100 kDa Ultra filter (Amicon, UFC810096) according to the manufacturer\u0026rsquo;s instructions. PLVs were filter sterilized through 0.2 \u0026micro;m Acrodisc Supor filters (Amicon, UFC910008). Particle size, polydispersity index (PDI), and zeta potential was measured on final samples using the Malvern Zetasizer Range and a Universal 'Dip' Cell Kit (Malvern, ZEN1002) following the manufacturer\u0026rsquo;s instructions. The nucleic acid encapsulation efficiency and concentration was calculated using a modified Quant-IT PicoGreen dsDNA assay (Thermo Fisher Scientific, Edmonton, Canada).\u003c/p\u003e\n\u003cdiv\u003e\n\u003ch2\u003eIn Vitro Transfection\u003c/h2\u003e\n\u003cp\u003eAll cells were counted using a hemocytometer prior to plating. All pDNA constructs were prepared with Fusogenix \u003cem\u003ein vitro\u003c/em\u003e transfection reagent (formulation 37N as described previously\u003csup\u003e46\u003c/sup\u003e, with slight modifications to the formulation to increase \u003cem\u003ein vitro\u003c/em\u003e transfection efficiency, stability, and tolerability). Lipid and pDNA were prepared in separate 1.5 ml tubes. pDNA was added to 10 mM acetate buffer (pH 4.0) to reach a final concentration of 100 ng/uL, and modified lipid formulation 37N was added to 10 mM acetate buffer (pH 4.0) at a 5:1 molar ratio of ionizable lipid:pDNA. The pDNA tube was added dropwise to the lipid tube, gently mixed by inverting, and left at room temperature before adding to cells. For the luciferase assay, 5,000 cells were seeded into 96-well plates (250\u0026micro;L per well) and immediately transfected with the PLV platform. Plates were incubated for 72 hours, following which, a luciferase reporter assay was used to measure expression levels of FLuc produced by p16 promoter. Cell culture media was removed from cells growing in a 96-well plate, and cells washed with 1x PBS. A 50-microliter aliquot of reporter lysis buffer (Promega, Cat# E397A) was added to the cells. The cells were mixed and incubated at room temperature for 15 mins on an orbital shaker. D-luciferin (150\u0026micro;g/mL, GOLDBIO, LUCK-100) was dissolved in 100 mM Tris-HCl (pH 7.8), 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 2 mM EDTA, 4 mM DTT, 250 \u0026micro;M acetyl-CoA, and 150 \u0026micro;M ATP. The luciferin substrate (100 \u0026micro;L) of was added via auto-injector to each well immediately before measurement (1\u0026ndash;2 second settling time). Luminescence was measured via the FLUOSTAR Omega fluorometer using the MARS data analysis software for analysis. Cells being transfected for cell death assays or Western blotting were seeded at 500,000 cells/well in a 6-well plate and immediately transfected with 10 \u0026micro;g pDNA (p53-iCasp9, p53-iCasp9-GFP, or p53-inactive). Seventy-two hours after transfection with p53-iCasp9-GFP or p16-iCasp9-GFP, H1299 cells have 100 nM RAPA (CID) or vehicle control added for 24 hours before microscopy. Images were analyzed for the number of GFP expressing cells using ImageJ\u003csup\u003e69\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003eWestern Blot\u003c/h2\u003e\n\u003cp\u003eCells were lysed in ice-cold Pierce RIPA buffer (Thermo Scientific, Cat. No. 89900) 48 hours after transfection with p53-iCasp9. Protein amount was determined using the Pierce BCA protein assay (Thermo Scientific, Cat. No. 23225). Equal amounts of total protein from each lysate were loaded onto Mini-PROTEAN 4\u0026ndash;20% Gradient TGX precast gels (BIO-RAD, Cat. No. 456\u0026ndash;1095). Separated proteins were transferred to nitrocellulose membranes (BIO-RAD, Cat. No. 1620112). Membranes were blocked with fluorescent Western blocking buffer (Rockland, Cat. No. MB-070) for 1 hour at room temperature. Primary antibodies were diluted 1:1000 in blocking buffer and added to the membranes overnight at 4˚C with shaking. Goat anti-rabbit Alexa Fluor 680 (Thermo Scientific, Cat. No. A27042), or goat anti-mouse Alexa Fluor 750 (Thermo Scientific, Cat. No. A-21084) were diluted 1:10000 in blocking buffer and added for 1 hour at room temperature in the dark. Membranes were visualized on the LI-COR Odyssey.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003eImaging Flow Cytometry: SA-\u0026beta;-Gal Staining\u003c/h2\u003e\n\u003cp\u003eUsing the method for SA-\u0026beta;-Gal staining developed by Biran et al.\u003csup\u003e70\u003c/sup\u003e with slight modifications. Cells are lifted, washed, and fixed with 2% paraformaldehyde for 5 minutes. Cells are then washed with 1mM MgCl2/PBS (pH 6 for human cells, pH 5.5 for mouse cells) twice, before resuspending in SA-\u0026beta;-Gal staining buffer: 1mg/ml X-Gal (Sigma Aldrich, Cat# B4252), 5mM K3[Fe(CN)6] (Sigma Aldrich, Cat# 244023), 5mM K4[Fe(CN)6]\u0026middot;3H2O (Sigma Aldrich, Cat# 455989) in 1mM MgCl2/PBS (pH 6 for human cells, pH 5.5 for mouse cells). Cells are incubated for 12 hours in a 37˚C incubator with no CO2 in the dark. Cells are washed twice with flow cytometry buffer and stained with Hoechst 33342 if analysis is to be done immediately. Cells were washed and resuspended in 50 \u0026micro;L of flow cytometry buffer and run on the Amnis ImageStream Mark II imaging flow cytometrer. Cells were gated based on their Area vs Aspect Ratio to identify single cells, then captured based on a positive nuclear stain. SA-\u0026beta;-Gal staining intensity is determined via the mean pixel intensity of the bright field channel (lower values\u0026thinsp;=\u0026thinsp;higher stain intensity), typically a value of -100 to -150 represents the upper cut-off for senescent cells. At least 10,000 events were captured before data was analyzed on the IDEAS imaging software. For SA-\u0026beta;-Gal kidney single cell suspensions, due to a high degree of debris impacting the analysis a mask recapitulating the area occupied by the nuclear stain is generated. This mask is then applied to the bright field channel and expanded 20% to ensure only nucleated cells are included in SA-\u0026beta;-Gal analysis. Cell area is calculated from pixel area with the conversion: 1\u0026micro;m2\u0026thinsp;=\u0026thinsp;4 pixels.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003eMouse Studies\u003c/h2\u003e\n\u003cp\u003eAll animal studies were carried out according to the guidelines of the Canadian Council on Animal Care (CCAC) and approved by the University of Alberta Animal Care and Use Committee, or the European Community Council Directives of 2010/63/UE and the protocol was approved according to current Italian law (D.Lgs. n. 26/2014) by the Organismo Preposto al Benessere Animale (OPBA, animal care and health committee) of IRCCS INRCA and by the General Direction of Animal Health and Veterinary Drugs of the Italian Ministry of Health with the authorization n\u0026deg; 622/2020-PR, where applicable. Mice were group-housed in IVCs under SPF conditions, with constant temperature and humidity with lighting on a fixed light/dark cycle (12-hours/12-hours) and ad libitum access to food and water. FAST-PLVs were delivered via intravenous injection via the lateral tail vein with a max volume of 200 \u0026micro;L. Rapamycin was dissolved in 5%PEG400/5%Tween-80 (in H\u003csub\u003e2\u003c/sub\u003eO) to give an amount equivalent to 0.1 mg/kg per 100\u0026micro;L and is administered via intraperitoneal injection. Mice are sacrificed via CO2 asphyxiation with cervical dislocation to confirm euthanasia. A total of 149 mice, 51 females and 98 males (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD age 24.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 for both sexes) entered the frailty study. After two months of adaptation to manipulations and tests, the mice were sequentially randomized into two age-matched experimental groups: a control group (A group), receiving a monthly i.v. injection of empty PLV, and a treatment group (B group), receiving a monthly i.v. injection of senolytics PLV. All experimenters were blinded to the treatment conditions until the end of the study. Non-invasive measurements of frailty (both clinical frailty index and fried phenotype) were performed once a month in all mice from the inclusion (3rd month, baseline) up to the end of the study. We also recorded the time-to-death data for each mouse and performed necropsies to establish pathologies at death. Mortality occurred when animals died suddenly or were euthanized due to severe illness.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003eImmunohistochemistry Staining\u003c/h2\u003e\n\u003cp\u003eHeat-induced antigen retrieval for IHC samples was conducted by immersing rehydrated slides in 10mM sodium citrate (pH 6) and heating until boiling occurred. Slides were blocked in 10% normal rabbit serum (Cat. No. 869019-M, Sigma, Oakville, Canada) with 1% bovine serum albumin (BSA, Cat. No. A9418, Sigma, Oakville, Canada) in TBS with 0.1% Tween-20 for one hour at ambient temperature. Anti-p19ARF antibody (Invitrogen, Cat# PA1-30670) was diluted 1:250 in blocking buffer and incubated on sections overnight. Endogenous peroxidase was blocked with 3% H2O2 in PBS. Goat anti-rabbit HRP (Agilent Dako, Cat# P044801-2) was added to slides for 1 hour. Slides were stained with EnVision FLEX DAB\u0026thinsp;+\u0026thinsp;Chromogen (Agilent Dako, Cat# GV82511-2).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003eMeasurement of clinical frailty index\u003c/h2\u003e\n\u003cp\u003eWe measured both the clinical frailty index (FI) and physical frailty in mice as previously described\u003csup\u003e71\u003c/sup\u003e with slight modifications. We measured the FI in mice based on the validated tool described previously\u003csup\u003e72\u0026ndash;74\u003c/sup\u003e. All measurements of frailty were performed within the SPF animal facility of INRCA in a dedicated area. The clinical FI score for each mouse was calculated using the checklist published previously\u003csup\u003e74\u003c/sup\u003e. Clinical assessment included evaluation of the integument, musculoskeletal system, vestibulocochlear and auditory systems, ocular and nasal systems, digestive system, urogenital system, respiratory system, signs of discomfort, as well as body weight and body surface temperature (collected with an infrared thermometer in the ventral zone of the mice). For each parameter, a score of 0 was given if there was no sign of a deficit, a score of 0.5 denoted a mild deficit and a score of 1 indicated a severe deficit. Deficits in body weight and body surface temperature were scored based on their deviation from average reference values obtained from the entire cohort. Values that differed from reference values by less than 1 SD were scored as 0. Values that were \u0026plusmn;\u0026thinsp;1 SD with respect to the reference value were given a frailty value of 0.25; values that differed by \u0026plusmn;\u0026thinsp;2 SD scored 0.5, those that differed by \u0026plusmn;\u0026thinsp;3 SD scored 0.75 and values that were \u0026gt;\u0026thinsp;3 SD above or below the mean received the maximal frailty value of 1. The sum of the values assigned to the 31 items on the checklist was then divided by 31 to yield a FI score between 0 and 1 for each animal.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003eMeasurement of physical frailty\u003c/h2\u003e\n\u003cp\u003eThe measurement of physical frailty in mice was performed following the same procedure described to translate the physical frailty screening performed in humans\u003csup\u003e75\u003c/sup\u003e to mice (\u003csup\u003e52,76,77\u003c/sup\u003e). In order to ensure testing reliability, we performed multiple measurements for each of the five criteria of the frailty assessment (shrinking, weakness, exhaustion, slowness and sedentarity) and the same testers performed all measurements.\u003c/p\u003e\n\u003cp\u003eEach criteria and the respective measurements are listed below:\u003c/p\u003e\n\u003cp\u003e1) Shrinking: was assessed through a composite score reflecting the body condition of the mice that included current body weight and body length of the mice. The weight was corrected to the previous measurement in the case we detected an increase in weight due to the presence of a tumor or distended abdomen. The body length (nose to base of tail) was measured during the locomotor activity test when the mouse was not in resting state. The locomotor activity area was calibrated with a ruler and the mean of 3 length measurements was calculated using the video analysis software Tracker (v. 5.1.5, https://physlets.org/tracker/).\u003c/p\u003e\n\u003cp\u003e2) Weakness: This criterion was assessed through a composite score reflecting the forelimb grip strength of the mice that included the measurements from 4 different tests.\u003c/p\u003e\na) Grip strength meter test (Ugo Basile, Varese, Italy) with a plastic grid\u003csup\u003e71,78\u003c/sup\u003e\u003cbr /\u003e\n\u003cp\u003eb) Grip strength meter test (Ugo Basile, Varese, Italy) with an iron bar.\u003c/p\u003e\n\u003cp\u003ec) Home cage lift test. The mouse was gently held by the base of the tail at the top of an empty cage placed above a scale with rapid response and the mean of the two most negative peaks from about 10 attempts was collected.\u003c/p\u003e\n\u003cp\u003ed) Gripping weights lift test with the modification described elsewhere (\u003csup\u003e71,79,80\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003e3) Exhaustion: This criterion was assessed through a composite score reflecting the endurance capacity of the mice that included treadmill distance (program: starting at 5 rpm for 2 min and increasing speed from 5 to 50 m/s in 2700 s), mean time to fall at rotarod test (program: starting at 5 m/s for 2 min and increasing speed from 5 to 40 rpm in 300 s) and the score of the gripping weights lift test normalized to body weight. This last measurement was indeed correlated with the other endurance measurements (data not shown) as the test includes an endurance component due to the continuous increase of the weight to be lifted by the mouse\u003csup\u003e81\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e4) Slowness: This criterion was assessed through a composite score reflecting four different measurements related to the speed of the mice during their normal locomotion.\u003c/p\u003e\na) We analyzed the distribution of the time spent by the mouse in different speed intervals in an Open Field test (whole test duration 5 min). The speed intervals considered where: I1 (0\u0026ndash;1 cm/s), I2 (1\u0026ndash;5 cm/s), I3 (5\u0026ndash;10 cm/s), I4 (10\u0026ndash;15 cm/s), I5 (15\u0026ndash;20 cm/s), I6 (20\u0026ndash;25 cm/s), I7 (25\u0026ndash;30 cm/s), I8 (30\u0026ndash;35 cm/s), I9 (35\u0026ndash;40 cm/s), I10 (40\u0026ndash;90 cm/s). We recorded the highest speed interval that the mouse ran for at least 3 s and assigned as value of the test the mean speed of the interval (e.g. 12.5 for I4 and 37.5 cm/s for I9) as previously described\u003csup\u003e79,80\u003c/sup\u003e. Locomotor activity was conducted by a 5-min open field test on a white wood-chamber (72\u0026times;72\u0026times;30 cm) surmounted by a Logitech Brio Ultra HD Webcam 4K 1080 P 60FPS (Logitech Lausanne Switzerland). Tracking and analysis was performed with Biobserve Viewer3 (Biobserve GmbH, Germany)\u003csup\u003e79,80\u003c/sup\u003e. b) An additional measurement for slowness was obtained by recording the maximum speed recorded at rotarod test.\u003cbr /\u003e\n\u003cp\u003ec) As previously reported, we also assessed slowness by including the measurement of the mean stride length of the mice with the footprint test\u003csup\u003e82\u003c/sup\u003e as well as\u003c/p\u003e\n\u003cp\u003ed) by measuring the distance between two consecutive hindlimbs paws during a straight walk in the open filed arena (manual tracking of the paws was performed with Tracker v. 5.1.5, https://physlets.org/tracker/).\u003c/p\u003e\n\u003cp\u003eIndeed, there is a strong rationale in support of the relationship between walking speed and stride length, especially in older individuals\u003csup\u003e83\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e5) Sedentarity (low activity): This criterion was assessed by recording two measurements related to the active behavior of the mice during a locomotor activity test:\u003c/p\u003e\na) total track length (total distance in cm) and\u003cbr /\u003e\n\u003cp\u003eb) the time the mice were not resting (speed above 1 cm/s) during the locomotor activity test. Both measurements were recorded automatically by Biobserve Viewer3 (Biobserve GmbH, Germany).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003eDevelopment of the composite functional scores and detection of the frailty phenotype\u003c/h2\u003e\n\u003cp\u003eThe results from the multiple measurements related to the same criterion were combined in a unique score following this procedure. All variables were normalized with the MIN-MAX procedure Z= (Xi \u0026ndash; Min) / (MAX \u0026ndash; MIN) (where Xi is the measurement, Z is the normalized data, MIN and MAX are the minimum and maximum values for X recorded in the population of mice) separately for each sex and the variables assigned to the same criterion were averaged to create a composite score for each criteria. This provided five quantitative composite scores, namely body condition score, strength score, endurance score, speed score and activity score. An overall score representative of physical decline (named Physical Function score) was computed as the mean of the composite scores of the five criteria.\u003c/p\u003e\n\u003cp\u003eTo avoid bias due to potential outliers we used the 95th percentile as maximum valuefor all measurements excluding weight. In this last case, we used as MAX the reference weights for mice aged 10\u0026ndash;20 months in our colony (28.4 and 33.8 for females and males, respectively). Indeed, the weight of C57BL6/J mice increase until 10 months, then remains relatively stable from 10 to 20 months and only later starts to decline. Hence each measurement range from 1 (or slightly above in some exceptional individuals) to 0. Following the percentiles used by Fried et al. in humans\u003csup\u003e75\u003c/sup\u003e and by others in mice (\u003csup\u003e52,76,77,79\u003c/sup\u003e), mice that fell in the bottom 20% of our old cohort for the composite score computed for each criterion were considered positive for frailty for that given criterion. Mice with three or more positive frailty criteria were identified as frail.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003eSA-\u0026beta;-Gal Staining on Frozen Mouse Tissues\u003c/h2\u003e\n\u003cp\u003eTissues were snap frozen in liquid nitrogen and stored in liquid nitrogen until use. Section of 10 \u0026micro;m-thickness were prepared with a cryostat and mounted on slides. Senescence-associated \u0026beta;-galactosidase (SA- \u0026beta;-Gal) staining was performed using a staining kit (Sigma-Aldrich, St.Louis, USA), according to the manufacturer's instructions. Nuclei were counterstained with Nuclear Fast Red (Sigma-Aldrich) and images were acquired using a Zeiss AxioCam HRc mounted on a Leitz Laborlux S light microscope. The percentage of senescent cells was determined by counting of total and SA-\u0026beta;-Gal-positive cells with the positive cell selection tool available in QuPath v. 0.2.3 \u003csup\u003e84\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eA two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test or a one-way analysis of variance (ANOVA) was performed when comparing two groups or more than two groups, respectively. Statistical analysis was performed using Microsoft Excel and Prism 7.0 (GraphPad). Data are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;s.d. The difference was considered significant if \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 unless otherwise indicated).\u003c/p\u003e\n\u003cp\u003eFrailty data were analyzed by generalized linear mixed models (SPSS 26.0, IBM) to account for the longitudinal design of the frailty study in mice. The identifier of each mouse, age group, sex, age of mouse at inclusion, and time were indicated in the model. The linear model was developed assuming normal distribution with the identity link function. The Satterthwaite approximation and robust estimator were used to account for unbalanced data and violation of the assumptions.\u003c/p\u003e\n\u003cp\u003eDifferential patterns of survival due to the treatment were estimated by Kaplan-Meier and Cox-regression (SPSS 26.0, IBM) stratified by sex and taking also accounting for possible confounder variables (age at inclusion and frailty index at baseline).\u003c/p\u003e\n\u003cp\u003eComparisons of SA-\u0026beta;-Gal Staining between control and treatment groups were performed by generalized linear mixed models (SPSS 26.0, IBM) including tissue and treatment as fixed factors, as well as their interaction. The linear model was developed assuming normal distribution with identity link function. The Satterthwaite approximation and robust estimator were used to account for unbalanced data and violations of the assumptions.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll processed data are available in the main text or the extended data materials and source data.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by an operating grant to John D. Lewis from the Canadian Institutes of Health Research (CIHR), in partnership with the Institute of Aging: Research Nova Scotia, reference number VR1-172710. Dr. Lewis holds the Bird Dogs Chair in Translational Oncology funded by the Alberta Cancer Foundation. Research in the laboratory of Dr. Demaria was supported by a VIDI grant from the Nederlandse organisatie voor gezondheidsonderzoek en zorginnovatie (ZonMw) domain of the Dutch Research Council (NWO; #09150172010029) and by a sponsored research agreement with Oisin Biotechnologies. \u0026nbsp;We thank Katia Carmine-Simmen for her technical support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChristensen K, Doblhammer G, Rau R, Vaupel JW (2009) Ageing populations: the challenges ahead. Lancet 374:1196\u0026ndash;1208\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWorld Health (2011) Global health and aging. NIH Publication 11\u0026ndash;7737\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL\u0026oacute;pez-Ot\u0026iacute;n C, Blasco MA, Partridge L, Serrano M, Kroemer G (2013) The hallmarks of aging. 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Diseases 7:17\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBankhead P et al (2017) QuPath: Open source software for digital pathology image analysis. Sci Rep 7\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Senescence, Aging, Senolytic, Senolysis, Gene therapy, Nucleic Acid Delivery, Proteolipid Vehicle, PLV, Inducible Caspase 9, iCasp9, Suicide Gene Therapy","lastPublishedDoi":"10.21203/rs.3.rs-5575296/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5575296/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eApproaches to eliminate senescent cells \u003cem\u003ein vivo\u003c/em\u003e using transgenic mouse models have demonstrated significant improvements in lifespan, reduction in cancer incidence, and amelioration of age-related degeneration. These approaches require, however, that the organism be genetically engineered from the embryo and/or repeatedly dosed for the organism\u0026rsquo;s lifespan, making them challenging to implement in humans using current technologies. To overcome these limitations, we developed a clinically viable senolytic gene therapy consisting of a suicide gene, inducible caspase 9 (iCasp9), under control of the early senescence and tumor suppressive p53 promoter or the late senescence p16\u003csup\u003eInk\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003ea\u003c/sup\u003e promoter. \u003cem\u003eIn vitro\u003c/em\u003e, this gene therapy selectively activates in senescent cells and induces caspase-9-dependent apoptosis. When formulated in the FAST-PLV platform and administered systemically to aged mice, the burden of senescent cells was significantly reduced in various tissues, leading to a 123% increase in post-treatment survival for animals given a combination of p16 and p53 targeted senolytic gene therapies. Treated mice showed significantly reduced frailty, increased physical function, and improved heart health. Gross necropsy indicated a 3-fold reduced tumor incidence. In summary, we demonstrate a novel and redosable senolytic genetic medicine approach that improves healthspan by targeting senescent cells based on their transcriptional activity.\u003c/p\u003e","manuscriptTitle":"Systemic Senolysis in Naturally Aged Mice Using a FAST-PLV Gene Therapy Approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-12 09:28:46","doi":"10.21203/rs.3.rs-5575296/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2ed45754-a110-44d4-b542-ee2d97b8dbfe","owner":[],"postedDate":"December 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":41463365,"name":"Biological sciences/Cell biology/Senescence"},{"id":41463366,"name":"Biological sciences/Drug discovery/Biologics/Gene therapy"},{"id":41463367,"name":"Biological sciences/Physiology/Ageing"}],"tags":[],"updatedAt":"2026-01-20T21:40:08+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-12 09:28:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5575296","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5575296","identity":"rs-5575296","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}