Methods of delaying cellular senescence by inhibiting target genes

Abstract

The present invention relates to compositions including targeting agents capable of inhibiting the activity of a target gene regulated by ZBTB2 and selected from NDST3, BATF, AIM2, IFIH1, DDX58, JAM2, 0MA1, and any combination thereof, and their use in methods for inhibiting, delaying, or reversing cellular senescence and for treating aging-related diseases.

Description

[0001] METHODS OF DELAYING CELLULAR SENESCENCE BY INHIBITING TARGET GENES

[0002] FIELD OF THE INVENTION

[0003] The present invention is generally directed to methods and constructs for inhibiting, delaying, or reversing cellular senescence. More specifically, the invention relates to methods and constructs for inhibiting the activity of target genes to inhibit, delay, or reverse cellular senescence and aging-related diseases.

[0004] BACKGROUND OF THE INVENTION

[0005] Aging is a complex process that is characterized by a gradual decline in the body's ability to repair and maintain itself which is influenced by a combination of genetic, environmental, and lifestyle factors. Aging thus results in general deterioration of health, including reduced muscle mass and bone density, decreased cognitive function, and an increased risk of chronic diseases. A large number of molecular, cellular, tissular, and systemic biological processes have been associated with aging, such as accumulation of DNA damage, telomere shortening, mitochondrial dysfunction, epigenetic reprogramming, chronic inflammation, cellular senescence, impaired protein homeostasis, altered cellular communication, and some others. Whereas the causal relationships between these mechanisms and aging are poorly understood, one of the most prominent features of an aging mammalian organism is the accumulation of senescent cells in various tissues and organs.

[0006] Cellular senescence is a fundamental biological process in which cells permanently lose the ability to proliferate. Senescent cells accumulate with age in many tissues, including the skin, liver, lung, and kidney, and are associated with a variety of age-related diseases such as cancer, atherosclerosis, and osteoarthritis. Major mechanisms causing cellular senescence are associated with DNA damage, production of reactive oxygen species (ROS), shortening of telomeres, epigenetic alterations, and activation of oncogenes, which closely resembles the processes associated with aging in general. There exists strong evidence suggesting a driving role of cellular senescence in aging. Thus, studies have shown that the removal of senescent cells can delay the onset of aging-related diseases and increase lifespan. Furthermore, it appears that the fraction of senescent cells in tissues is a biomarker of aging that displays the best correlation with organismal mortality among other hallmarks and biomarkers of aging for both primates and humans. Interestingly, both the mortality and the fraction of senescent fibroblasts typically show exponential growth with age. Senescent cells are characterized by the activation of the senescence-associated secretory phenotype (SASP). The S ASP is a hallmark feature of senescence and refers to the release of pro- inflammatory cytokines, chemokines, growth factors, and other molecules by senescent cells. These SASP factors can promote inflammation, alter tissue architecture, and drive the growth of neighboring cells, including potentially pre-cancerous cells. Therefore, the SASP serves as a mediator, propagating the harmful impacts of senescent cells, both within the local tissue microenvironment and systemically. These effects contribute to the development of pathological conditions and aging-related diseases.

[0007] Senescent cells can be cleared by the immune system, but with age the clearance of these cells becomes less efficient, leading to the accumulation of senescent cells in tissues and organs. Consequently, interventions that can achieve targeted clearance of senescent cells in an aging organism have been proposed and widely acknowledged as an anti-aging treatment with very high potential. Thus, it has been shown that elimination of senescent p2C cells in mice can reduce the incidence of aging-related diseases and morbidities. In humans, the clearance of senescent cells has been shown to hold promise in a clinical trial to ameliorate inflammatory kidney conditions.

[0008] Cellular senescence has been the focus of research for many decades now. It appears that there is no single mechanism that drives senescence. Instead, a wide range of mechanisms and pathways have been implicated. Broadly, two types of stimuli that lead to cellular senescence can be identified, which are a) intrinsic and b) extrinsic. The former is typically associated with the exhaustion of replicative potential leading to the attrition of telomeres and the permanent cell cycle arrest via the pl6arf4 or associated pathways. This type of senescence is also often referred to as replicative senescence. The extrinsic pathways are more diverse and can be triggered by oncogenes, DNA damage, e.g. from chemical genotoxins or ionizing radiation, ROS production, abnormal epigenetic regulation, etc. While the original stimuli leading to senescence differ, the pathways often converge on common cell cycle arrest signalling cascades involving p21, p!9, p53 and Rb. Cellular and tissue context (type of cells, microenvironment) define a particular set of molecular changes in a cell that may eventually contribute to senescence, thus making it difficult to identify generic genetic determinants and drivers of senescence. Identification of molecular targets that can delay or even reverse senescence is a promising strategy to combat senescence in tissues and thus to improve health and increase the life span and health span in humans. Several studies have successfully demonstrated that reprogramming of senescent cells using the cyclic overexpression of Oct4, Sox2, Klf4, and c-Myc (OSKM) ameliorates a range of physiological, cellular, and molecular markers of senescence in both in vitro (Lapasset et al. 2011. Genes Dev 25(21 ) :2248-2253) and in vivo conditions, with the mouse study also demonstrating an increase in life span of treated animals (Ocampo et al. 2016. Cell 167(7):1719-1733 el712). These results indirectly support the possibility of reversing senescence by the regulation of gene expression. However, the effects of transitory induction of the OSKM are mediated by epigenetic reprogramming primarily aiming at the generation of induced pluripotent stem cells. Furthermore, this method is associated with the risk of teratoma and cancer formation when applied in vivo (Abad et al., 2013. Nature 502:340-345).

[0009] There is therefore a continuous need to identify improved gene targets that may be manipulated in order to inhibit senescence.

[0010] SUMMARY OF INVENTION

[0011] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other advantages or improvements.

[0012] In some embodiments, there is provided a method for inhibiting, delaying, or reversing cellular senescence, the method including contacting an aged cell with a composition including a targeting agent capable of inhibiting activity of a target gene regulated by ZBTB2 and selected from NDST3, BATF, AIM2, IFIH1, DDX58, JAM2, OMA1, and any combination thereof.

[0013] In some embodiments, the aged cell is a cell capable of differentiation. In some embodiments, the aged cell is a fibroblast. In some embodiments, the aged cell is from a tissue selected from blood, bone marrow, brain, connective tissue, heart, intestine, kidney, liver, lung, mucosa, muscle, pancreatic, and skin.

[0014] In some embodiments, the aged cell is a human cell. In some embodiments, the aged cell is from an aged subject or a subject suffering from an aging-related disease. In some embodiments, the aged subject is at least 50 years old.

[0015] In some embodiments, the targeting agent includes a small molecule drug.

[0016] In some embodiments, the targeting agent includes a nucleic acid molecule. In some embodiments, the nucleic acid molecule includes a targeting nucleotide sequence encoding an inhibitory RNA specific for the target gene. In some embodiments, the inhibitory RNA is selected from shRNA, siRNA, and miRNA. In some embodiments, the nucleic acid molecule is included in a vector suitable for delivery of the nucleic acid molecule to the cell. In some embodiments, the vector is selected from a viral vector, a non- viral vector, a retroviral vector, and a lentiviral vector.

[0017] In some embodiments, the method is an in vitro or ex vivo method.

[0018] In some embodiments, the method is an in vitro method. In some embodiments, the method is an ex vivo method.

[0019] In some embodiments, there is provided a knock-down (KD) cell produced by the method disclosed herein.

[0020] In some embodiments, the KD cell includes rejuvenated features including at least one of: a reduced SA-b-gal activity level; a reduced nuclear size; an increased cell motility; higher average expression levels of K67+and / or EdU+; lower average p21+expression levels; lower levels of at least one cell cycle regulation selected from CDKN1A / P21, CDKN2D / P19 and one SASP cytokine gene; and lower levels of at least one senescence-associated secretory phenotype (SASP) cytokine selected from IL-6, DPP4, GRO, MMP-9, RANTES, and VEGF; in the KD cell compared to the aged cell. In some embodiments, the rejuvenated features include SA-b-gal activity level being at least 20% lower in the KD cell than in the aged cell. In some embodiments, the rejuvenated features include nuclear size being at least 20% lower in the KD cell than in the aged cell. In some embodiments, the rejuvenated features include cell motility being at least 20% higher in the KD cell than the aged cell motility. In some embodiments, the rejuvenated features include average expression levels of K67+and / or EdU+being at least 20% higher in the KD cell than in the aged cell. In some embodiments, the rejuvenated features include average p21+expression levels being at least 20% lower in the KD cell than in the aged cell. In some embodiments, the rejuvenated features include levels of at least one cell cycle regulation selected from CDKN1A / P21, CDKN2D / P19 and one SASP cytokine gene in the KD cell being at least 20% lower than corresponding levels in the aged cell. In some embodiments, the rejuvenated features include levels of at least one senescence-associated secretory phenotype (SASP) cytokine selected from IL-6, DPP4, GRO, MMP-9, RANTES, and VEGF in the KD cell being at least 20% lower than the corresponding levels in the aged cell.

[0021] In some embodiments, there is provided a composition including the KD cell disclosed herein, or a targeting agent capable of inhibiting the activity of a target gene regulated by ZBTB2 and selected from NDST3, BATF, AIM2, IFIH1, DDX58, JAM2, 0MA1, and any combination thereof, for use in inhibiting, delaying, or reversing cellular senescence in a subject over the age of 50, and / or who is suffering from an aging-related disease.

[0022] In some embodiments, there is provided a method of treating aging-related diseases in a subject in need thereof by inhibiting, delaying, or reversing cellular senescence, the method including administering to the subject a composition including the KD cell disclosed herein or a targeting agent capable of inhibiting activity of a target gene regulated by ZBTB2 and selected from NDST3, BATF, AIM2, IFIH1, DDX58, JAM2, 0MA1, and any combination thereof, wherein the subject is over the age of 50, and / or is suffering from an aging-related disease. In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the figures and by study of the following detailed descriptions.

[0023] BRIEF DESCRIPTION OF DRAWINGS

[0024] The invention will now be described in relation to certain examples and embodiments with reference to the following illustrative figures.

[0025] Figs. 1A-1C show effects of target genes knockdown on cellular senescence Fig. 1A. mRNA expression levels of the target genes 2 weeks after transduction of normal human fibroblasts from aged donors with targeting shRNA lentivirus relative to control (same cells transduced with non-targeting lentivirus), as measured by RT-qPCR. Fig. IB. The % of SA-b-gal positive cells compared to young and aged. shNT indicates control transduction with a nontargeting construct. Fig. 1C similar to Fig. IB, separate results for AIM2. Significance was determined using a 2-tailed t-test and is designated as *, ** and *** for P<0.05, <0.01 and <0.001, respectively, in comparison to aged shNT.

[0026] Figs. 2A-2B show effect of target genes knockdown on morphological and functional cell properties of aged cells. Pooled cells were transduced with control (shNT) or targeting shRNA lentiviral vectors and the nuclear size Fig. 2A and cell motility Fig. 2B, were quantified 2 weeks post-transduction. Statistical significance was tested using the Mann-Whitney Rank Sum Test for nuclear size, and One-Way ANOVA for multiple comparisons of cell motility. The significant difference is designated as and *** forP<0.01 and P.0.001, respectively, in comparison to Aged shNT group.

[0027] Figs. 3A-3C shows the effects of target genes knockdown on cell proliferation features of aged cells . Pooled cells were transduced with control (shNT) or targeting lentiviral shRNA vectors and the fraction of Ki67-positive Fig. 3A, EdU positive Fig. 3B, and p21-positive Fig. 3C cells were quantified 2 weeks post-transduction. Statistical significance was tested using one-way ANOVA for multiple comparisons and designated as * and *** for P<0.05 and P<0.001, respectively, in comparison to the Aged shNT group.

[0028] Figs. 4A-4F show effects of target genes knockdown on the senescence markers CDKN1A / P21 - Fig. 4A, CDKN2D / P19 - Fig. 4B, and SASP (DPP4) - Fig. 4C; and the sternness (differentiation) markers (CMYC) - Fig. 4D, SOX2 - Fig. 4E, and OCT4 / POU5F1 - Fig. 4F. Pooled cells were transduced with control (shNT) or targeting lentiviral shRNA vectors and mRNA levels of the indicated genes were measured using RT-qPCR. Results were normalized to the expression of the house-keeping genes ACTB and GUSB. Comparisons were performed by one-way ANOVA against control Aged shNT cells, *<0.05, ** p<0.005, *** p<0.0005, **** p<0.0001.

[0029] Fig- 5 shows the effects of target genes knockdown on the mRNA levels of SASP cytokine genes (CCL5, CXCL1, DPP4, IL-6, MMP9, and VEGFA)) in aged cells. Pooled cells were transduced with control (shNT) or targeting shRNA lentiviral vectors and the levels of the indicated cytokine genes mRNA were measured using qPCR arrays. Results are presented as a heatmap with the numbers relative to the aged shNT cells.

[0030] Figs. 6A-6G show a multivariate PLS-DA analysis of the integral anti-aging effect exerted by the KD interventions Clustering of the experimental replicates in the space of reduced dimensionality is shown in the top panels for NDST3 (Fig. 6A), BATF (Fig. 6B), AIM2 (Fig. 6C), IFIH1 (Fig. 6D), DDX58 (Fig. 6E), JAM2 (Fig. 6F), and 0MA1 (Fig. 6G)). Feature correlation circle reflecting the contribution of individual readouts in the discrimination of clusters in the reduced dimensionality space are shown in the bottom panels; the closer the parameter to the circle of radius 1.0, the greater its contribution or importance.

[0031] DETAILED DESCRIPTION OF THE INVENTION

[0032] In the following description, various aspects of the disclosure will be described. For explanation, specific configurations and details are outlined to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to one skilled in the art that the disclosure may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.

[0033] As described in the present invention, the inventors have identified several target genes that were previously unknown to be related to senescence, including NDST3, BATF, AIM2, IFIH1, DDX58, JAM2, 0MA1, for which reduced expression led to rejuvenation effects. As described below, knock-down (KD) cells for these target genes were produced by transduction of primary cell cultures of human fibroblasts obtained from aged subjects with lentiviral vectors carrying shRNAs specific to these genes, and various cellular senescence-related characteristics of the KD cells were compared to fibroblasts from young and aged human subjects.

[0034] The cellular senescence-related characteristics tested included senescence markers (SA-b- gal, Figs. 1B-C), nuclear size (Fig. 2A), cell motility (Fig. 2B), proliferation properties as determined by % Ki67+, EdU+, and p21+cells (Figs. 3A, 3B, and 3C, respectively), levels of cell cycle arrest regulation, inflammation, and de-differentiation markers (Fig. 4), and levels of the expression of SASP (senescence-associated secretory phenotype) cytokine genes (Fig. 5). As can be seen from the figures, KD of the target genes had rejuvenating effects on the aged cells, resulting in their functions being generally “younger” than the reference aged cells. Results of integrative multivariate analysis using partial-least squares discriminant analysis (PLS-DS) (Fig. 6) clearly confirmed that the KD aged cells had characteristics of young cells.

[0035] Accordingly, these genes may serve as target genes for down-regulation, in order to inhibit, delay, or reverse cellular senescence.

[0036] It was further found by the inventors (see Example 8) that all 7 target genes (NDST3, B ATF, AIM2, IFIH1, DDX58, JAM2, 0MA1) were regulated by the transcription factor ZBTB2. This common regulation may be relevant to their common function of promoting senescence.

[0037] Accordingly, in some embodiments, the present invention relates to compositions including a targeting agent capable of reducing expression of a target gene regulated by ZBTB2 for use in inhibiting, delaying, or reversing cellular senescence-related or aging-related diseases, or in treating a subject in need thereof.

[0038] In some embodiments, there is provided a method for inhibiting, delaying, or reversing cellular senescence, the method including contacting an aged cell with a composition including a targeting agent capable of inhibiting the activity of a target gene regulated by ZBTB2 and selected from NDST3, BATF, AIM2, IFIH1, DDX58, JAM2, 0MA1, and any combination thereof.

[0039] Cellular senescence is a process which causes cells to lose the ability to proliferate. Senescent cells are characterized by the activation of the S ASP, a hallmark feature of senescence referring to the release of pro -inflammatory cytokines, chemokines, growth factors, and other molecules by senescent cells. These SASP factors can promote inflammation, alter tissue architecture, and drive the growth of neighboring cells, including potentially pre-cancerous cells. Cellular senescence appears to be related to aging of an individual carrying senescent cells, inter alia, by a variety of aging-related diseases such as cancer, atherosclerosis, osteoarthritis, hypertension, hearing loss, obesity, cataracts and refractive errors, chronic obstructive pulmonary disease, diabetes, depression, dementia, Myocardial Ischemia, Stroke, Multiple Sclerosis (MS), Alzheimer’s disease (AD), Amyotrophic Lateral Sclerosis (ALS), Motoneuron Disease (MND), Huntington disease, Parkinson’s disease (PD), osteoporosis, Age-related Macular Degeneration (AMD), Chronic Obstructive Pulmonary Disease (COPD), idiopathic pulmonary fibrosis (IPF), Sarcopenia, Systemic Lupus Erythematosus (SLE), Schnitzler’s syndrome, Rheumatoid Arthritis (RA), Polymyalgia Rheumatica (PMR), Giant Cell Arteritis, Hashimoto's thyroiditis, Graves' disease, Sjogren's Syndrome, Inflammatory Bowel Syndrome (IBD), and other autoimmune diseases that can cause an early onset of age related phenotypes or relevant symptoms in adulthood.

[0040] The phrase “inhibiting, delaying, or reversing cellular senescence”, as used herein, means causing aged cells in a subject, or obtained from a subject, to present with cellular senescence- related features characteristic of cells from a younger subject than the subject from whom the aged cells were obtained or are part of. In other words, treating the cells with the compositions of the invention causes rejuvenation of the cells. It is appreciated that since cellular senescence is related to aging, treating cellular senescence in a subject is expected to result in treatment of aging -related diseases in the subject, as further described below.

[0041] The cellular senescence-related features may be any cellular structural or functional features which are related to senescence. Non-limiting examples for cellular senescence-related features include SA-b-gal senescence marker; nuclear size; cell motility; proliferation properties; levels of cell cycle arrest regulation, inflammation, and de-differentiation markers; levels of SASP cytokines, telomere length, karyomegaly, DNA damage foci, heterochromatic foci, and dysfunctional mitochondria.

[0042] The term “aged cell” relates to a cell which has been obtained from an aged individual, and / or that has at least one characteristic of cellular senescence or of aging, as further defined below.

[0043] The aged cell is a cell which is contacted with the compositions of the invention. The aged cell may be any mammalian cell. In some embodiments, the aged cell is a human cell In some embodiments, the aged cell is a cell capable of differentiation. In some embodiments, the aged cell is a cell in a cell culture. In some embodiments, the aged cell is an isolated cell. In some embodiments, the aged cell is part of an organism, such as a human. In some embodiments, the aged cells are obtained from, or are part of, an aged subject, as defined below. In some embodiments, the aged cells are obtained from, or are part of, a subject suffering from an aging- related disease, as defined below. Non-limiting examples of aged cell types include cells from blood, bone marrow, brain, connective tissue, heart, intestine, kidney, liver, lung, mucosa, muscle, pancreatic, and skin cells. Additional cell types suitable for use with the invention include fibroblasts, T cells, natural killer (NK) cells, mesenchymal stem cells (MSCs), induced pluripotential stem cells (iPSCs), embryonic stem cells (ESCs), Hematopoietic stem cells (HSCs), B cells, CAR-T cells, and macrophages.

[0044] The term “aged” with reference to a subject, relates to a human subject that is at least about 50 years old. In some embodiments, the aged subject is at least about 55, 60, 65, 70, 75, or 80 years old. In a parallel way, when the term “aged” is used to describe cells, it relates to cells obtained from, or part of, an aged subject.

[0045] The term “contacting” relates to adding the compositions described herein to the cells by any suitable method, for delivering the targeting agent into the cell. The contacting may refer to contacting the cells in vitro, such as contacting isolated cells in a test tube or in a cell culture, or to the administration of the composition to a subject in vivo, thereby delivering the composition to the cells of the subject. The contacting may be part of a method of delivery of DNA or RNA to cells, such as transduction or transformation.

[0046] The targeting agent is an agent that is capable of specifically inhibiting the activity of a target gene or of a target gene product. Inhibiting activity relates to an intervention which results in lowering or abolishing activity of the targeted gene by any suitable way, including inhibiting activity at the protein level (non-limiting examples include degradation of a protein, suppressing its catalytic functions and abilities to bind partner proteins or complexes), inhibiting activity at the RNA level (non-limiting examples include degradation of the targeted gene mRNA, suppression of its translation by any means), inhibiting expression of the gene, and reprogramming at the DNA level, such as by generating a knock-out (KO) or a knock-down (KD) of the gene.

[0047] In some embodiments, targeting the target gene with the targeting agent results in a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the activity of the target gene compared to the activity of the target gene in the aged cell. The activity of the target gene may be measured depending on the target gene, based on the gene activity.

[0048] In some embodiments, targeting the target gene with the targeting agent results in a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the expression of the target gene compared to expression of the target gene in the aged cell. The expression of the target gene may be measured by any method suitable for measuring gene expression, including but not limited to northern blot, western blot, RT-qPCR, or flow cytometry.

[0049] In some embodiments, the targeting agent includes a small molecule drug, designed for specifically inhibiting the target gene. Small molecules can be designed to target genes by specifically interacting with their products, such as proteins or mRNA. The process begins with a deep understanding of the target's structure and function, often aided by computational modeling and structural biology techniques. Screening and functional assays are typically used to identify lead compounds.

[0050] In some embodiments, the targeting agent includes a nucleic acid molecule including a targeting nucleotide sequence specific to the sequence of the target gene. In some embodiments, the nucleic acid molecule is a DNA molecule. In some embodiments, the nucleic acid molecule is an RNA molecule.

[0051] In some embodiments, the targeting nucleotide sequence encodes a guide RNA for use in a genome editing system such as CRISPR.

[0052] In some embodiments, the targeting nucleotide sequence encodes an inhibitory RNA specific for the target gene. In some embodiments, the inhibitory RNA is selected from shRNA, siRNA, and miRNA.

[0053] In some embodiments, the targeting nucleotide sequence has a length of about 10-500, 10- 100, 10-50, 10-40, 10-30, or 10-25 nt.

[0054] In some embodiments, the targeting nucleotide sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to a sequence of the target gene. In some embodiments, the targeting nucleotide sequence is identical to a sequence of the target gene. In some embodiments, the targeting nucleotide sequence is complementary to a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to an mRNA sequence of the target gene.

[0055] In some embodiments, the targeting nucleotide sequence includes a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to any one of SEQ ID NO: 1-7. In some embodiments, the targeting nucleotide sequence includes a sequence at least 85% identical to any one of SEQ ID NO: 1 7. In some embodiments, the targeting nucleotide sequence includes a sequence at least 95% identical to any one of SEQ ID NO: 1-7.

[0056] In some embodiments, the nucleic acid molecule is included in a vector for delivery of the nucleic acid molecule to the cell. The vector may be any vector suitable for delivery to mammalian cells in vitro or in vivo. In some embodiments, the vector is selected from a viral vector, a non- viral vector, a retroviral vector, and a lentiviral vector.

[0057] Methods of delivery are also known, and non-limiting examples include encapsulation in liposomes or nanoparticles, gene gun biolistic particle delivery methods, and peptide-based delivery methods.

[0058] The term “regulated by” means that the target gene is responsive to a transcription factor (TF). Evidence for responsiveness of the target gene may be obtained, e.g., by experimental evidence showing a correlation between the TF and the target gene expression, or by analysis of the target gene promoter and finding of sequence elements responsive or predicted to bind to the TF.

[0059] ZBTB2 (zinc finger and BTB domain containing 2) is a transcription factor that belongs to the broad family of zinc finger proteins, characterized by their ability to bind DNA, and regulate the expression of genes. It contains a BTB (Broad-Complex, Tramtrack, and Bric-a-Brac) domain and a POZ (Pox virus and Zinc finger) domain. The BTB / POZ domain plays a crucial role in ZBTB2's interactions with other proteins, such as Spl, underscoring its involvement in transcriptional regulation of gene networks of various cellular processes, including proliferation, differentiation, and apoptosis. As explained in Example 8, ZBTB2 was found to regulate all 7 target genes of the invention.

[0060] In some embodiments, the target gene is NDST3. In some embodiments, the target gene is BATF. In some embodiments, the target gene is AIM2. In some embodiments, the target gene is IFIH1. In some embodiments, the target gene is DDX58. In some embodiments, the target gene is JAM2. In some embodiments, the target gene is 0MA1.

[0061] While various methods of inhibiting activity or expression are available, as described above, these methods may lead to the generation of a cell carrying a knocked-down (or a knocked-out) target gene. Two non-limiting examples for such methods include: reprogramming at the DNA level, by modifying the gene sequence to inactivate it, e.g. by gene editing; and inserting an RNA interference agent that is expressed in the cell to inhibit expression of the target gene. One more specific example is the ex vivo editing of T cells as part of adoptive T cell therapy (ACT).

[0062] Accordingly, in some embodiments, when the targeting agent includes a nucleic acid molecule, the methods described herein lead to generating a KD cell for the target gene.

[0063] In some embodiments, the method is an in vitro method. When the method is conducted in vitro, the cell is an isolated cell, not part of a body. In some embodiments, the KD cell is an isolated cell. In some embodiments, the KD cell is in suspension. In some embodiments, the KD cell is in a cell culture. In some embodiments, the targeting agent is a nucleic acid. In some embodiments, the in vitro method generates a KD cell that may be used in an ex vivo method by administering the KD cells to a subject from whom the aged cells were obtained

[0064] In some embodiments, the method is an in vivo method, wherein the contacting includes administering to a subject the composition including the targeting agent, wherein the subject is at least 50 years old, and / or is suffering from an aging-related disease.

[0065] In some embodiments, the method is an ex vivo method, further including administering to a subject the composition including the KD cell, wherein the subject is at least 50 years old, and / or is suffering from an aging-related disease. It is noted that the term ex vivo implies that the aged cells used to generate the KD cells were obtained from the subject, or are autologous to the subject. Nevertheless, the method also encompasses a scenario in which the KD cells were not generated from cells of the subject, and are allogeneic to the subject.

[0066] In some embodiments, there is provided a KD cell produced by the methods disclosed herein.

[0067] Definitions and embodiments mentioned above and which may be relevant to the KD cells also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis). In some embodiments, the KD cell is an isolated cell. In some embodiments, the KD cell is in suspension. In some embodiments, the KD cell is in a cell culture.

[0068] In some embodiments, the KD cell exhibits rejuvenated features.

[0069] The term “rejuvenated features” relates to cellular senescence-related characteristics, which in the KD cell are closer to young cells (or more different from aged cells) compared to the aged cells. Some non-limiting examples are provided below.

[0070] In some embodiments, the rejuvenated features include a lower SA-b-gal activity level in the KD cells compared to the aged cells. In some embodiments, the lower SA-b-gal activity level is at least about 10%, 20%, 30%, 50%, 60%, or 70% lower in the KD cells compared to the aged cells.

[0071] In some embodiments, the rejuvenated features include a lower nuclear size in the KD cells compared to the aged cells. In some embodiments, the lower nuclear size is at least about 10%, 20%, 30%, 50%, 60%, or 70% lower in the KD cells compared to the aged cells.

[0072] In some embodiments, the rejuvenated features include a higher cell motility in the KD cells compared to the aged cells. In some embodiments, the higher cell motility is at least about 10%, 20%, 30%, 50%, 60%, or 70% higher in the KD cells compared to the aged cells.

[0073] In some embodiments, the rejuvenated features include higher average K67+and / or EdU+expression levels in the KD cells compared to the aged cells. In some embodiments, the higher average K67+and / or EdU+expression levels are at least about 10%, 20%, 30%, 50%, 60%, or 70% higher in the KD cells compared to the aged cells.

[0074] In some embodiments, the rejuvenated features include lower average p21+expression levels in the KD cells compared to the aged cells. In some embodiments, the lower average p21+expression levels are at least about 10%, 20%, 30%, 50%, 60%, or 70% lower in the KD cells compared to the aged cells.

[0075] In some embodiments, the rejuvenated features include lower levels of at least one cell cycle regulation gene or inflammation gene, and de-differentiation marker selected from CDKN1A / P21, CDKN2D / P19, DPP4, CMYC, SOX2, and OCT4 / POU5F1 in the KD cells compared to the aged cells. In some embodiments, the lower levels of at least one cell cycle regulation, inflammation, and de-differentiation marker selected from CDKN1A / P21, CDKN2D / P19, DPP4, CMYC, SOX2, and OCT4 / POU5F1 are at least about 10%, 20%, 30%, 50%, 60%, or 70% lower in the KD cells compared to corresponding levels in the aged cells.

[0076] In some embodiments, the rejuvenated features include lower levels of mRNA of at least one senescence-associated secretory phenotype (SASP) cytokine gene selected from CCL5 / RANTES, CXCL1 / GRO, DPP4, IL-6, MMP-9, and VEGFA in the KD cells compared to the aged cells. In some embodiments, the lower levels of at least one senescence-associated secretory phenotype (SASP) cytokine selected from CCL5 / RANTES, CXCL1 / GRO, DPP4, IL-6, MMP-9, and VEGFA are at least about 10%, 20%, 30%, 50%, 60%, or 70% lower in the KD cells compared to the aged cells.

[0077] In some embodiments, there is provided a composition including a targeting nucleotide sequence including a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to any one of SEQ ID NO: 1-7, and a pharmaceutically acceptable carrier. In some embodiments, there is provided a composition including a targeting nucleotide sequence including a sequence at least 85% identical to any one of SEQ ID NO: 1-7, and a pharmaceutically acceptable carrier. In some embodiments, there is provided a composition including a targeting nucleotide sequence including a sequence at least 95% identical to any one of SEQ ID NO: 1-7, and a pharmaceutically acceptable carrier.

[0078] Definitions and embodiments mentioned above and which may be relevant to the compositions also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

[0079] In some embodiments, the targeting nucleotide sequence is an inhibitory RNA. In some embodiments, the inhibitory RNA is an miRNA, an siRNA, or an shRNA.

[0080] In some embodiments, there is provided a composition including the KD cell disclosed herein or a targeting agent capable of inhibiting activity of a target gene regulated by ZBTB2 and selected from NDST3, BATF, AIM2, IFIH1, DDX58, JAM2, 0MA1, and any combination thereof, for use in treating an aging-related disease in subject in need thereof by inhibiting, delaying, or reversing cellular senescence.

[0081] Definitions and embodiments mentioned above and which may be relevant to the use embodiments also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above mutatis mutandis).

[0082] In some embodiments, the composition is a pharmaceutical composition, further including a pharmaceutically acceptable carrier.

[0083] In some embodiments, the pharmaceutically acceptable carrier is a buffer, diluent, adjuvant, excipient, or vehicle suitable for administration of the targeting agent. In some embodiments, the pharmaceutically acceptable carrier may be suitable for intravenous infusion. In some exemplary embodiments, the carrier may be DMSO (for example, at about 10%). In some embodiments, the pharmaceutically acceptable carrier may include a binder, such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monohydrate; a disintegrating agent, such as alginic acid, maize starch and the like; a lubricant or surfactant, such as magnesium stearate, or sodium lauryl sulphate; and a glidant, such as colloidal silicon dioxide.

[0084] In some embodiments, the subject is over the age of 50, and / or is suffering from an aging- related disease.

[0085] In some embodiments, there is provided a method of treating an aging-related disease in a subject in need thereof by inhibiting, delaying, or reversing cellular senescence in the subject, the method including administering to the subject a composition including a targeting agent capable of inhibiting activity of a target gene regulated by ZBTB2 and selected from NDST3, BATF, AIM2, IFIH1, DDX58, JAM2, 0MA1, and any combination thereof, wherein the subject is over the age of 50, and / or is suffering from an aging-related disease.

[0086] Definitions and embodiments mentioned above and which may be relevant to the treatment methods also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

[0087] The term “aging-related disease” encompasses diseases which have a prevalence that increases with age, or induce early onset of aging-related phenotypes, premature aging, or that appear mainly over a certain age.

[0088] In some embodiments, the aging-related disease is selected from diseases including cancer, atherosclerosis, osteoarthritis, hypertension, hearing loss, obesity, cataracts and refractive errors, chronic obstructive pulmonary disease, diabetes, depression, dementia, Myocardial Ischemia, Stroke, Multiple Sclerosis (MS), Alzheimer’s disease (AD), Amyotrophic Lateral Sclerosis (ALS), Motoneuron Disease (MND), Huntington disease, Parkinson’s disease (PD), osteoporosis, Age- related Macular Degeneration (AMD), Chronic Obstructive Pulmonary Disease (COPD), idiopathic pulmonary fibrosis (IPF), Sarcopenia, Systemic Lupus Erythematosus (SLE), Schnitzler’s syndrome, Rheumatoid Arthritis (RA), Polymyalgia Rheumatica (PMR), Giant Cell Arteritis, Hashimoto's thyroiditis, Graves' disease, Sjogren's Syndrome, Inflammatory Bowel Syndrome (IBD), and an autoimmune disease that can cause an early onset of age related phenotypes or relevant symptoms in adulthood.

[0089] Additional more specific aging-related diseases intended to be included in the present invention are diseases of accelerated aging, in which the body ages faster than normal, presenting with features that are typical to individuals of a higher chronological age.

[0090] In some embodiments, the aging -related disease is further selected from additional diseases including progeria, progeroid syndrome, Hutchinson-Gilford Progeria Syndrome (HGPS), atypical and typical Werner Syndrome, and Cockayne Syndrome. HGPS is a rare genetic condition characterized by rapid aging in childhood. Patients typically exhibit growth delays, loss of body fat and hair, aged-looking skin, stiffness of joints, and cardiovascular disease, leading to an early death usually from heart attack or stroke. WS is an adult-onset premature aging syndrome that becomes apparent during adolescence or early adulthood. It is characterized by short stature, early graying and loss of hair, skin atrophy, cataracts, diabetes mellitus, osteoporosis, and an increased risk of cancer. AWS presents similarly to the typical form but with a later onset and a milder phenotype. CS is a disorder characterized by poor growth, premature aging, photosensitivity, progressive neurological impairment, and a shortened lifespan.

[0091] The term “treating ”, as used herein, refers to means of obtaining a desired physiological effect, in this case, partially or completely curing the infection and / or symptoms thereof. The term may relate to ameliorating or inhibiting the infection, i.e. arresting its development or curing it completely by eradicating the virus.

[0092] The administering may be by any method or route suitable for treating the aging-related disease. The dosage and frequency of administration may be determined by a physician based on the type and severity of the disease or condition.

[0093] The administration may be a systemic or local. The route of administration may be, for example, intravenous, subcutaneous, intramuscular, intratumoral, intranasal, or topical administration.

[0094] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

[0095] The term “nt” means nucleotide

[0096] The term "a" and "an" refers to one or more than one (i.e., to at least one) of the grammatical objects of the article. By way of example, “an element” means one element or more than one element.

[0097] The term "about", when referring to a measurable value such as an amount, a ratio, and the like, is meant to encompass variations of ±10% of the indicated value, as such variations are also suitable to perform the disclosed invention. Any numerical values appearing in the application are intended to be construed as if preceded by “about” unless indicated otherwise.

[0098] While certain embodiments of the invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions, and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the present invention as described by the claims, which follow.

[0099] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0100] EXAMPLES

[0101] Materials and Methods

[0102] Cells

[0103] The study used primary human dermal fibroblasts (HDFs) that were purchased from commercial suppliers (Thermo Fisher Scientific and Lonza) or isolated in-house from surplus human skin as previously described (Hill et al. 2015. Mol Cancer Ther 14(1 l):2665-2673) following informed consent. Cells from 4 young and 4 aged female donors were pooled to make the cultures that are referred within this study as ‘Young’ and ‘Aged’ (Table 1). Cells were grown in DMEM (Sigma Aldrich) supplemented with 10% fetal bovine serum (FBS) (Sigma Aldrich), 100 U / mL penicillin / streptomycin (Sigma Aldrich) and 2 mM L-glutamine (Sigma Aldrich) in a humidified atmosphere containing 5% CO2 at 37°C. Cells were passaged when approached confluency (typically every 3-4 days for young cells, and about one week for aged cells) at a density of 6,666 cells per cm2, and the media was changed every 2-3 days.

[0104] Table 1. Characteristics of cells

[0105] Viral transduction

[0106] RNA interference to target genes of interest in this study was achieved using the SMARTvector® Lentivirus shRNA constructs (from SMARTvector) containing a TurboGFP reporter and a hEFla promoter that were ordered from and synthesized by Horizon Discovery (targeting vectors with Catalog numbers and shRNA sequences are listed in Table 2).

[0107] Lentiviral particles were produced in HEK293FT cells which were seeded in antibiotic-free medium supplemented with 0.1 mM MEM non-essential amino acids (Invitrogen) (6xl06per 10 cm dish). The following day the cells were transfected with OptiMEM™ (Invitrogen, 11058021) containing Lipofectamine 3000 (33 pl per dish), lentiviral expression vectors (5 pg) and 2nd generation packaging system plasmids, psPAX (3 75 pg, gift from Didier Trono (Addgene plasmid #12260)) and pCMV-VSV-G (2 5 pg, gift from Bob Weinberg (Addgene plasmid #8454)) After 24 h, media was replaced with fresh media without antibiotics. 48 h after transfection, media from HEK293FT cells was filtered through 0.45 pm PVDF filter to remove cell debris. Fresh polybrene (Hexadimethrine bromide) was prepared in H2O (10 mg / ml) and sterilised through a 02 pm PVDF filter Polybrene was added to the virus containing media (10 pg / ml). The media was then concentrated using Lenti-X (Takarabio) by adding the solution (Media:Lenti-X, 3: 1), mixing thoroughly and placing at 4°C for 48 hours. The mixture was then centrifuged at 1,500 x g for 45 minutes. The supernatant was removed, and the virus pellet was resuspended in complete DMEM. Viral transduction was performed by mixing concentrated virus with complete DMEM and adding to 70% confluent HDFs. Media containing virus was replaced after 24 h with fresh media containing 8 pg / ml of puromycin (Invitrogen) for selection of transduced cells. Media was replaced every 2-3 days for 10-12 days by keeping the antibiotic selection. Transduced HDFs were then maintained in the presence of lower puromycin concentrations (4 pg / ml) until seeding for experimental purposes.

[0108] Table 2. Characteristics of targeting shRNA sequences

[0109] Senescence-associated beta-galactosidase assay (SA-b-gal)

[0110] SA P-GAL staining solution was made up before fixation. For 10 ml, solution A was made up as follows; 1.5 ml 1 M NaCl, 20 pl 1 M MgCh, 800 pl 0.5 M citric acid, 1.2 ml 0.1 M sodium phosphate and 6.48 ml distilled H2O. The solution was adjusted to pH 6.0 using 5 M NaOH. For the final staining solution, 8.8 ml of solution A was mixed with 0.2 ml X-Gal (5-bromo-4-chloro- 3-indolyl-beta-D-galacto-pyranoside) and 1 ml Iron-bru (50 mM Potassium Ferrocyanide, 50 mM Potassium Ferricyanide in lx PBS). If volumes other than 10 ml were required, then all component volumes were adjusted accordingly.

[0111] Cells were seeded on coverslips in 24-well plates. Cells were washed twice with lx PBS and then fixed with 4% formaldehyde in PBS for 10 minutes. Fixed cells were washed three times with lx PBS, before adding to each well 0.5 ml of SA p-GAL staining solution. Plates were incubated for 24 h at 37 °C in darkness. The SA P-GAL staining solution was removed, and the coverslips were washed twice with lx PBS and mounted on slides using Prolong™ Gold Antifade Mountant with DAPI (Invitrogen). Slides were left to dry overnight at room temperature in darkness SA p- Gal staining was imaged using an inverted DM5500 microscope (Leica). SA P-GAL activity positive and negative cells were quantified by counting the proportion of cells displaying a strong stain above my determined threshold. Cell counting was performed using FIJI / ImageJ (www.fiji.se).

[0112] Immunofluorescence

[0113] Cells were seeded on coverslips in 24-well plates at -20,000 cells per well. After treatments, the media was aspirated, and the cells were washed twice in lx PBS. The cells were then fixed in 4% formaldehyde in PBS for 10 minutes at room temperature followed by three washes with lx PBS. Cells were permeabilized with 0.5% Triton X-100 (Sigma Aldrich) in PBS for 4 min at room temperature. Coverslips were then blocked for 1 hour in 5% normal goat serum (Sigma Aldrich) in PBS with 0.05% Tween- 20 (Sigma Aldrich) at room temperature. The blocking solution was removed, and the coverslips were incubated with primary antibodies overnight at 4°C in darkness. This was performed by placing coverslips face down onto 30 pl of primary antibody on a parafilm surface, in a foil-covered container. Damp blue roll was included in the container to prevent the coverslips from drying out. The following morning, cells were washed three times with lx PBS and incubated with the appropriate secondary antibodies for 1 hour at room temperature in darkness (Thermo Fisher Scientific, A31556 and A21235, 1: 1,000). This was performed by placing coverslips face down onto 30 pl of secondary antibody on a parafilm surface, in a foil- covered container. Damp blue roll was included in the container to prevent the coverslips from drying out. Cells were washed three times with lx PBS, and the coverslips were mounted onto slides using Prolong™ Gold Antifade Mountant with DAPI (Invitrogen). The slides were left to dry overnight at room temperature in darkness. Fluorescence images on fixed cells were obtained using an inverted DM5500 microscope (Leica). The following primary antibodies were used: rabbit a-Ki67 (Abeam, ab 15580, 1 :250), rabbit a-p21 (CST, 2947, 1:1,000). Quantification of the proportion of cells with positive antibody staining was performed using FIJFImageJ (www.fiji.se). Furthermore, DAPI staining provided a method for quantifying the average nuclear size by measuring the average area of DAPI.

[0114] Cell motility assay

[0115] Cells were seeded in glass-bottomed multiwell plates (Greiner, 662892) at 5,000 cells per well, 24 hours prior to imaging. Images were captured using a Zeiss CellDiscoverer 7 with a 5x / 0.35NA lens with a 2x optovar using oblique mode brightfield with a Hamamatsu Fusion camera every 300 seconds for 18h (1 ms exposure time), capturing 2 random fields per well. Cells were maintained at 37°C, 5% CO2 throughout. 6 cells were analyzed per field over 5 min using the Manual Tracking plugin in FIJI / ImageJ (www.fiji.se), and mean movement per cell was recorded.

[0116] RT-qPCR

[0117] To measure mRNA levels of marker genes, RT-qPCR was used. RNA was purified from cell pellets collected 2 weeks post-transduction using the RNeasy® Mini Kit (Qiagen,#74104) with beta-Mercaptoethanol kit following manufacturer’s instructions. The concentration and quality of the extracted RNA were assessed using NanoDrop™ One (Thermo scientific). Subsequently, 250-600 microgram in 8pl of total RNA from each sample was reverse transcribed into cDNA using the following protocol: 8 pl of RNA is mixed with 0.5pl Random primers (Thermo #48190011, diluted xlO to 0.3pg / pl), 1 u 1 dNTP mix (lOmM each, Promega dGTP #15518301, dCTP #U122D, dATP #15820001, dTTP #15955002), and 3.5pl nuclease free water (Promega, #P119C); heated at 65°C for 5 minutes; and incubated on ice for 1 minute. 7ul of a mix including: 4pl 5x buffer (Invitrogen, #Y02321), Ipl DTT (O.lmM, Invitrogen, #Y00147), lpl RNase inhibitor (Invitrogen, #100000840), and lpl Superscript™ III RT (Invitrogen, #55575) are added, and the reaction is incubated at 25°C for 5 minutes, 50°C for 1 hour, and 70°C for 20 minutes. The reaction products were stored at -80°C. Real-time PCR reactions were performed using PcwerSYBR® Green mix (Applied Biosystems, #4367659) on an Applied Biosystems StepOnePlus™ Detection System. Primer sets to detect genes of interest, CDKN1A / P21 (#PPH00211E-200), CDKN2D / P19 (#PPH00210C- 200), DPP4 (#PPH00035B-200), CMYC (#PPH00100B-200), SOX2 (#PPH02471A-200), and POU5F1 / OCT4 (#PPH02394E-200), were purchased from Qiagen. PCR cycling conditions were set as follows: an initial denaturation at 95°C for 5 minutes, followed by 40 cycles of 95°C for 15 seconds, 58°C for 15 seconds, and 72°C for 30 seconds. To ensure reproducibility and precision, each analysis was executed in three technical replicates. For the quantification of gene expression, the AACt method was employed, with normalization to the housekeeping gene ACTB .

[0118] Taqman PCR arrays

[0119] Taqman PCR assays were run according to manufacturer instructions on a custom 384-well TaqMan Array Cards (Thermofisher #4346798) using the primer / probe sets in Table 3 5Thermosfisher# 4331 182). Two hundred ng of cDNA was added per fill reservoir, with TaqMan Fast Advanced Master Mix (Thermofisher #4444557). Array cards were run on QuantStudio 7 Flex, and QuantStudio 12K Flex according to manufacturer thermal protocols as follows: UNG incubation for 2min at 50°C, enzyme activation for lOmin at 92 °C, and 40 cycles of [denature for 1 sec at 95 °C + annealing / extension for 20 sec at 60 °C]. Results were analysed using AACt with ACTB and GUSB used as the reference genes.

[0120] Table 3: Primer / Probe sets (for human sequences) in custom TaqMan Array Cards

[0121] EdU assay

[0122] Proliferating cells were quantified using the Invitrogen Click-iT EdU Alexa Fluor 594 Imaging Kit (C10339, Thermofisher). The assay was performed according to manufacturer instructions, with the following modifications: a 4 hour incubation time with EdU wass used (final concentration lOuM), and Hoescht was substituted with DAPI in the Prolong Gold Antifade Mounting media (P36934, Thermo fisher). Cells were plated at 25,000 cells / well for aged cells, and 20,000 cells / well for Young cells in 24-well plates.

[0123] Multivariate integrative data analyses

[0124] Partial Least Squares-Discriminant Analysis (PLS-DA) was employed as a multivariate statistical technique to perform an integrative analysis of data derived from six distinct cell assays. The fractions of SA-b-gal+, Ki67+, Edu+, and p21+ cells, as well as nuclear size and cell motility are the readouts that all examine various aspects of cellular senescence and how they are affected by our genes knock-down (KD) interventions. Therefore, the PLS-DA approach was utilized to identify general patterns observed between the control Aged and Young cells and how the KDs modify these in a synthetic multivariate space, and correlations within the multivariate dataset, enabling the discrimination of different treatment groups and the identification of key variables driving these distinctions. Our methodology was underpinned by a comprehensive data normalization and quality control process to ensure comparability across assays. For the statistical analysis, the R environment was used, specifically leveraging the mixOmics package, renowned for its robust multivariate analysis capabilities. This approach was instrumental in managing and interpreting the complex dataset derived from the cell assays. Additionally, a custom R script was utilized, which applied the NIPALS algorithm to effectively handle missing values within our dataset. This script decomposed the data matrix, performed imputation, and ensured the integrity and reliability of our data prior to analysis. The robustness of the PLS-DA model was assessed using cross-validation and permutation tests. Additionally, sample plots were generated to provide a visual summary of the data distribution and group separations. Lastly, correlation circles were used to illustrate the relationships between the variables and the principal components. All these plots are included in the Figures and are crucial in interpreting the complex relationships within the multivariate data and for the overall visualization of the anti-aging effect of the genes KD interventions.

[0125] Statistical analyses

[0126] Depending on the assay / readout, and hence variable distribution, different statistics were used to test the difference between groups. For SA-b-gal assay, a 2-tailed 2-sample equal variance t-test was used to compare control Young shNT vs. Aged shNT groups, and a 2-tailed paired t-test for intervention significance. The 2-tailed paired t-test was also used for the SASP cytokine array data. Mann-Whitney Rank Sum Test was used for nuclear size, and One-Way ANOVA for multiple comparisons for cell motility, Ki67+, EdU+ and p21+ cell fractions readouts. Example 1: General design of the Knock-down (KD) experiments

[0127] Frozen human dermal fibroblast (HDF) cells were thawed, and then cells were pooled and allowed to grow for about 5 days to alleviate regrowing stress. Cells were then transduced with lentiviruses generated as described in the “Viral transduction” section above and carrying the shRNA gene-targeting constructs shown in Table 2. Transduction with viruses carrying nontargeting scrambled sequences was used for control. About 14 days following transduction, assays were performed to evaluate the effect of the KD: SA-b-gal assay, cell morphology readouts, cell cycle and proliferation assays, RT-qPCR, Taqman qPCR array for SASP cytokine genes as further described in the methods and below in the results.

[0128] An important feature of the cell model used is the use of HDF that were aged in vivo (from aged donors) as opposed to aging in vitro via continuous culture and passaging of commercial primary cultures of human fibroblasts isolated from embryos of neonates, such as IMR90, HFL1, MRC5 and AG1522. Aging in vitro eventually results in replicative senescence that may not be representative of physiological in vivo aging due to the lack of tissue microenvironment and hypertoxic conditions. This adds confidence in translatability of our results to future applications in humans to inhibit, delay, or reverse cellular senescence.

[0129] Example 2: Effect on a SA-b-gal marker of senescence

[0130] KD efficiency was first validated using RT-qPCR. The measured mRNA levels of the target genes are shown in Fig. 1A, confirming that transduction with the shRNA vectors resulted in the suppression of the target genes mRNA levels, whereas the control non-targeting transduction (shNT) did not affect the mRNA levels. Consequently, any changes in senescence readouts can be attributed to the role of the KD effect.

[0131] SA-b-gal activity, originating in numerous lysosomes of aged cells, is considered a classical marker of senescent cells and has widely been used in aging in vitro and in vivo studies. As can be seen from Fig. IB, the fraction of SA-b-gal+ cells in the pooled culture from aged donors was about 20%, suggesting that the majority of the cells had not reached senescence. Although this is higher than in the culture of fibroblasts from young donors (10%), this suggests that the cultures from aged donors have not reached significant senescence levels and still have proliferative potential. As further seen from Fig. IB, aged cells with targeted KDs had overall decreased fractions of SA-b-gal-i- cells compared to the aged control, however, this decrease reached statistical significance only for DDX58, 0MA1 and AIM2. Note that a separate experiment with its controls was carried out for the AIM2 KD, resulting in differing values of SA-b-gal-i- cells which is shown in a smaller panel in Fig. 1C. There, although higher levels of SA-b-gal+ cells were observed (-50%) in aged control transduced cells, the effectiveness of AIM2 KD was significant. In summary, the KD of the 7 target genes tended to rejuvenate the aged cells using the classical marker of cellular senescence, the fraction of SA-b-gal cells in a cell population.

[0132] Example 3: KD of the target genes enhances morphological and functional properties of aged cells

[0133] Next, morphological features and functional characteristics commonly associated with cellular aging were examined, including nuclear size and cell motility. Nuclear size was measured using brightfield microscopy. As shown in Fig. 2A, control transduced cells from Young vs Aged donors differed substantially in the nucleus size, with the aged cells displaying larger nuclear size. All 7 gene KDs resulted in a significant reduction of the nuclear size compared with the Aged shNT control. In the case of DDX58, the nuclear size was reduced down to one recorded in Young shNT control cells.

[0134] Regarding cell motility, as shown in Fig. 2B, the motility of the control Young cells was significantly higher (2-fold) than that of the control aged cells. This is a common feature observed in senescent fibroblasts and reflects the general functional decline of the cells. Downregulation of the 7 target genes enhanced the motility of aged cells. For AIM2 KD the enhancement was 100% back to the levels observed in young cells. Restoration between 50_ 80% was seen for NDST3, BATF, AIM2, IFIH1, DDX58, JAM2, 0MA1. Moderate enhancement was achieved by the DDX58 KD, and none by JAM2 KD.

[0135] Overall, the results of measuring key morphological and functional features of cells suggest that targeted genes KD tend to rejuvenate the fibroblast cultures established from aged donors.

[0136] Example 4: KD of the target genes enhances proliferative properties of aged cells

[0137] Key functional change observed in senescence is the decline of proliferative potential of cells. Therefore, to assess the efficiency of the gene KD interventions, the changes in proliferative properties in control Aged vs Young cells, and in aged cells with KD of the 7 target genes were explored next. Post-intervention assessments were conducted after two weeks, focusing on the analysis of Ki67+, EdU+, and p21+cell fractions via immunofluorescence microscopy.

[0138] Ki-67 is a protein that is present in dividing cells, specifically in the nuclei during the active phases of the cell cycle (Gl, S, G2, and mitosis), but absent in the resting phase (GO). It is also commonly used for the assessment of proliferative activity in tumors. As seen from Fig. 3A, KD of BATF, AIM2, DDX58, and 0MA1 significantly increased the fraction of Ki67+cells in aged cell culture. This increase resulted in the levels observed in control cells from Young donors for AIM2 and DDX58, suggesting a rejuvenation effect.

[0139] Fig. 3B shows that 2 out of 7 KD interventions lead to significant increases in the EdU+cells, suggesting reactivation of the entry of cells into S-phase from GO / Gl-phase in these cultures. Interestingly, BATF, DDX58, and 0MA1 KDs did not affect the EdU+cell fraction, while they did increase the fraction of Ki67+cells (Fig. 3A). As seen in Fig. 3B, IFIH1 KD resulted in higher fractions of EdU+cells but did not affect the Ki67 levels (Fig. 3A). AIM2 resulted in a significant increase in both tests. These complex results may reflect the complex regulation of the progression of cells along the phases of the cell cycle. Indeed, while EdU labels only cells completing the S- phase, Ki67 may be present in S, G2, M and even G1 cells with changes in its nuclear distribution pattern which was not accessed in this study. Additionally, as evident from the SA-b-gal results, the control Aged shNT cells were not nearing 100% senescence and thus retained measurable proliferative capacity suggesting that even subtle improvements by the interventions present significant interest.

[0140] Fig. 3C shows the fraction of cells with high levels of p21, a known cyclin-dependent kinase inhibitor that plays a critical role in cell cycle regulation upon various stress and norm conditions and is intricately linked to cellular senescence. As seen in the Figure, there was a drastic difference (>5-fold) between control young and aged cells. Remarkably, all KD interventions resulted in a decrease in the fraction of p21+cells in aged fibroblasts, with NDST3, IFIH1 and JAM2 KD cells displaying levels similar to those observed in Young control cells. The reduction of p21+cells suggests that the targeted gene knockdowns effectively mitigated the senescent phenotype, favoring a proliferative state over senescence. This aligns with the understanding that while high levels of p21 contribute to senescence, its downregulation can promote cell cycle progression and proliferation.

[0141] Concurrently, the increase in Ki67+and EdU+cells, yet not for all KDs studied, further corroborates the shift towards a more proliferative state. However proliferative activity in each of the KD cell cultures generated may possess context-dependent features, expressed as distinct EdU and Ki67 expression patterns. For example, NDST3 KD resulted in the reduction of p21+ cells but did not affect EdU and Ki-67 readouts. On the other hand, a more subtle reduction of p21+ cell fraction by AIM2 KD was also associated with strong increases in EdU-i- and Ki67+ cells. This may suggest that the removal of p21 -dependent repression of the cell cycle creates a condition that favors proliferation, but the actual initiation of proliferation and its pattern depends on other factors. In any case, these data collectively suggest the KD of each of the eight gene targets overall restores the proliferative potential of the primary fibroblasts from Aged donors. This further highlights the potential of genetic interventions to KD the 7 target genes to modulate cellular aging processes, possibly reversing or delaying the onset of cellular senescence.

[0142] Example 5: Effects of target genes KD on the expression of select gene markers of cell cycle, inflammation, and de-differentiation

[0143] The molecular markers of cell cycle regulation, inflammation, and de-differentiation were examined by measuring the mRNA expression levels of select relevant genes. The expression of p21 (CDKN1A) and pl9 (CDKN2D), known cell cycle control genes active in senescent cells, was assessed. Dipeptidyl peptidase-4 (DPP4) upregulation is linked to increased pro-inflammatory cytokines and chemokines, highlighting DPP4's involvement in the inflammatory aspects of SASP. The reversal of senescence, in theory, might lead to a state of de-differentiation, providing cells with a more plastic and less differentiated state, which could be a step toward neoplastic transformation, especially if additional oncogenic stimuli are present. To assess the potential of the genetic KD anti-senescence interventions to induce new undesired cellular phenotype that might be associated with a risk of neoplastic transformation, three classical molecular markers of de-differentiated state were used: 0CT4 (POU5F1), MYC and S0X2.

[0144] As can be seen from Fig. 4A and Fig. 4B, the Aged shNT cells had at least two-fold higher levels of P21 and P19 mRNA levels. Remarkably, all 7 KD interventions resulted in a significant reduction of the mRNA levels of the two genes. Notably, qPCR results shown in Fig. 4A for P21 are consistent with the immunofluorescence microscopy results for P21+ cells shown in Fig. 3C. Four out of 7 KD interventions lead to a reduced mRNA levels of DPP4 compared with the control shNT cells from Aged donors. These decreases were greatest for BATF KD (~5 fold), followed by NDST3 and JAM2 (- 4 and 3-fold, respectively). About a 2-fold reduction was seen for the 0MA1 KD cells. This pro- inflammatory gene was expectedly expressed at much higher levels in the control Aged vs. Young cells, demonstrating the physiological appropriateness of this marker of cellular senescence. Together, these results suggest that classical molecular markers of senescence-associated with cell cycle arrest and pro-inflammatory signaling can be ameliorated by the intervention of targeting the 7 genes used in this study. These rejuvenation-related changes at the level of mRNA are consistent with cellular phenotypes resulting from these interventions described above.

[0145] The levels of mRNA of 3 classical markers of de-differentiation, CMYC, S0X2 and OCT4 / POU5F1, were examined in an attempt to assess the potential of the KD interventions to induce undesired consequences related to cell neoplastic transformation. Surprisingly, these genes were expressed at higher levels in the Aged control cells (Aged shNT) compared with the control Young shNT cells (Figs. 4D, 4E and 4F). This phenomenon seems to be unexplored, except for a similar relationship between OCT4 and senescence (Jaganathan and Bonnet, 2012, Cytotherapy 14(9):1054-63), but might suggest that by up-regulating the expression of these genes the cells are trying to maintain their proliferation state countering the process of the accumulation of senescence in the cell population. As can be seen from Figs. 4D, 4E and 4F, all 7 KDs resulted in an overall down-regulation of CMYC, SOX2 and OCT4 / POU5F1 mRNA levels relative to the Aged control cells towards the levels seen in Young cells. The effect was very strong for CMYC and OCT4 / POU5F1 for all 7 KDs; in some instances, the expression went back to the levels observed in Young cells. More variable results were seen for SOX2 gene expression, whereby NDST3 and BATF did not alter the mRNA levels of SOX2, and AIM2 KD causing the levels to go even below that of the Young cells. In summary, these results suggest that the KD interventions targeting 7 genes of interest in cells from Aged donors result in modulation of the expression of classical de-differentiation markers in such a way that they resemble those found in Young cells.

[0146] Example 6: Target genes KD alleviate the expression of some SASP cytokine genes in aged cells

[0147] One of the prominent features of senescent cells, which is relevant to their in vivo functional and physiological roles, is a very specific repertoire of cytokines that they secrete at a high rate and that are collectively called SASP. Eight cytokines were measured at the mRNA levels from the control and KD cells: CCL5 / GRO, CXCL1 / RANTES, DPP4, IL-1A, IL-6, DPP4, MMP-9, and VEGFA.

[0148] IL-1A and IL-10 were found to be expressed at very low levels so detection was unreliable (data not shown) and they were thus excluded from further considerations. Among the remaining 6 cytokines, 5 showed canonical increased levels in control aged cells vs. control Young cells, except for CXCL1 / RANTES which was expressed 2-fold higher in the young cells (Fig. 5). As can be seen from the heatmap in Fig. 5, the overall pattern of expression of the cytokine genes is such that the KD interventions in aged cells tend to reconstitute levels back to ones found in Young cells. This is not the case for DPP4 which was not overall modified (except for JAM2 which reduced, and DDX58 which increased its expression). Noted changes outside the anticipated pattern were also observed, such as increased CCL5 in DDX58 KD cells, and increased MMP-9 in DDX58 and 0MA1 KD cells. This complex regulation with distinct expression patterns may reflect the context-dependence and complex anti- and pro-inflammatory regulation circuits that are highly dynamic and responsive to external stimuli. However, the overall pattern seen in this experiment suggests the reversal of the SASP gene expression profile in aged cells as a result of the KD interventions to that typical of young control cells. This further underscores the cell rejuvenation potential of the target gene manipulations described herein.

[0149] Example 7: Multivariate data integration demonstrates rejuvenation effect

[0150] In the assessment of the effect of target genes KD on cellular senescence phenotype a panel of assays that span several levels of biological organization / phenotypes was used: sub-cellular morphology (nuclear size), sub-cellular function (lysosomal SA-b-gal expression), cellular phenotype, and cellular function (p21, EdU, Ki67, cell motility). Responses that were observed followed no common pattern, with KD interventions ameliorating cellular senescence markers in one assay, but not the other. These responses also varied with the targeted gene. While these results reflect the complex mechanistic biology of cellular aging, it is somewhat difficult to assess an overall efficiency of the anti-aging potential of the interventions used. To facilitate such assessment and to produce a visual means of visualizing the overall picture of the anti-aging effects of the KD interventions, an integration of the data was conducted by using PLS-DA, a supervised statistical method used for classification and dimensionality reduction of complex multimodal datasets.

[0151] In these analyses, data obtained using SA-b-gal, nuclear size, cell motility (also labelled as cell velocity), Ki67, EdU and P21 assays is inputted. The sample plots shown in Figs. 6A-6H (top plots in each panel) show clustering of samples within sets of three experimental groups: Young shNT, Aged shNT and aged cells with a KD. It is evident that the Young shNT (plus cluster) and Aged shNT (triangle cluster) cells clustered very distinctly so that they are separated by a substantial space in each plot. It is also evident that each of the 7 KD interventions resulted in a separation of the KD cells (circle clusters) from the parental triangle clusters. This suggests the overall integrative change of the properties of these cells towards young phenotype, a process called rejuvenation. The PLS-DA derived sample plots also allow for the assessment of the efficiency of the interventions. Thus, clusters of NDST3 and BATF KD cells got separated from the parental cluster completely and even slightly overlapped with the cluster of control young cells.

[0152] Another important result of the PLS-DA analyses is the so-called correlation circle that allows to correlate individual features (variables) used in the analyses with a particular clustering pattern. For example, cell motility, EdU and Ki67 positively contributed the most to young cells cluster, whereas SA-b-gal, p21 and nuclear size contributed to the aged cells clustering. The radial position of these individual features on the correlation circle and their distance to the outer circle can be correlated with the effects of the KD interventions and their strengths.

[0153] The inclusion of the individual molecular markers (gene marker expression) and the SASP cytokine array results were intentionally avoided in the PLS -DA analyses. Whereas all readouts included in the PLS-DA represent a physiologically relevant endpoint and / or phenotype, the individual marker gene / protein readouts may not have equally substantial weight in the overall senescence phenotype.

[0154] Example 8: Common regulation of the target genes

[0155] Transcription factors (TFs) are pivotal in gene regulation, binding to specific TF-binding DNA sequences (TFBS) to promote or inhibit transcription. The presence of shared TFBS among a group of genes often indicates a common regulatory mechanism, suggesting these genes may be co-regulated by the same TFs. Analyzing TFBS also aids in understanding the functional interplay within cellular pathways. Genes sharing TFBS are frequently involved in related biological processes or pathways leading to a widespread use of this feature to understand biological mechanism and generate hypotheses. This association can be particularly illuminating in dissecting complex biological processes like development, differentiation, disease pathogenesis and aging. By grouping genes based on shared TFBS, new facets of these processes, potentially revealing novel therapeutic targets or biomarkers can be unraveled. Furthermore, genes with common TFBS often exhibit correlated expression patterns.

[0156] The hTFtarget database for regulations of human transcription factors and their targets (Zhang er al. 2020. Genomics, proteomics & bioinformatics 18(2): 120-128) was used to interrogate the TFBS commonalities between the eight target genes. It was found that all 7 target genes were regulated by the transcription factor ZBTB2.

Claims

CLAIMS1. An in vitro or ex vivo method for inhibiting, delaying, or reversing cellular senescence, the method comprising contacting an aged cell with a composition comprising a targeting agent capable of inhibiting activity of a target gene regulated by ZBTB2 and selected from NDST3, BATF, AIM2, IFIH1, DDX58, JAM2, 0MA1, and any combination thereof.

2. The method of claim 1, wherein the aged cell is from a tissue selected from blood, bone marrow, brain, connective tissue, heart, intestine, kidney, liver, lung, mucosa, muscle, pancreatic, and skin.

3. The method of claim 1 or 2, wherein the aged cell is a human cell.

4. The method of claim 3, wherein the aged cell is from an aged subject or a subject suffering from an aging-related disease.

5. The method of claim 4, wherein the aged subject is at least about 50 years old.

6. The method of claim any one of claims 1-5, wherein the targeting agent comprises a small molecule drug.

7. The method of claim any one of claims 1-5, wherein the targeting agent comprises a nucleic acid molecule.

8. The method of claim 7 wherein the nucleic acid molecule comprises a targeting nucleotide sequence encoding an inhibitory RNA specific for the target gene.

9. The method of claim 8, wherein the inhibitory RNA is selected from shRNA, siRNA, and miRNA.

10. The method of any one of claims 7-9, wherein the nucleic acid molecule is comprised in a vector suitable for delivery of the nucleic acid molecule to the cell.

11. The method of claim 10, wherein the vector is selected from a viral vector, a non-viral vector, a retroviral vector, and a lentiviral vector.

12. A knock-down (KD) cell produced by the method of any one of claims 7-11.

13. The KD cell of claim 12, wherein the KD cell comprises rejuvenated features including at least one of: a reduced SA-b-gal activity level; a reduced nuclear size; an increased cell motility;higher average expression levels of K67+and / or EdU+; lower average p21+expression levels; lower levels of at least one cell cycle regulation selected from CDKN1A / P21, CDKN2D / P19 and one SASP cytokine gene; and lower levels of at least one senescence-associated secretory phenotype (SASP) cytokine selected from IL-6, DPP4, GRO, MMP-9, RANTES, and VEGF; in the KD cell compared to the aged cell.

14. The KD cell of claim 13, wherein the rejuvenated features include SA-b-gal activity level being at least 20% lower in the KD cell than in the aged cell.

15. The KD cell of claim 13 or 14, wherein the rejuvenated features include nuclear size being at least 20% lower in the KD cell than in the aged cell.

16. The KD cell of any one of claims 13-15, wherein the rejuvenated features include cell motility being at least 20% higher in the KD cell than the aged cell motility.

17. The KD cell of any one of claims 13-16, wherein the rejuvenated features include average expression levels of K67+and / or EdU+being at least 20% higher in the KD cell than in the aged cell.

18. The KD cell of any one of claims 13-17, wherein the rejuvenated features include average p21+expression levels being at least 20% lower in the KD cell than in the aged cell.

19. The KD cell of any one of claims 13-18, wherein the rejuvenated features include levels of at least one cell cycle regulation selected from CDKN1A / P21, CDKN2D / P19 and one SASP cytokine gene in the KD cell being at least 20% lower than corresponding levels in the aged cell.

20. The KD cell of any one of claims 13-19, wherein the rejuvenated features include levels of at least one senescence-associated secretory phenotype (SASP) cytokine selected from IL-6, DPP4, GRO, MMP-9, RANTES, and VEGF in the KD cell being at least 20% lower than the corresponding levels in the aged cell.

21. A composition comprising the KD cell of any one of claims 12-20, or a targeting agent capable of inhibiting activity of a target gene regulated by ZBTB2 and selected from NDST3, BATE, AIM2, IFIH1, DDX58, IAM2, OMA1, and any combination thereof, for use in treating an aging-related disease by inhibiting, delaying, or reversing cellular senescence in a subject over the age of 50, and / or who is suffering from an aging-related disease.

22. A method of treating an aging-related disease in subject in need thereof by inhibiting, delaying, or reversing cellular senescence, the method comprising administering to the subject a composition comprising the KD cell of any one of claims 12-20 or a targeting agent capable of inhibiting activity of a target gene regulated by ZBTB2 and selected from NDST3, BATF, AIM2, IFIH1, DDX58, JAM2, 0MA1, and any combination thereof, wherein the subject is over the age of 50, and / or is suffering from an aging-related disease.

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