Compositions comprising SASP modulators and senescence attenuators, and uses thereof for modulating cellular senescence

Abstract

To provide compositions and methods for modulating cellular senescence of a cell or induction of the senescence-associated secretory phenotype (SASP) in a cell.SOLUTION: There is provided a composition for treating (i) a disease or condition associated with premature senescence of cells and / or (ii) a metabolic disease or condition in a subject, comprising a semaphorin 3A (SEMA3A) antagonist and a carrier, wherein the SEMA3A antagonist is (a) a soluble NRP1 polypeptide that binds to SEMA3A and comprises an amino acid sequence having at least 95% identity to a specific sequence, or (b) an anti-SEMA3A antibody or an antigen-binding fragment thereof.SELECTED DRAWING: Figure 20

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a PCT application having serial number PCT / CA2017 / *, filed on September 22, 2017, published in English under PCT Article 21(2), which itself claims the benefit of U.S. Provisional Application No. 62 / 474,827, filed on September 23, 2016, U.S. Provisional Application No. 62 / 398,797, filed on September 23, 2016, and U.S. Provisional Application No. 62 / 398,183, filed on September 22, 2016, each of which is incorporated herein by reference in its entirety. REFERENCE TO SEQUENCE LISTING

[0002] This application contains a sequence listing in computer readable form entitled "12810_651_SL_ST25.txt", which was created on September 22, 2017, and has a size of approximately 249KB. The computer readable form is incorporated herein by reference. FIELD OF THEINVENTION

[0003] The present invention relates to compositions and methods for modulating cellular senescence. More specifically, the present invention relates to modulating the senescence-associated secretory phenotype (SASP) in the prevention and treatment of diseases and conditions associated with cellular senescence, such as vascular eye diseases. [Background technology]

[0004] Cellular senescence is generally defined as the state of cells in which they are still viable and metabolically active, but lose the ability to proliferate.Cellular senescence can be caused by various stimuli or factors, such as telomere shortening due to DNA end replication, DNA damage, changes in the activity of tumor suppressor genes and oncogenes, oxidative stress, inflammation, chemotherapeutic agents, and exposure to UV and ionizing radiation (Kuilman et al., Genes & Development.(2010)24:2463-2479).

[0005] Three cellular pathways leading to the senescent phenotype have been described: replicative senescence, premature senescence and postdifferential senescence (SAD). Replicative senescence is a type of senescence that occurs after a large number of cell divisions. For example, when grown in culture, primary cells undergo cellular senescence after about 50 cell divisions. This barrier to further proliferation is thought to occur when each successive cell division shortens the telomeres of cells, thus reaching a point where the cells trigger a DNA damage response (the so-called "Hayflick limit"), ultimately causing the induction of growth arrest and senescence.

[0006] Cellular senescence can also be induced in the absence of telomere loss or dysfunction. This type of cellular senescence is called premature cellular senescence and can result from a variety of stimuli, such as DNA damage resulting from chemotherapy, radiation therapy, exposure to DNA damaging compounds, or from sunlight and UV radiation, oxidative stress, inflammation, strong mitogenic signaling, and ribosomal stress. DNA damage can take the form of chromosomal dysfunction, such as unequal chromosomal segregation during mitosis, DNA strand breaks, or aneuploidy resulting from chemical modification of DNA (e.g., alkylation). Premature cellular senescence can also be induced by a DNA damage response (DDR), which may or may not reflect actual DNA damage.

[0007] In recent years, it has become clear that the aging process involves more than a simple arrest of cell proliferation, as it has been shown that in several diseases, terminally differentiated postmitotic cells acquire a senescence-like phenotype (including SASP). This third type of aging has been termed postdifferentiation senescence (SAD) and can be induced by a variety of stressors, including genotoxic, proteotoxic, oxidative and ribosomal stressors (see, e.g., Naylor RM et al., 2013 Clin. Pharmacol Ther. 93(1):105-116).

[0008] Not all senescent cells express all possible senescence markers. Nevertheless, hallmarks of senescent cells include (i) growth arrest, (ii) enlarged and flattened cell morphology, (iii) DNA damage foci in the nucleus, (iv) secretion of growth factor proteases, cytokines and other factors defined as the senescence-associated secretory phenotype (SASP), (v) senescence-associated β-galactosidase (SA-β-gal) activity (which in part reflects increased lysosomal mass), (vi) expression of the tumor suppressor p16INK4a (which may activate pRB and lead to the formation of senescence-associated heterochromatin foci (SAHF)), and (vii) an increase in the number and size of PML nucleoli. Furthermore, although diverse factors are known to induce cellular senescence, two tumor suppressor pathways, p53 / p21 and p16INK4 / pRB, have been shown to play key roles in regulating cellular senescence.

[0009] Recent studies have extended the involvement of cellular senescence to complex physiological processes such as embryogenesis and tissue repair (19-22). Conversely, in chronic diseases and aging, the increase in senescent cells exacerbates tissue dysfunction (23-25). Depending on the condition, cellular senescence has been shown to be either beneficial or detrimental (see Rodier and Campisi, JCB, 2011, 192(4):547-556 and Naylor et al., Clin Pharmacol Ther. 2013 93(1):105-116 for review on cellular senescence).

[0010] Cellular senescence has been implicated causally in the pathogenesis of various age-related diseases and conditions, such as thinning of the epidermis, skin wrinkling, hair loss and graying, loss of muscle thickness and strength (sarcopenia), increased incidence of inflammation, metabolic disorders, loss of endurance, atherosclerosis, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), neurodegenerative diseases, osteoarthritis, osteoporosis, Parkinson's disease, and cataracts. In addition, cellular senescence is believed to contribute to the damage to healthy tissues experienced during and after chemotherapy and / or radiation therapy, as well as the poor health effects following chemotherapy and / or radiation therapy.

[0011] Cellular senescence can also be beneficial. For example, its role as an anti-cancer mechanism in response to DNA damage has been established for decades. Furthermore, senescent cells have been shown to be important for efficient tissue repair and wound healing. Indeed, many factors of the SASP (e.g., growth factors and proteases involved in wound healing, attractants of immune cells that kill pathogens, and proteins that recruit stem or progenitor cells) are important for tissue repair. Thus, the SASP may also function to communicate cellular damage / dysfunction to surrounding tissues and stimulate repair when necessary. Recent studies support this concept. For example, studies have shown that senescent cells are rapidly established near wounds to help initiate the inflammatory response (via the SASP) that initiates the healing process during the proliferative phase. This rapid senescence attracts and activates immune cells to fight infection and remove dead cells and debris. During the remodeling phase, senescent cells play a role in dissolving fibrous proteins that are present during the proliferative phase, limiting the formation of scars. Beneficial effects of cellular senescence have also been reported in liver fibrosis, myocardial infarction and cardiac fibrosis, atherosclerosis, and pulmonary hypertension.

[0012] Therefore, preventing cells from undergoing cellular senescence, or preventing DNA damage, DNA damage response pathways or chromatin changes that activate senescence (e.g., may lead to SASP), reversing or limiting cellular senescence, and / or reducing paracrine senescence in cells undergoing cellular senescence, is advantageous for preventing or treating diseases and conditions in which senescence is detrimental. Conversely, promoting cellular senescence in diseases and conditions that are positively affected by cellular senescence can improve recovery or reduce the severity of such diseases or conditions.

[0013] Obesity and the subsequent sequelae of metabolic syndrome, type 2 diabetes mellitus (TIIDM), and cardiovascular complications have become a global epidemic. Global obesity has more than doubled since 1980, with more than 1.9 billion adults being overweight in 2014, of which 600 million were obese (World Health Organization (WHO), 2015).

[0014] Overweight and obesity are defined as abnormal or excessive fat accumulation that may be harmful to health. Body mass index (BMI) is a simple index of weight relative to height that is commonly used to classify overweight and obesity in adults. This index is calculated as a person's weight in kilograms divided by their height in meters squared (kg / m 2 The WHO definition is: (i) BMI of 25 kg / m 2 and (ii) a BMI of 30 kg / m 2 If it is greater than or equal to 100%, it is obese. BMI is the same for men and women and for all ages in adults, making it a useful population-level measure of overweight and obesity. However, BMI is considered a rough guide because it may not represent the same degree of obesity in different individuals.

[0015] Fat accumulation is observed in a range of conditions such as obesity, non-alcoholic fatty liver disease (NAFLD), metabolic syndrome, and lipodystrophy syndrome. Elevated BMI (overweight or obesity) is a major risk factor for the following diseases and conditions: cardiovascular disease (CVD, primarily heart disease and stroke), insulin resistance (increasing the risk of developing TIIDM). Excess fat accumulation also increases the risk of suffering from other diseases or conditions such as musculoskeletal disorders (particularly osteoarthritis), and some cancers (endometrial, breast, and colon). The risk of these diseases generally increases with increasing BMI.

[0016] Metabolic syndrome, also known as syndrome X, affects obese humans as well as those with increased amounts of abdominal fat and is characterized by insulin resistance, dyslipidemia (hypertriglyceridemia, low serum HDL cholesterol, elevated LDL cholesterol) and hypertension. These conditions are related to each other and share underlying mediators, mechanisms and pathways. Altered fat distribution, increased WHR (waist-to-hip ratio) and accumulation of visceral fat are associated with increased metabolic risk index.

[0017] Although most conditions associated with metabolic syndrome are asymptomatic, a large waist is a clear sign. Several organizations have criteria for diagnosing metabolic syndrome. The NCEP ATP III definition is one of the most widely used criteria for metabolic syndrome. This definition incorporates the key features of hyperglycemia / insulin resistance, visceral obesity, atherogenic dyslipidemia, and hypertension / endothelial dysfunction. According to guidelines used by the National Institutes of Health, a subject has metabolic syndrome if three or more of the following characteristics are present or the subject is taking medications to control those characteristics: (i) visceral obesity (i.e., a large waist circumference, e.g., at least 35 inches (89 centimeters) for women and 40 inches (102 centimeters) for men; (ii) high triglyceride levels--150 milligrams per deciliter (mg / dL), or 1.7 millimoles per liter (mmol / L), or the amount of this type of fat found in the blood; (iii) reduced high-density lipoprotein (HDL) cholesterol--less than 40 mg / dL (1.04 mmol / L) of "good" cholesterol in men and less than 50 mg / dL (1.3 mmol / L) in women; (iv) elevated blood pressure--130 / 85 millimeters of mercury (mmHg) or higher; and (v) elevated fasting blood glucose--100 mg / dL (5.6 mmol / L) or higher.

[0018] The currently accepted mechanism of obesity-induced metabolic syndrome is that adipose lipid accumulation triggers the release of cytokines, inducing M1 activation and systemic inflammation (Olefsky and Glass, 2010). Chronic inflammation and macrophage activation are hypothesized to cause insulin resistance (Osborn and Olefsky, 2012), but it remains controversial whether adipose tissue inflammation is an adaptive response that can efficiently store excess nutrients (Wernstedt Asterholm et al., 2014) and whether proper angiogenesis is essential for adipose tissue expansion (Cullberg et al., 2013). Both adipocyte oxygen consumption (Lee et al., 2014) and adipose tissue vascular remodeling (Sung et al., 2013) control the inflammatory state of adipose tissue, which subsequently leads to insulin insensitivity and hyperglycemia.

[0019] Despite the increasing social awareness of obesity-related problems, the proportion of overweight and obese subjects continues to rise.Therefore, there is still a need to develop new approaches for the prevention and / or treatment of diseases and conditions associated with fat accumulation, taking into account their high prevalence and associated morbidity and mortality.

[0020] This specification refers to several documents, the contents of which are incorporated herein by reference in their entirety. Summary of the Invention

[0021] Disruption of the vascular bed in ischemic retinopathy, whether it is glycemia-driven in diabetic retinopathy (DR) or oxygen-driven in retinopathy of prematurity (ROP), results in hypoxic / ischemic central nervous system (CNS) tissue subjected to a constellation of biochemical and inflammatory stressors that impair cellular function. These avascular zones are a source of proangiogenic factors that mediate pathological angiogenesis (4), as evidenced by the clinical success of laser photocoagulation to ablate these regions (5). While much effort has been focused on understanding the next wave of pathological preretinal angiogenesis, relatively little is known about the cellular processes at play during the precursor state of neural tissue hypoxia. A more complete understanding of the cellular responses at play during the early stages of retinal ischemia may provide therapeutic avenues to benefit a portion of the 93 million DR and 15 million preterm infants born annually who present with neovascular retinal disease (6-8).

[0022] Central neurons such as retinal ganglion cells (RGCs), which are directly aligned to the degenerated vasculature in ischemic retinopathies, require a steady metabolic supply for proper function. Interestingly, during the progression of DR, there is a disconnect between the extent of overt retinal vascular pathology (9) and the relatively subtle, long-lasting morphological and functional abnormalities observed in RGCs (10–12). Furthermore, although there is evidence supporting RGC apoptosis in DR (13–15), the magnitude and kinetics of neuronal cell death remain controversial (16–18). The relative resilience of retinal ganglion neurons in DR suggests that they receive metabolic supply from alternative vascular plexuses or mount defensive mechanisms that render them less susceptible to ischemia-induced cell death.

[0023] Mechanisms induced to maintain neural tissue integrity during ischemic insults confer critical survival advantages and allow timely repair and restoration of function (44, 61, 62). Mechanisms leading to cellular senescence may have evolved in parallel with apoptotic mechanisms to limit carcinogenesis (21), but for postmitotic CNS neurons such as those found in the retina, cellular senescence may prevent stressor-induced neurodegeneration. Although studied in the context of aging and disease for nearly 60 years (63), our studies reported herein present a novel role for cellular senescence in weather-resistant ischemia in the CNS. These studies further uncover a previously undescribed role for SEMA3A in regulating senescence, including paracrine senescence in pathological processes, and reveal therapeutic benefits in modulating SEMA3A activity in aging-associated diseases and conditions.

[0024] More specifically, in one aspect, the applicants have identified an unexpected mechanism triggered by neurons in the avascularized retinal zone, in which neurons enter an early cellular senescence state and adopt a senescence-associated secretory phenotype (SASP) by activating the endoribonuclease activity of the ER stress effector inositol-requiring enzyme-1α (IRE-1α). Factors produced via the SASP, such as secreted embryonic patterning cue semaphorin 3A (SEMA3A), allow senescence to spread to neurons, microglia and the overlying vasculature (paracrine senescence) in the ischemic retina, which contributes to destructive preretinal angiogenesis.

[0025] 1. Compositions comprising SASP modulators and aging attenuators, and uses thereof, for treating and preventing ocular diseases. In a first aspect, the data described herein show that the senescence pathway is initially engaged as a homeostatic mechanism in the retina to withstand hypoxic stress. However, when it persists, the senescence pathway becomes pathological and tissue integrity is compromised. A consequence of cellular senescence is the SASP, which impedes proper vascular regeneration through the secretion of inflammatory factors. In particular, as shown herein, analysis of patients suffering from proliferative diabetic retinopathy showed SASP-related cytokines in the vitreous. Moreover, pharmacological inhibition of the SASP with the well-known biguanide metformin, or pharmacological or genetic interference with IRE1α, limits senescence, enhances reparative vascular regeneration, and prevents destructive neovascularization (Figure 6H-J), stalling retinal pathology in vivo. These data provide evidence for a previously undescribed paradigm implicating cellular and paracrine senescence in pathological angiogenesis and reveal the therapeutic benefit of ocular delivery of modulators of the SASP to treat retinopathies and ocular vascular diseases (angiopathies) such as age-related macular degeneration (AMD) and macular edema.

[0026] Thus, according to the present invention, there is provided a method of treating or preventing a vascular eye disease or disorder (ocular vasculopathy, particularly an aging-related eye disease or disorder, e.g., retinopathy), comprising reducing (attenuating / inhibiting) cellular senescence in the eye of a subject. Cellular senescence may be reduced by contacting the subject's cells with one or more compounds that reduce cellular senescence (senescence inhibitors). In an embodiment, the senescence inhibitor reduces or inhibits SASP in ocular cells.

[0027] The present invention further provides a method for inhibiting retinal angiogenesis (pathological neovascularization), comprising administering to a subject an aging inhibitor (e.g., a SASP inhibitor). In an embodiment, the retinal angiogenesis is secondary to ischemia.

[0028] The present invention also provides a method of promoting ocular vascular repair and / or reducing ocular ischemia comprising administering to a subject an agent that inhibits aging (e.g., a SASP inhibitor).

[0029] The present invention also provides a method for preventing or reducing ocular cell aging, comprising administering to a subject a senescence inhibitor. In embodiments, the senescence inhibitor is a SASP inhibitor.

[0030] The present invention also provides a method of preventing or reducing senescence of an ocular cell, comprising contacting an ocular cell with a senescence inhibitor. In embodiments, the senescence inhibitor is a SASP inhibitor.

[0031] In an embodiment, the aging is paracrine aging. In an embodiment, the aging is post-differentiation aging. In an embodiment, the aging is premature aging. In an embodiment, the premature aging is characterized by increased expression and / or RNAse activity of IRE1α. In an embodiment, the aging is retinal aging. In an embodiment, the aging is characterized by (i) increased expression and / or activity of P16INK4a, Tp53, IRE1α, Cdkn1a, Cdkn2a, and / or senescence-associated β-gal activity, (ii) expression of γH2Ax and / or PML, and / or (iii) expression of a senescence-associated secretory phenotype (SASP). In an embodiment, the SASP includes secretion of IL-1β, IL-6, Pai1, TGFβ1, IRE1α, and / or VEGF-α. In an embodiment, the SASP is secondary to cellular ischemia.

[0032] In embodiments, the cell is a human cell. In embodiments, the cell is a retinal cell. In embodiments, the cell is an endothelial cell. In embodiments, the cell is a microglial cell. In embodiments, the cell is a neuron. In embodiments, the cell is a retinal ganglion cell. In embodiments, the cell is a retinal ganglion neuron. In embodiments, the cell is a vascular cell. In embodiments, the cell is a vascular endothelial cell. In embodiments, the cell is not a vascular cell (i.e., located in an avascular region / zone). In embodiments, the cell is a fibroblast. In embodiments, the cell is a macrophage.

[0033] In embodiments, administration is topical or local ocular administration. In embodiments, topical ocular administration is subconjunctival (sub-Tenon), intravitreal, retrobulbar, posterior juxtascleral or intracameral administration. In embodiments, topical ocular administration is intravitreal administration. In certain embodiments, topical ocular administration is intravitreal injection.

[0034] The present invention also relates to a composition comprising an aging inhibitor or a SASP inhibitor for use in the methods of the present invention. In an embodiment, the composition is an ophthalmic composition. In an embodiment, the composition comprises a suitable pharmaceutical carrier, diluent, or excipient. In an embodiment, the suitable pharmaceutical carrier, diluent, or excipient is not normally found in admixture with the inhibitor disclosed herein (i.e., the non-natural carrier or composition is not found in nature, i.e., synthetic or artificial). In an embodiment, the composition is for treating or preventing a vascular eye disease or disorder. In an embodiment, the composition is for inhibiting retinal angiogenesis. In an embodiment, the composition is for promoting ocular vascular repair and / or reducing ocular ischemia. In an embodiment, the composition is for preventing or reducing ocular cellular aging. In an embodiment, the composition is for use in the preparation of a medicament for (i) treating or preventing a vascular eye disease or disorder, (ii) inhibiting retinal angiogenesis (e.g., pathological retinal neovascularization), (iii) promoting ocular vascular repair and / or reducing ocular ischemia, and / or (iv) preventing or reducing ocular cellular aging.

[0035] In an embodiment, the SASP inhibitor is not an inhibitor of IRE1α. In an embodiment, the SASP inhibitor is a biguanide compound. In an embodiment, the biguanide compound is metformin, phenformin, buformin, proguanil, chlorproguanil, synthalin A or synthalin B. In an embodiment, the biguanide compound is metformin. In an embodiment, the SASP inhibitor is an inhibitor of IRE1α.

[0036] In embodiments, the vascular eye disease or disorder is diabetic retinopathy, retinopathy of prematurity, ischemic retinopathy, hypertensive retinopathy, drug-induced retinal vasculopathy, diabetic macular edema, age-related macular degeneration, juvenile macular degeneration, retinal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, choroidal neovascularization, polypoidal choroidal vasculopathy, physical injury to the eye, glaucoma, rhegmatogenous retinal detachment (RRD), retinal vasculitis, retinal macroaneurysm, retinal microaneurysm, Fuchs' dystrophy, ischemic optic neuropathy, macular telangiectasia, optic neuritis, Usher syndrome, retinitis pigmentosa, uveitis, ischemic optic neuropathy (ION), or Stargardt's disease. In one embodiment, the vascular eye disease or disorder is diabetic retinopathy, retinopathy of prematurity, diabetic macular edema, age-related macular edema, retinal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, or choroidal neovascularization. In one embodiment, the vascular eye disease is diabetic retinopathy, retinopathy of prematurity, dry (atrophic) age-related macular degeneration, wet (exudative) age-related macular degeneration, branch retinal vein occlusion, or macular telangiectasia.

[0037] In embodiments, the subject treated with the SASP inhibitor or composition of the present invention has been diagnosed with one of the above vascular eye diseases or disorders. In embodiments, the subject has been diagnosed with diabetic retinopathy, retinopathy of prematurity, diabetic macular edema, age-related macular degeneration, pathological retinal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, choroidal neovascularization, polypoidal choroidal vasculopathy, or macular telangiectasia.

[0038] 2. Compositions and methods for modulating cellular senescence comprising IRE1α or SEMA3A modulators. (i) IRE1α modulator The invention also provides a method for inhibiting or preventing (i) cellular senescence of a cell, or (ii) a senescence-associated secretory phenotype (and / or induction thereof) of a cell, comprising reducing expression, activation or activity of IRE1α. The invention also relates to a method for inhibiting or preventing (i) cellular senescence of a cell, or (ii) induction of a senescence-associated secretory phenotype in a cell of a subject, comprising administering to the subject an IRE1α inhibitor. In embodiments, the method of the invention reduces activation of IRE1α, activity of SA-β-gal, and / or expression of Pai1, IL-6, Il-1b, TGF-b, tp53, XBP1(s), and / or Vegfa in a cell.

[0039] The present invention further relates to a composition comprising an IRE1α inhibitor for (i) inhibiting or preventing cellular senescence in a cell, or (ii) inducing a senescence-associated secretory phenotype in a cell comprising the IRE1α inhibitor. In an embodiment, the composition is for (i) inhibiting or preventing cellular senescence in a cell, or (ii) inducing a senescence-associated secretory phenotype in a cell. In an embodiment, the composition is for use in the preparation of a medicament for (i) inhibiting or preventing cellular senescence in a cell, or (ii) inducing a senescence-associated secretory phenotype in a cell.

[0040] In embodiments, the IRE1α inhibitor is an antisense or shRNA against IRE1α, 4u8c, bortezomib, N-[(2-hydroxy-1-naphthalenyl)methylene]-2-thiophenesulfonamide (STF-083010), or MKC-3946. In embodiments, the inhibitor reduces activation of IRE1α, activity of SA-β-gal, and / or expression of Pai1, IL-6, Il-1b, TGF-b, tp53, XBP1(s), and / or Vegfa in a cell.

[0041] The present invention also provides a method for stimulating or inducing (i) cellular senescence or (ii) cellular senescence-associated secretory phenotype (SASP) (and / or induction thereof), comprising increasing IRE1α levels or activity. The present invention also provides a method for improving wound repair, comprising increasing IRE1α levels or activity, in which (i) cellular senescence or (ii) senescence-associated secretory phenotype (SASP) in the cell is increased or induced. The present invention further provides a method for stimulating or inducing (i) cellular senescence or (ii) senescence-associated secretory phenotype in a cell of a subject, comprising increasing IRE1α levels or activity. In an embodiment, the method comprises contacting the cell with a compound that increases IRE1α levels or activity. In an embodiment, the method increases IRE1α activation, SA-β-gal activity, and / or Pai1, IL-6, Il-1b, TGF-b, tp53, XBP1(s), and / or Vegfa expression in the cell.

[0042] In embodiments, IRE1α activity includes IRE1α ribonuclease activity and kinase activity.

[0043] The present invention further provides a composition comprising a compound that increases IRE1α level or activity. In an embodiment, the composition is for inducing (i) cellular senescence, or (ii) a senescence-associated secretory phenotype in a cell. In an embodiment, the composition is for treating or preventing liver fibrosis, renal fibrosis, pulmonary hypertension, myocardial infarction, or cardiac fibrosis. In an embodiment, the composition is for improving wound healing. In an embodiment, the composition is used for the preparation of a medicament for inducing (i) cellular senescence, or (ii) a senescence-associated secretory phenotype in a cell. In an embodiment, the composition is used for the preparation of a medicament for treating or preventing liver fibrosis, renal fibrosis, pulmonary hypertension, myocardial infarction, or cardiac fibrosis. In an embodiment, the composition is for use in the preparation of a medicament for improving wound healing.

[0044] In embodiments, the compound that increases IRE1α levels or activity is Apy29 or sunitinib.

[0045] In embodiments, the cell is a terminally differentiated cell. In embodiments, the cell is a neuron, a microglial cell, or an endothelial cell. In embodiments, the cell is a retinal cell. In embodiments, the cell is a myeloid cell. In embodiments, the cell is a retinal ganglion cell. In embodiments, the cell is a retinal ganglion neuron. In embodiments, the cell is a vascular cell. In embodiments, the cell is a vascular endothelial cell. In embodiments, the cell is not a vascular cell (i.e., located in an avascular region / area). In embodiments, the cell is a fibroblast. In embodiments, the cell is a macrophage. In embodiments, the cell is a monocyte. In embodiments, the cell is a hepatocyte. In embodiments, the cell is a hepatic stellate cell. In embodiments, the cell is a human cell. In embodiments, the cell is a human microvascular endothelial cell. In certain embodiments, the cell is not an ocular cell.

[0046] In an embodiment, the aging is paracrine aging. In an embodiment, the aging is post-differentiation aging. In an embodiment, the aging is premature aging. In an embodiment, the premature aging is characterized by increased expression and / or RNAse activity of IRE1α. In an embodiment, the aging is retinal aging. In an embodiment, the aging is characterized by (i) increased expression and / or activity of P16INK4a, Tp53, IRE1a, Cdkn1a, Cdkn2a, and / or senescence-associated β-gal activity, (ii) expression of γH2Ax and / or PML, and / or (iii) expression of senescence-associated secretory phenotype (SASP). In an embodiment, the SASP includes secretion of IL-1β, IL-6, Pai1, TGFβ1, IRE1a and / or VEGFa. In an embodiment, the SASP is secondary to cellular ischemia. In embodiments, the cells are derived from a subject suffering from or at risk of suffering from sarcopenia, neurodegeneration, epidermal thinning, skin wrinkling, hair loss, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), atherosclerosis, osteoarthritis, osteoporosis or Parkinson's disease, intestinal disease, glaucoma, intervertebral disc degeneration, cerebral aneurysm, aortic aneurysm, pancreatic fibrosis, or cystic fibrosis. In embodiments, the cells are derived from a subject who has undergone or is undergoing cancer treatment. In embodiments, the cells are not retinal cells. In embodiments, the cellular senescence is not associated with retinal vascular disease (i.e., it does not occur in the context of a retinal disease). In embodiments, the cellular senescence is not associated with vascular eye disease (i.e., it does not occur in the context of an eye disease). In embodiments, the cellular senescence is not associated with diabetic retinopathy (i.e., it does not occur in the context of diabetic retinopathy). In embodiments, the cellular senescence is not associated with macular degeneration. In embodiments, the cells are derived from a subject having or at risk of having liver fibrosis, renal fibrosis, pulmonary hypertension, myocardial infarction, or cardiac fibrosis. In embodiments, the subject is wounded (e.g., has a skin / tissue wound (e.g., a cut)).

[0047] (ii) SEMA3A modulators In a further aspect, the data presented herein provide evidence of a previously undescribed role for SEMA3A in regulating aging, including paracrine aging in pathological processes, and reveal therapeutic benefits in modulating SEMA3A activity in aging-related diseases and conditions. Indeed, the data demonstrate that SEMA3A activates the ER stress effector inositol-requiring enzyme-1α (IRE-1α) as well as the senescence effectors p53 and p16.

[0048] Thus, in one aspect, there is provided a method of modulating (i) cellular senescence, or (ii) cellular senescence-associated secretory phenotype (SASP) (and / or induction thereof), comprising modulating SEMA3A levels or activity. In embodiments, modulating SEMA3A levels or activity comprises contacting the cell with a SEMA3A antagonist or a SEMA3A agonist.

[0049] In a related aspect, the invention provides a method of inhibiting or preventing (i) cellular senescence, or (ii) cellular senescence-associated secretory phenotype (SASP) (and / or induction thereof), comprising decreasing the level or activity of SEMA3A, In embodiments, decreasing the level or activity of SEMA3A comprises contacting the cell with a SEMA3A antagonist.

[0050] The invention further provides a method of inhibiting or preventing (i) cellular senescence, or (ii) senescence-associated secretory phenotype (SASP) (and / or induction thereof) in a cell of a subject, comprising decreasing the level or activity of SEMA3A, In embodiments, decreasing the level or activity of SEMA3A comprises administering to the subject (or contacting the cells of the subject) a SEMA3A antagonist.

[0051] In a further aspect, the present invention relates to a SEMA3A antagonist. In an embodiment, the SEMA3A antagonist is (a) a SEMA3A antibody, (b) a SEMA3A antisense or shRNA, and / or (c) a soluble NRP1 polypeptide, or a functional fragment thereof (NRP1 trap).

[0052] The present invention also relates to a composition comprising the above-mentioned SEMA3A antagonist. Such antagonist or the composition comprising it can be used in the above-mentioned method (for example, for use in inhibiting or preventing (i) cellular senescence or (ii) the induction of cellular senescence-associated secretory phenotype (SASP)).

[0053] In a related aspect, the invention relates to the use of a SEMA3A antagonist or composition of the invention in the preparation of a medicament for inhibiting or preventing (i) cellular senescence, or (ii) cellular senescence-associated secretory phenotype (SASP) (and / or induction thereof). In an embodiment, the methods and compositions described herein are for treating or preventing a disease or condition associated with aging, which is sarcopenia, neurodegeneration (e.g., Alzheimer's disease), epidermal thinning, skin wrinkling, hair loss, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), atherosclerosis, osteoarthritis, osteoporosis, Parkinson's disease, intestinal disease, glaucoma, intervertebral disc degeneration, cerebral aneurysm, aortic aneurysm, pancreatic fibrosis, or cystic fibrosis metabolic syndrome and / or obesity. In an embodiment, the cell is not a retinal cell. In an embodiment, the cell is not a retinal ganglion cell. In an embodiment, the cell is not derived from the eye of a subject (ocular cell). In embodiments, the cellular senescence is not associated with retinal vascular disease.

[0054] In embodiments, the above methods reduce activation of IRE1α and expression of Pai1, IL-6, Il-1b, TGF-b, tp53, XBP1(s), and / or Vegfa in cells.

[0055] In embodiments, the cell is a terminally differentiated cell. In embodiments, the cell is a neuron, a microglial cell, or an endothelial cell. In embodiments, the cell is a retinal cell. In embodiments, the cell is a myeloid cell. In embodiments, the cell is an adipose tissue cell. In embodiments, the cell is a retinal ganglion cell. In embodiments, the cell is a retinal ganglion neuron. In embodiments, the cell is a vascular cell. In embodiments, the cell is a vascular endothelial cell. In embodiments, the cell is not a vascular cell (i.e., located in an avascular region / area). In embodiments, the cell is a fibroblast. In embodiments, the cell is a macrophage. In embodiments, the cell is a monocyte. In embodiments, the cell is a hepatocyte. In embodiments, the cell is a hepatic stellate cell. In embodiments, the cell is a human cell. In embodiments, the cell is a human microvascular endothelial cell. In certain embodiments, the cell is not an ocular cell. In embodiments, the senescence described above is paracrine senescence. In embodiments, the senescence is secondary to cellular ischemia. In embodiments, the SASP is secondary to cellular ischemia. In embodiments, the aging is secondary to glucose intolerance. In embodiments, the SASP is secondary to glucose intolerance.

[0056] In embodiments, the cells are derived from a subject suffering from or at risk of suffering from an age-related disease or condition that is sarcopenia, neurodegeneration (e.g., Alzheimer's disease), epidermal thinning, skin wrinkling, hair loss, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), atherosclerosis, osteoarthritis, osteoporosis, Parkinson's disease, intestinal disease, glaucoma, intervertebral disc degeneration, cerebral aneurysm, aortic aneurysm, pancreatic fibrosis, or cystic fibrosis, metabolic syndrome, and / or obesity. In embodiments, the cells are not retinal cells. In embodiments, the cells are not retinal ganglion cells. In embodiments, the cells are not derived from the subject's eye (ocular cells). In embodiments, the cellular senescence is not associated with retinal vascular disease. In embodiments, the cellular senescence is not associated with ocular disease (ocular cell senescence). In embodiments, the cellular senescence is not associated with Alzheimer's disease. In embodiments, the cellular senescence is not associated with diabetes. In embodiments, the cellular senescence is not associated with cancer. In embodiments, the cellular senescence is not associated with septic shock.

[0057] In another aspect, the present invention relates to a method for stimulating or inducing (i) cellular senescence, or (ii) a senescence-associated secretory phenotype in a cell, comprising contacting a cell with a SEMA3A polypeptide or a functional variant or fragment thereof.

[0058] Also provided is a method for stimulating or inducing (i) cellular senescence in a cell, or (ii) a senescence-associated secretory phenotype in a cell of a subject, comprising administering to a subject an effective amount of a SEMA3A polypeptide, or a functional variant or fragment thereof.

[0059] In a further aspect, the present invention relates to a method for improving wound healing in a tissue comprising a cell, the method comprising contacting the cell with a SEMA3A polypeptide or a functional variant or fragment thereof.

[0060] In related aspects, the present invention provides SEMA3A polypeptides, or functional variants or fragments thereof, nucleic acids encoding same, vectors for delivering and / or expressing SEMA3A polypeptides, or functional variants or fragments thereof, and host cells comprising such polypeptides or functional variants or fragments, nucleic acids and / or vectors.

[0061] The present invention also relates to compositions comprising the above-mentioned SEMA3A polypeptides, or functional variants or fragments thereof, nucleic acids, vectors and / or host cells. Such compositions, SEMA3A polypeptides or functional variants or fragments thereof, nucleic acids, vectors and host cells can be used in the above-mentioned methods (e.g., (a) for inducing (i) cellular senescence or (ii) a senescence-associated secretory phenotype in cells, (b) in the preparation of a medicament for inducing (i) cellular senescence or (ii) a senescence-associated secretory phenotype in cells, or (c) for improving wound healing).

[0062] In an embodiment, the cell in the method of stimulating or inducing (i) cellular senescence, (ii) SASP, or (iii) wound healing is a terminally differentiated cell. In an embodiment, the cell is a neuron, a microglial cell, or an endothelial cell. In an embodiment, the senescence is paracrine senescence. In an embodiment, the SASP is secondary to cellular ischemia. In an embodiment, the cell is derived from a subject having or at risk of having liver fibrosis, pulmonary hypertension, myocardial infarction, cancer, renal fibrosis, or cardiac fibrosis.

[0063] In embodiments, the methods described above reduce the activation of IRE1α and the expression of Pai1, IL-6, Il-1b, TGF-b, tp53, XBP1(s), and Vegfa in cells.

[0064] The present invention also provides nucleic acids encoding the polypeptides (e.g., NRP1 trap, SEMA3A, IRE1α, etc.), antisense, shRNA, etc. disclosed herein, as well as vectors and host cells for delivering and / or expressing the nucleic acids, polypeptides, antisense, shRNA, etc. disclosed herein.

[0065] Other objects, advantages and features of the present invention will become more apparent upon reading the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

[0066] 3. Modulation of Lipid Parameters In another aspect, the present invention relates to a method of altering lipid parameters in a subject, the method comprising administering to the subject: (a) a soluble NRP1 polypeptide or fragment thereof, (b) an NRP1 antibody, or (c) a composition comprising (a) and / or (b) together with a pharma- ceutically acceptable carrier. The alteration of the lipid parameters is (a) lowering total cholesterol levels, (b) lowering non-HDL cholesterol levels, (c) lowering triglyceride levels, (d) lowering the ratio of total cholesterol:HDL cholesterol, (e) reducing circulating free fatty acids, (f) increasing HDL cholesterol, or (f) any combination of (a)-(e).

[0067] In another aspect, the invention relates to a method for preventing or treating a disease or condition associated with fat accumulation in a subject, the method comprising administering to the subject: (a) a soluble NRP1 polypeptide or fragment thereof, (b) an NRP1 antibody, or (c) a composition comprising (a) and / or (b) together with a pharma- ceutical acceptable carrier.

[0068] In one embodiment, the disease or condition associated with adiposity is high body mass index (BMI), obesity, metabolic syndrome, NAFLD, cardiovascular disease (CVD), hypertension and / or type II diabetes mellitus (TIIDM).

[0069] In one embodiment, the cardiovascular disease is congestive heart failure, hypercholesterolemia, and / or atherosclerosis.

[0070] In another aspect, the invention relates to a method for altering a body composition parameter in a subject, the method comprising administering to the subject: (a) a soluble NRP1 polypeptide or fragment thereof, (b) an NRP1 antibody, or (c) a composition comprising (a) and / or (b) together with a pharma- ceutically acceptable carrier. The body composition parameter is visceral fat area (VFA), body mass index (BMI), waist-to-hip ratio (WHR), waist-to-height ratio (WHeR), waist circumference (WC), upper arm circumference (AC), conicity index, body fat percentage (PBF), triceps skinfold, subscapular skinfold, white adipose tissue (WAT) levels, and / or brown adipose (BAT) tissue levels.

[0071] In embodiments, the soluble NRP1 polypeptide or fragment thereof comprises or consists of an NRP1 polypeptide trap as set forth in Table 2, or as set forth in Figure 7 or Figure 9A.

[0072] In one embodiment, the soluble NRP1 polypeptide or fragment thereof is administered systemically.

[0073] In another aspect, the invention relates to a composition comprising (a) a soluble NRP1 polypeptide or fragment thereof, (b) an NRP1 antibody, or (c) a composition comprising (a) and / or (b) together with a pharma- ceutically acceptable carrier, for altering a lipid parameter in a subject, the alteration of the lipid parameter being (a) lowering total cholesterol levels, (b) lowering non-HDL cholesterol levels, (c) lowering triglyceride levels, (d) lowering the ratio of total cholesterol:HDL cholesterol, (e) reducing circulating free fatty acids, (f) increasing HDL cholesterol, or (f) any combination of (a)-(e).

[0074] In another aspect, the present invention relates to a composition comprising (a) a soluble NRP1 polypeptide or fragment thereof, (b) an NRP1 antibody, or (c) a composition comprising (a) and / or (b) together with a pharma- ceutical acceptable carrier, for preventing or treating a disease or condition associated with fat accumulation in a subject.

[0075] In one embodiment, the disease or condition associated with adiposity is high body mass index (BMI), obesity, metabolic syndrome, NAFLD, cardiovascular disease (CVD), hypertension and / or type II diabetes mellitus (TIIDM).

[0076] In one embodiment, the cardiovascular disease is congestive heart failure, hypercholesterolemia, and / or atherosclerosis.

[0077] In another aspect, the present invention relates to a composition comprising (a) a soluble NRP1 polypeptide or fragment thereof, (b) an NRP1 antibody, or (c) a composition comprising (a) and / or (b) together with a pharma- ceutically acceptable carrier, for altering body composition parameters in a subject. Body composition parameters were visceral fat area (VFA), body mass index (BMI), waist-to-hip ratio (WHR), waist-to-height ratio (WHeR), waist circumference (WC), upper arm circumference (AC), conicity index, percentage of body fat (PBF), triceps skinfold, subscapular skinfold, white adipose tissue (WAT) level, or brown adipose (BAT) tissue level.

[0078] In one embodiment, the soluble NRP1 polypeptide or fragment thereof comprises or consists of an NRP1 polypeptide trap as set forth in Table 2, or as set forth in Figure 7 or Figure 9A.

[0079] In one embodiment, the soluble NRP1 polypeptide or fragment thereof is for systemic administration.

[0080] In another aspect, the present invention relates to the use of (a) a soluble NRP1 polypeptide or fragment thereof, (b) an NRP1 antibody, or (c) a composition comprising (a) and / or (b) together with a pharma- ceutically acceptable carrier, for altering lipid parameters in a subject. The modification of lipid parameters is (a) lowering total cholesterol levels, (b) lowering non-HDL cholesterol levels, (c) lowering triglyceride levels, (d) lowering the total cholesterol:HDL cholesterol ratio, (e) decreasing circulating free fatty acids, (f) increasing HDL cholesterol, or (f) any combination of (a)-(e).

[0081] In another aspect, the invention relates to the use of a soluble NRP1 polypeptide or fragment thereof, (b) an NRP1 antibody, or (c) a composition comprising (a) and / or (b) together with a pharma- ceutically acceptable carrier, for preventing or treating a disease or condition associated with fat accumulation in a subject.

[0082] In one embodiment, the disease or condition associated with adiposity is high body mass index (BMI), obesity, metabolic syndrome, NAFLD, cardiovascular disease (CVD), hypertension and / or type II diabetes mellitus (TIIDM).

[0083] In one embodiment, the cardiovascular disease is congestive heart failure, hypercholesterolemia, and / or atherosclerosis.

[0084] In another aspect, the present invention relates to the use of (a) a soluble NRP1 polypeptide or fragment thereof, (b) an NRP1 antibody, or (c) a composition comprising (a) and / or (b) together with a pharma- ceutically acceptable carrier, to alter body composition parameters in a subject. Body composition parameters were visceral fat area (VFA), body mass index (BMI), waist-to-hip ratio (WHR), waist-to-height ratio (WHeR), waist circumference (WC), upper arm circumference (AC), conicity index, percentage of body fat (PBF), triceps skinfold, subscapular skinfold, white adipose tissue (WAT) level, or brown adipose (BAT) tissue level.

[0085] In one embodiment, the soluble NRP1 polypeptide or fragment thereof comprises or consists of an NRP1 polypeptide trap as set forth in Table 2, or as set forth in Figure 7 or Figure 9A.

[0086] In one embodiment, the soluble NRP1 polypeptide or fragment thereof is for systemic administration.

[0087] In another aspect, the present invention relates to the use of (a) a soluble NRP1 polypeptide or fragment thereof, (b) an NRP1 antibody, or (c) a composition comprising (a) and / or (b) together with a pharma- ceutically acceptable carrier for preparing a medicament for altering lipid parameters in a subject. The modification of lipid parameters is (a) lowering total cholesterol levels, (b) lowering non-HDL cholesterol levels, (c) lowering triglyceride levels, (d) lowering the total cholesterol:HDL cholesterol ratio, (e) decreasing circulating free fatty acids, (f) increasing HDL cholesterol, or (f) any combination of (a)-(e).

[0088] In another aspect, the present invention relates to the use of a composition comprising (a) a soluble NRP1 polypeptide or fragment thereof, (b) an NRP1 antibody, (c) (a) and / or (b) together with a pharma- ceutically acceptable carrier for the preparation of a medicament for preventing or treating a disease or condition associated with fat accumulation in a subject.

[0089] In one embodiment, the disease or condition associated with adiposity is high body mass index (BMI), obesity, metabolic syndrome, NAFLD, cardiovascular disease (CVD), hypertension and / or type II diabetes mellitus (TIIDM).

[0090] In one embodiment, the cardiovascular disease is congestive heart failure, hypercholesterolemia, and / or atherosclerosis.

[0091] In another aspect, the present invention relates to the use of (a) a soluble NRP1 polypeptide or fragment thereof, (b) an NRP1 antibody, or (c) a composition comprising (a) and / or (b) together with a pharma- ceutically acceptable carrier, for the preparation of a medicament for altering a body composition parameter in a subject. Body composition parameters were visceral fat area (VFA), body mass index (BMI), waist-to-hip ratio (WHR), waist-to-height ratio (WHeR), waist circumference (WC), upper arm circumference (AC), conicity index, percentage of body fat (PBF), triceps skinfold, subscapular skinfold, white adipose tissue (WAT) level, or brown adipose (BAT) tissue level.

[0092] In one embodiment, the soluble NRP1 polypeptide or fragment thereof comprises or consists of an NRP1 polypeptide trap as set forth in Table 2, or as set forth in Figure 7 or Figure 9A.

[0093] In one embodiment, the medicament is for systemic administration. [Brief description of the drawings]

[0094] [Figure 1-1] Figure 1: Retinal ischemia induces cellular senescence and a senescence-associated secretory phenotype. (A) Schematic of a mouse model of oxygen-induced retinopathy (OIR). (B) Representative gene set enrichment analysis (GSEA) corresponding to inflammatory and apoptotic features from large-scale genome-wide RNA sequencing of P14 normoxic and OIR retinas. Gene expression profiles positively and negatively correlated with the OIR phenotype are depicted. [Figure 1-2] Retinal ischemia induces cellular senescence and a senescence-associated secretory phenotype. (C) Heatmap and GSEA clusters of Fridman's senescence-associated genes (28) in P14 OIR (left column) versus normoxic retina (right column). (NES=normalized enrichment score; FDRq=false discovery rate), color scale representing log2 expression (fold change) from low expression (-2, left) to high expression (2, right). [Figure 1-3] Figure 1: Retinal ischemia induces cellular senescence and a senescence-associated secretory phenotype. (D) Immunoblots of retinal cell lysates from P14 OIR and normoxic mice show induction of senescence markers. (E) RT-qPCR shows induction of expression of senescence-associated genes, Cdkn1a (p21) and Cdkn2a (p16), in P14 OIR versus normoxic retinas. [Figure 1-4] Retinal ischemia induces cellular senescence and a senescence-associated secretory phenotype. (F) Representative isolectin B4 (IB4) and senescence-associated β-galactosidase staining (SA-β-gal) of P14 normoxic and OIR flat-mount retinas. [Figure 1-5] Figure 1: Retinal ischemia induces cellular senescence and a senescence-associated secretory phenotype. (G) Quantification (percentage) of senescence-associated β-galactosidase staining (SA-β-gal) in P14 OIR vs normoxic flat-mount retinas. Higher magnification images of boxed central (c) / peripheral (p) and vascular / avascular zones of retinas are shown (central***P<0.0001 compared to peripheral OIR retinas (n=10); avascular†††P<0.0001 compared to central P14 normoxic retina (n=7)). (H) Representative IB4 and SA-β-gal staining of sagittal sections of P14 normoxic and OIR retinas. A higher magnification image of the boxed central avascular retinal zone is shown in the right panel. For orientation, retinal ganglion cells (GCL), inner nuclear layer (INL) and outer nuclear layer (ONL) are shown. (I) Quantification of the percentage area stained with SA-β-gal in sagittal retinal sections at P14 (* P < 0.05 ( N = 10) compared with normoxic retina, † P < 0.05 ( n = 7) compared with other P14 OIR layers of the retina, ns = not significant). [Figure 1-6] FIG. 1: Retinal ischemia induces cellular senescence and a senescence-associated secretory phenotype. (J) TUNEL staining of P14 OIR and normoxic retinas. Scale bar is 500 μM (H). High magnification images and (H, J) Scale bar is 200 μM. Data are presented as mean ± SEM. [Figure 2-1] Cellular senescence propagates during retinopathy progression. (A) Isolectin B4 (IB4) staining of retinas during OIR progression (P14, P17 and P21). (B) Schematic of the propagation of cellular senescence throughout OIR. Bottom panel shows SA-β-gal stained flat-mounted OIR retina (scale bar 50 μm). Higher magnification images reveal distinct senescent cell populations (scale bar 25 μm). [Figure 2-2] Cellular senescence propagates during retinopathy progression. (C) Representative confocal micrographs of P14 OIR retinas show intense staining of senescence markers (γH2AX (top row) and Pai1 (middle row)) in RGCs (Brn3a staining (second row)). Insets are high magnification images of boxed areas. [Figure 2-3] Cellular senescence propagates during retinopathy progression. (D) Representative confocal micrographs of P17 OIR flat-mounted retinas reveal that senescence markers, PML (bottom row) and γH2AX (top row), co-label with microglia (IBA1) and vascular IB4. [Figure 2-4] Cellular senescence propagates during retinopathy progression. (E) Heatmap and GSEA clusters of paracrine senescence-related genes in P14 OIR vs normoxic retinas for two different samples. (F) RT-qPCR shows that expression of Pai1, Il1β, Tgf-β1, I16, Vegf-a, Ire1α and Tp53 is induced in P14 OIR vs normoxic retinas. β-actin was used as a reference gene. In C and D, scale bars are 100 μm. Data are presented as mean ± SEM. [Figure 3-1]Figure 2: SEMA3A mediates senescence and paracrine senescence. (A) Western blot analysis of SEMA3A protein expression levels during OIR compared to normoxic controls at P10, P14, P17 and P21. β-actin is used as a loading control. (B) Immunoblot analysis of SEMA3A protein levels during Ras-induced senescence in mouse embryonic fibroblasts (MEFs). Cell lysates from MEFs retrovirally transduced with H-RasV12 oncogene or control empty vector harvested 14 days after selection. (C) SEMA3A transcript levels in human normal diploid fibroblasts (IMR90) retrovirally transduced with empty vector, MEK1 alone or MEK and human papillomavirus oncoproteins E6 and E7. Data are from GEO profile GSE2487 (77) (**P<0.05CT compared to MEK, and ††P<0.05 compared to MEK / E6 / E7). (D) SEMA3A transcript levels in proliferative or senescent cultured primary human activated hepatic stellate cells (HSCs) (**P=0.027). [Figure 3-2] Figure 2: SEMA3A mediates senescence and paracrine senescence. (E) Retinas from P5 mouse pups that received intravitreal injections of recombinant SEMA3A (100 ng / ml) at P2 and harvested at P5 were subjected to immunoblot analysis for senescence markers and sagittal cryosections were subjected to SA-β-gal staining (F). (G) Cell cycle distribution profile of SEMA3A-treated (100 and 500 ng / ml) HRMEC (human retinal microvascular endothelial cells) obtained by FACS analysis 7 days after treatment (P<0.0001; two-way ANOVA), (H) Transendothelial resistance measured in real time by electrical cell impedance sensing (ECIS) demonstrated that SEMA3A reduces endothelial cell proliferation (3-7 days) (0.048>P>0.009). [Figure 3-3]Figure 2: SEMA3A mediates senescence and paracrine senescence. (I) HRMEC, retinal neurons (661W), and J774 (macrophage-like cells) were stimulated with recombinant SEMA3A (100 ng / ml) or vehicle (CT) for 7 days, stained with SA-β-gal, and quantified in (J). (n=3 separate experiments), **P<0.005 and ***P<0.0001 by two-tailed Student's t-test J. (K) Conditioned medium (CM) was collected from senescent retinal neurons (661W) and macrophage-like J774 cells grown for 7 days after H2O2 stimulation (150 μM, 2 h). Senescent cells were stained with SA-β-gal (right), and the levels of secreted SEMA3A (S3A) protein in CM were assessed by immunoblotting (left). (L) SA-β-gal staining of neuronal 661W cells infected with the indicated vectors (Lv.sh_GFP or Lv.sh_SEMA3A) and treated with H2O2 or vehicle (CT). Senescence was assessed after 7 days of treatment. (M) Quantification of the percentage of SA-β-gal positive cells treated as in (J) (n=3 independent experiments). *P<0.005, H2O2-treated sh_GFP cells compared to untreated sh_GFP cells; and †P<0.005, H2O2-treated sh_S3A cells compared to H2O2-treated sh_GFP cells. Two-tailed Student's t-test. Data are presented as mean ± SEM. [Diagram 3-4] Figure 2: SEMA3A mediates senescence and paracrine senescence. (N) Induction of paracrine senescence in HRMEC (CM from senescent or non-senescent neuronal progenitor cells (see (661W) figure (I))) was assessed after 7 days by SA-β-gal staining. (O) Quantification (percentage) of SA-β-gal positive cells using CM from senescent 661W cells treated as in (I). [Figure 3-5] Figure 2: SEMA3A mediates senescence and paracrine senescence. (P) IB4 and SA-β-gal staining of retinas from P14 OIR mice intravitreally injected with Lv.sh_S3A or Lv.sh_GFP at P12. (Q) Quantification (***P<0.0001). [Figure 4] Heatmap and GSEA for signatures of misfolded protein response in P14 OIR vs normoxic retinas. (A) Gene expression profiles positively and negatively correlated with OIR phenotype. (B) Representative SA-β-gal and IB4 staining of LysM-Cre / ROSA26EYFPfl / fl retinas at P14 and P17 OIR. (C) Representative γH2AX staining and EYFP in the central region from LysM-Cre / ROSA26EYFPfl / fl at P14 and P17 OIR. White sagittas indicate co-labeling of γH2AX and EYFP+ microglia. [Diagram 5] Figure 2: SEMA3A is secreted by senescent cells and induces paracrine senescence in human retinal microvasculature endothelial cells. Relative mRNA levels of SEMA3A in P14 (A) and P17, P21 (B) retinas (normoxia vs. OIR) measured by RT-qPCR. (C) Western blot analysis of SEMA3A protein expression showing the efficiency of Sh-RNA downregulation in neuronal cells (661W). (D) Schematic illustrating conditioned medium (CM) experiments to obtain the contribution of paracrine senescence. CM from retinal neuronal progenitor cell lines expressing sh_GFP or sh_SEMA3A were senescent with H2O2, and CM was harvested and applied to HRMEC. Induction of paracrine senescence in HRMEC was assessed after 7 days by SA-β-gal staining. (E) Levels of secreted SEMA3A in CM from retinal neuronal progenitor cells infected with Lv.sh_GFP or Lv.sh_S3A were assessed by immunoblot. (F) Immunoblot analysis of p53 expression in HRMECs exposed to CM from senescent (or non-senescent) retinal neuronal progenitor cells (treated with H2O2 or vehicle). (G) Immunoblot analysis of HRMECs exposed to CM from retinal neuronal progenitor cells transfected with sh_GFP or sh_SEMA3A (sh_S3A) and treated with H2O2 or vehicle. (H) Immunoblot analysis of the ER stress effector, p-IRE1αS724, in HRMEC lysates harvested after 7 days of treatment with recombinant SEMA3A (100 ng / ml) or CM from senescent J774 cells (Sen-J774). [Figure 6-1] Figure 2 shows that SEMA3A induces senescence in J774 macrophages / monocytes. (A) P14 retinal lysates from mice receiving intravitreal injections of Lv.sh_GFP or Lv.sh-IRE1α were immunoblotted for anti-IRE1α and Cre. (B) SA-β-gal staining of J774 macrophages treated with SEMA3A (100 ng / ml, 7 days) or H2O2 as positive controls. Boxed areas show higher magnification views. (C) Immunoblot analysis of J774 macrophages exposed to SEMA3A (100 ng / ml, 7 days) or H2O2 as positive controls, and HRMEC treated with conditioned medium (CM) from the respective J774 conditions indicated by dashed arrows. (D) Immunoblot for IRE1α demonstrates the efficiency of lentivirus-mediated depletion of IRE1α in HRMEC. [Figure 6-2] Figure 2 shows that SEMA3A induces senescence in J774 macrophages / monocytes. SA-β-gal staining of HRMEC infected with and then treated with recombinant SEMA3A (100 ng / ml, 7 days) or vehicle (CT). (F) Relative mRNA expression levels of Cdkn1a, Cdkn2a in J774 treated with SEMA3A (100 ng / ml, 7 days) or vehicle (CT). [Figure 7]Figure 1: RNAse activity of IRE1α contributes to senescence. (A) Immunoblots for senescence markers in J774 cell lysates stimulated with SEMA3A (S3A) (100 ng / mL) or vehicle for 3 or 7 days show that p-IRE1αS724, p53, Pai1 are induced (n=3). (B) RT-qPCR in J774 macrophages revealed increased expression of Pai1, I16, I11β, Tgf-β1, and Tp53 mRNA after exposure to SEMA3A (100 ng / mL) for 7 days. (C) Representative confocal immunofluorescence staining of γH2AX and DAPI of J774 macrophages stimulated with SEMA3A (100 ng / mL) or vehicle for 7 days (scale bar is 100 μm). Higher magnification of boxed areas is shown (scale bar is 50 μm). (D) Immunoblot of XBP1(s) (spliced ​​isoforms) and (E) PCR of XBP1(s) and unspliced ​​XBP1(u) in J774 cell lysates treated with vehicle, S3A (100 ng / ml), and / or 4μ8c (1 ng / ml) at day 7 after treatment. (F) RT-qPCR of Pai1, I16, I11β, Tgf-β1, Tp53, Ire1α, and Tnfα levels in J774 stimulated with SEMA3A alone or 4μ8c. β-actin was used as a reference gene. ***P<0.0001, *P<0.005. Scale bar: 100 μm for C&G. Higher magnification in C is 50 μm. Data are presented as mean ± SEM. [Figure 8-1]Figure 1 shows that metformin abrogates SASP and pathological retinal angiogenesis. (A) Angiograms, spectral domain optical coherence tomography (SD-OCT) and 3D retinal maps obtained from patients selected for this study. Control patients (CT) (n=10) with non-vascular ocular pathology were compared with patients with proliferative diabetic retinopathy (PDR) (n=10). Table 1 (Example 6) shows the characteristics of the patients. (B) Multiplex assessment of the vitreous humor of patients for cytokines involved in paracrine aging shows induction in VEGF-A, Pai1, IL-6 and IL-8. Results are expressed as fold change normalized to CT patients. Points represent individual values; **P<0.001, ***P<0.0001. [Figure 8-2] Metformin abrogates SASP and pathological retinal angiogenesis. (C) Immunoblot analysis of p-NFκBS536 and p-IRE1αS724 in retinal lysates from P14 and P17 OIR mice intravitreally injected with metformin or vehicle at P12. (D) RT-qPCR for levels of Cdkn1a, Cdkn2a and Il6 (β-actin was used as reference gene) measured in retinas of P14 OIR mice intravitreally injected with metformin or vehicle at P12. Representative P14 (E) and P17 (F) OIR flat-mount retinas labeled with IB4 and SA-β-gal in mice intravitreally injected with metformin or vehicle at P12. [Figure 8-3]Metformin abrogates SASP and pathological retinal angiogenesis. (G) Quantification of percentage of SA-β-gal stained area in P14 and P17 OIR mice treated as in E and F (**P=0.0042 (n=9) at P14, **P=0.0013 (n=11) at P17. Compare metformin vs. vehicle injected retinas). (H) Representative IB4 stained flat-mount retinas from P14 and P17 OIR mice intravitreally injected with metformin or vehicle at P12. Avascular area quantified at P14 (I) and P17 (J) post-OIR. Preretinal neovascularization was assessed at P17 OIR (K). Results are expressed as percentage of avascular or neovascular area of ​​total retinal area (***P<0.0001 and ***P<0.001; Metformin vs. vehicle-injected retinas (n=13)). Horizontal lines represent mean percentages and points represent individual values. Scale bar is 500 μM. Data are expressed as mean±SEM. [Figure 9-1] Figure 1: Aflibercept abrogates pathological angiogenesis without promoting vascular repair or cellular senescence. Representative P14 (A) and P17 (C) OIR flat-mount retinas labeled with IB4 and SA-β-gal in mice intravitreally injected with aflibercept or vehicle at P12. Quantification of the percentage of SA-β-gal stained area in P14 (B) and P17 (D) OIR mice treated as in A or C (P = 0.3087 (n = 13–14) at P14, P = 0.1580 (n = 13) at P17, comparing aflibercept vs. vehicle-injected retinas at P14 in OIR (P = 0.4897, n = 11–13). [Figure 9-2]Figure 1: Aflibercept abrogates pathological angiogenesis without promoting vascular repair or cellular senescence. Representative P14 (A) and P17 (C) OIR flat-mount retinas labeled with IB4 and SA-β-gal in mice intravitreally injected with aflibercept or vehicle at P12. Quantification of the percentage of SA-β-gal stained area in P14 (B) and P17 (D) OIR mice treated as in A or C (P=0.3087 (n=13-14) at P14, P=0.1580 (n=13) at P17, comparing aflibercept vs. vehicle injected retinas). Quantification of avascular area at P14 (P=0.4897, n=11-13) (E), and P17 (P=0.9502, n=6-7) (F) in OIR. Preretinal neovascularization was assessed at P17 OIR (G). Results are expressed as the percentage of avascular or neovascular area relative to the total retinal area (***P<0.0207, n=5-6) comparing aflibercept with vehicle-injected retinas (n=5-6). Horizontal lines represent the mean percentages and points represent individual values. Scale bar, 500 μM. Data are expressed as mean ± SEM. [Figure 10-1] Figure 1 shows that retinal cell senescence is induced during OIR and in the STZ model of type I diabetes. (A) Isolectin B4 (IB4) and TUNEL staining of whole eye cryosections at P14 OIR and normoxia depicted in Figure 1I. [Figure 10-2] Figure 2 shows that retinal cellular senescence is induced during OIR and in the STZ model of type I diabetes.(B) Representative confocal immunofluorescence of γH2AX (green; left column), p-IRE1αS724 (green; middle column) and Pai1 (green; right column), isolectin B4 (IB4) (red), and DAPI (blue) in cryosectioned P14 normoxic and OIR retinas. [Figure 10-3]Figure 2: Retinal cellular senescence is induced during OIR and in the STZ model of type I diabetes. (C) Representative confocal immunofluorescence for PML (green; left column), p16 (green; right column), IB4 and DAPI in cryosectioned P14 OIR eyes. (D) Representative confocal immunofluorescence for PML, isolectin B4 (IB4) and DAPI in flat-mounted retinas at P21 OIR. [Figure 10-4] Figure 1: Retinal cell senescence is induced during OIR and in the STZ model of type I diabetes. (E) Representative SA-β-gal staining of frozen sections from citrate and STZ retinas. Retinal ganglion cells (GCL), inner nuclear layer (INL) and outer nuclear layer (ONL) are shown for orientation. (F) Representative confocal immunofluorescence of α-SMA or NG2, isolectin B4 (IB4) on flat-mounted retinas from adult mouse citrate (control) or STZ (diabetes). [Figure 11] Figure 1. Vascular accessibility during OIR. Representative confocal immunofluorescence of retinal flat mounts stained for α-SMA (A) or NG2 (B), isolectin B4 (IB4) at P17 and P21 during normoxia and OIR. Scale bar is 200 μM. [Figure 12] Figure 2: Metformin inhibits senescence during OIR. (A) Representative SA-β-gal staining of cryosections from P14 and P17 OIR retinas injected with metformin (10 μg / μl, P12) or vehicle (PBS). (B) Representative confocal immunofluorescence of TUNEL (left) and DAPI (middle) on cryosectioned P14 OIR retinas treated or not with metformin. Retinal ganglion cells (GCL), inner nuclear layer (INL) and outer nuclear layer (ONL) are shown for orientation. (C) Western blot analysis of cleaved caspase-3 protein expression levels during OIR (P14, P17 and P21). β-actin is used as a loading control. Scale bars are 200 μm. [Figure 13]Figure 1 shows genes that are not affected by metformin and aflibercept in OIR. (A) RT-qPCR of Vegf-a, Vegf-c, Vegfr-1 and Vegfr-2 levels measured in the retina of P14 OIR mice that were intravitreally injected with metformin or vehicle at P12 (β-actin was used as reference gene). (B) RT-qPCR of Cdkn2a, Tp53, Il1β, Tgf-β1 and Sema3a levels measured in the retina of P14 OIR mice that were intravitreally injected with aflibercept or vehicle at P12 (β-actin was used as reference gene). [Figure 14] FIG. 10: How SA-β-gal quantification is performed. Schematic representation of quantification of SA-β-gal staining on flat-mounted retinas (or sagittal eye sections) using Image J software analysis. [Figure 15] 1 shows structures of exemplary biguanide compounds and inhibitors of IRE1α: (A) biguanide (CAS#56-03-1), (B) metformin (N,N-dimethylimidodicarbonimidic diamide; CAS#657-24-9), (C) buformin (1-butylbiguanide, CAS#692-13-7), and (D) phenformin (2-(N-phenethylcarbamimidoyl)guanidine, CAS#114-86-3), (E) "Compound 3" IRE1α inhibitor that inhibits the RNAse activity of IRE1α (Wang et al., 2012, Nat. Chem. Bio. 8(12):982-989). [Figure 16] 1 shows the amino acid sequence of human SEMA3A precursor protein (SEQ ID NO:50), which is further processed to the mature form by removing the signal peptide (amino acids 1-21). [Figure 17-1]FIG. 1 shows alignment of the NRP1 protein sequence (SEQ ID NO: 47) with the rat (Accession No. EDL96784, NP_659566, SEQ ID NO: 48), human (Accession No. NM003873, SEQ ID NO: 96), and mouse (Accession No. NP_032763, SEQ ID NO: 48) NRP1 consensus sequence. The NRP1 signaling domain (amino acids 1-20 / 1-21 / 1-27), subdomain a1 (about aa22 to about aa148), subdomain a2 (about aa149 to about aa275), subdomain b1 (about aa276 to about aa428), and subdomain b2 (about aa429 to about aa589), domain c (about aa590 to about aa859), the transmembrane domain (about aa860 to about aa883), and the cytoplasmic domain (about aa884 to about aa923) have been identified. [Figure 17-2] FIG. 1 shows alignment of the NRP1 protein sequence (SEQ ID NO: 47) with the rat (Accession No. EDL96784, NP_659566, SEQ ID NO: 48), human (Accession No. NM003873, SEQ ID NO: 96), and mouse (Accession No. NP_032763, SEQ ID NO: 48) NRP1 consensus sequence. The NRP1 signaling domain (amino acids 1-20 / 1-21 / 1-27), subdomain a1 (about aa22 to about aa148), subdomain a2 (about aa149 to about aa275), subdomain b1 (about aa276 to about aa428), and subdomain b2 (about aa429 to about aa589), domain c (about aa590 to about aa859), the transmembrane domain (about aa860 to about aa883), and the cytoplasmic domain (about aa884 to about aa923) have been identified. [Figure 18-1] 18A-G show amino acid sequence alignments between exemplary traps of the present invention (see Tables 2 and 9 for the SEQ ID NOs corresponding to each trap shown). [Figure 18-2] 18A-G show amino acid sequence alignments between exemplary traps of the present invention (see Tables 2 and 9 for the SEQ ID NOs corresponding to each trap shown). [Figure 18-3]18A-G show amino acid sequence alignments between exemplary traps of the present invention (see Tables 2 and 9 for the SEQ ID NOs corresponding to each trap shown). [Figure 18-4] 18A-G show amino acid sequence alignments between exemplary traps of the present invention (see Tables 2 and 9 for the SEQ ID NOs corresponding to each trap shown). [Figure 18-5] 18A-G show amino acid sequence alignments between exemplary traps of the present invention (see Tables 2 and 9 for the SEQ ID NOs corresponding to each trap shown). [Figure 18-6] 18A-G show amino acid sequence alignments between exemplary traps of the present invention (see Tables 2 and 9 for the SEQ ID NOs corresponding to each trap shown). [Figure 18-7] 18A-G show amino acid sequence alignments between exemplary traps of the present invention (see Tables 2 and 9 for the SEQ ID NOs corresponding to each trap shown). [Figure 19] 1 shows human soluble neuropilin-1 (NRP1) protein sequences: (A) NRP1 isoform b / s12 (644 amino acids; ref. no. NP_001019799.1; NM_001024628.2; Uniprot: O-14786-2, SEQ ID NO: 44), (B) NRP1 isoform c / Siv (609 amino acids; ref. no. NP_001019800.1; NM001024629.2; Uniprot: O14786, SEQ ID NO: 45), (C) NRP1 isoform SIII (704 amino acids; Ensembl: ENSP00000363956, SEQ ID NO: 46). [Figure 20] Figure 1. Attenuation of cellular senescence by a single injection of NRP1 Trap. (A) Representative P17 OIR flat-mount retina labeled with SA-β-gal in mice intravitreally injected with Trap M and G at P12. (B) Quantification of SA-β-gal staining reveals that cellular senescence is significantly attenuated when mice receive a single injection of Trap M or Trap G. [Figure 21-1] Figure 1: NRP1-expressing macrophages accumulate in adipose tissue during diet-induced obesity. (A) NRP1 expression levels in eosinophils (adipose tissue, peripheral blood), neutrophils (blood, synovial fluid, bone marrow), monocytes (classical: MHCII+, MHCII-, MHCII-LN, bone marrow; non-classical: MHCII intermediate, MHCII high, MHCII-, bone marrow), macrophages (adipose tissue, bone marrow, red pulp, lung resident, lung CD11b+, central nervous system, steady-state peritoneal (high), steady-state peritoneal (low), small intestinal serosa, small intestinal lamina propria) (n = 1–4 per group). [Figure 21-2] Figure 2: NRP1-expressing macrophages accumulate in adipose tissue during diet-induced obesity. (B) Adipose tissue macrophage (ATM) population FACS in high-fat diet (HFD) for 10 weeks, and normal diet (RD) C57BL / 6 age-matched controls in white adipose tissue (WAT), (C) NRP1 expression levels in macrophages (ATM) (n=9 per group). (D-G) mRNA expression of NRP1 ligands: (D) Sema3a, (E) Vegfa, (F) Vegfb, and (G) Tgfb1 in RD and HFD for 10 weeks in C57BL / 6 retroperitoneal white adipose tissue (RPWAT) (n=5 per group). Data are presented as mean ± SEM. Student's unpaired t-test (B-G) *p<0.05, **p<0.01, ***p<0.001. [Figure 22]Figure 1: NRP1 promotes FA uptake and phagocytosis. (A) Acute BODIPY™ uptake in control and LysM-Cre-NRP1fl / fl macrophages (n=7-8 per group), BODIPY™ uptake in (B) retroperitoneal white adipose tissue (RPWAT), (C) liver, (D) plasma, and (E) heart of HFD-fed control and LysM-Cre-NRP1fl / fl mice (n=6 / group). ORO (Oil Red O) staining of (F) control and (G) LysM-Cre-NRP1fl / fl macrophages incubated in adipocyte-conditioned medium. Quantification of ORO staining of LysM-Cre-NRP1fl / fl macrophages incubated in (H) control and adipocyte-conditioned medium (DMEM and insulin), (I) DMEM and insulin, (J) DMEM, (K) macrophage medium (F12) (n=18–35 per group). Data are presented as mean ± SEM. Student's unpaired t-test, *p<0.05, **p<0.01, ***p<0.001. [Diagram 23] Figure 1 shows that macrophages lacking NRP1 have reduced phagocytic capacity. Phagocytosis was measured using pHrodo green zymosan bioparticles conjugate in LysM-Cre-NRP1fl / fl and control macrophages. pHrodo fluorescence was detected in control and LysM-Cre-NRP1fl / fl macrophages (n=8 per group). Data presented as mean ± SEM. Student's unpaired t-test, **p<0.01. [Figure 24]Figure 9 shows that NRP1 polypeptide trap prevents weight gain in mice fed a high-fat diet (see Example 4). The effect of NRP1 trap on weight gain was assessed. Male mice were administered adenovirus expressing soluble NRP1 trap (trapM, Figure 9A) containing domains a1, a2 and b1 of NRP1; adeno-GFP; or saline (control), while mice were switched from normal diet to high-fat diet (HFD, T0). Weight gain was monitored over a 10-week period. Data are presented as mean ± SEM. Student's unpaired t-test, *p<0.05, **p<0.01, saline vs. adeno-trapM, two-way anova, Bonferroni post-hoc test, N=5. [Diagram 25] Glucose tolerance test in mice expressing TrapM. (A) Glycemia (mM) at different time points in mice fed a HFD after intraperitoneal injection of 2 g glucose per kg mouse. 6-8 week old C57B16 / J mice were intravenously injected with saline or Adeno-TrapM (0.25x1010 PFU / injection). Mice were fed a high fat diet immediately after injection. Glycemia was assessed at different time points after intraperitoneal injection of 2 g glucose per kg mouse. (B) Area under the curve shown in (A) **P<0.01 (Adeno-GFP vs. Adeno-TrapM) in a two-way Anova Bonferroni post-hoc test. N=5. [Figure 26] Analysis of the pharmacodynamic properties of TRAP M after systemic injection. C57B16 mice (6-8 weeks old) were tail vein injected with purified TRAP M (0.5 mg / kg dose) and serum samples were collected 6 h, 24 h and 48 h after injection. TRAP M captured from approximately 75 μl of serum using IMAC Sepharose was detected by immunoblotting with anti-human NRP1 (cubAI domain). [Figure 27]Cos cell expression of traps G, A, D and M after transduction with adenovirus origin. Cos cells were transduced with the indicated adenovirus origin. Traps G and M were purified from the supernatant of transduced cells using IMAC sepharose and traps A and D were concentrated using protein A / G sepharose. Traps were detected by immunoblotting with anti-human NRP1 (cubAI domain). Legend: NI) Supernatant from uninfected Cos cells, GFP supernatant from adeno-green fluorescent protein (GFP) infected Cos cells, S1) Lipofectamine™ 2000 transfected adenovirus origin, S2) Effectene transfected adenovirus origin, S3) PEI transfected adenovirus origin, A2) adenovirus origin amplification round 2, A3) adenovirus origin amplification round 3. Specifically, purification was performed using IMAC Sepharose (IMAC) or Protein A / G Sepharose (A / G). [Figure 28] Analysis of the pharmacological distribution of TrapM after systemic infection with adenovirus-TrapM constructs. C57B16 mice (6-8 weeks old) were tail vein injected with adenovirus-TrapM or control adenovirus-GFP progenitor. Mice were sacrificed 2 weeks after infection and serum, kidney and liver tissues were collected and stored at -80 °C until analysis. TrapM was captured from serum (~75 μl) or tissue lysates (~40 mg) from kidney or liver dissolved in PBS / 2% Triton x-100 using IMAC Sepharose and detected by immunoblotting with anti-human NRP1 (cubAI domain). Legend; 1) uninfected mice, 2) adeno-GFP infected mice, and 3) adeno-TrapM infected mice. [Figure 29] Expression of TrapM 2 weeks after adenovirus infection. TrapM was captured from blood (~25 μl) in PBS 2% Triton X-100 using IMAC Sepharose and detected by immunoblotting with anti-human NRP1 (cubAI domain). Legend: NI: non-infected mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0095] 1. The role of cellular senescence and SASP in ocular vascular diseases The data presented herein establishes a novel role for cellular senescence in weather-resistant ischemia and the regulation of angiogenesis in ocular vascular disease. In different retinopathy models, a transient accumulation of senescent cells was indeed established in different subcellular populations of the retina. More specifically, it was found that by adopting the SASP, retinal neurons stimulate the production of a series of paracrine factors and inflammatory cues that spread senescence to retinal microglia and endothelial cells and further aggravate pathological preretinal angiogenesis. Applicants have shown that regulating cellular senescence by inhibiting the SASP (e.g., by administering biguanide compounds (e.g., metformin) or by pharmacological or genetic inhibition of IRE1α) inhibits ischemia-induced senescence, increases vascular regeneration, and suppresses pathological neovascularization in vascular ocular disease. Thus, the SASP was shown to be involved in mediating pathological vascular growth by secreting proinflammatory cytokines that induce ischemic cells to enter a premature senescent state, drive paracrine senescence, exacerbate destructive angiogenesis, and impede restorative revascularization.

[0096] The data presented herein support that in the context of ocular vascular diseases such as retinopathy, cellular senescence plays a dichotomous role within the same disease, first protecting neurons from cell death and simultaneously preventing them from inducing a repair angiogenesis program.In addition, the paracrine senescence observed in retinopathy and its associated production of vascular regulators may prevent neovascularization into the physiologically avascular vitreous and promote early age-related complications in the retinal vasculature.This is particularly relevant in light of the increasing incidence of age-related neovascular eye diseases such as age-related macular degeneration and diabetic retinopathy.Therefore, preventing cellular senescence during the pathological angiogenesis stage by administering senescence modulators may therefore represent a simple therapeutic solution for disorders such as ocular vascular diseases and retinal vascular disorders.

[0097] 2. Method for treating or preventing vascular eye diseases associated with cellular senescence Thus, according to one aspect of the present invention, compositions and methods are provided for treating and / or preventing at least one symptom or sign of vascular eye disease or disorder in a subject. The method according to this aspect of the present invention comprises administering an inhibitor of SASP (e.g., a biguanide compound such as metformin) to a subject. In certain aspects, the inhibitor of SASP is administered locally (e.g., topically or intravitreally as opposed to systemically) to the subject's eye. In the case of biguanide compounds such as metformin, intraocular administration is particularly preferred, since systemic administration generally does not allow the compound to reach its target site due to the presence of the blood-retina barrier. In an embodiment, the vascular eye disease or disorder is secondary to cellular ischemia.

[0098] Vascular eye diseases or conditions that may benefit from the inhibition of SASP according to the present invention include any disease, disorder or condition characterized by abnormal angiogenesis (e.g., pathological angiogenesis and / or reduced revascularization). These diseases may be caused by a decrease (transient or sustained / chronic) in metabolic supply (e.g., oxygen, blood, nutrients) to cells that contribute to normal eye function (e.g., ocular vascular cells, retinal cells, neurons, microglia), which leads to the presence of senescent cells (or cells with a senescent phenotype). Such conditions may exist after an ischemic event, but are not limited to this. Thus, the term "vascular eye disease or disorder" or "vascular eye disease or condition" as used herein refers to a disease, disorder or condition that affects the normal physiological function of blood vessels in the eye. Non-limiting examples of such eye diseases or conditions include diabetic retinopathy, retinopathy of prematurity, ischemic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, choroidal neovascularization, polypoidal choroidal vasculopathy, physical damage to the eye, glaucoma, rhegmatogenous retinal detachment (RRD), retinal vasculitis, retinal macroaneurysm, retinal microaneurysm, Fuchs' dystrophy, ischemic optic neuropathy, juvenile macular degeneration, macular telangiectasia, optic neuritis, Usher syndrome, retinitis pigmentosa, uveitis, Stargardt's disease, and Leber's congenital amaurosis (LCA). In an embodiment, the vascular eye disease or disorder is ischemic retinopathy. In an embodiment, the ischemic retinopathy is associated with diabetic retinopathy, retinopathy or prematurity, ocular vein occlusion, central retinal vein occlusion, or branch retinal vein occlusion.

[0099] Compounds or agents that inhibit the SASP according to the present invention include biguanide compounds (e.g., metformin), mTor inhibitors (e.g., rapalogs, Torin 1), and / or inhibitors of IRE1α expression (e.g., antisense, shRNA, etc.), inhibitors of IRE1α activation (S724 phosphorylation), and / or inhibitors of IRE1α RNAse activity (e.g., pharmacological inhibitors / antagonists). In general, an "IRE1α inhibitor" that inhibits the SASP according to the present invention is one that ultimately reduces or abolishes IRE1α RNAse activity.

[0100] In certain embodiments, in the context of vascular eye diseases and disorders (e.g., involving proliferative retinopathy), compounds and agents that inhibit the SASP and prevent and / or attenuate cellular senescence increase physiological angiogenesis (i.e., beneficial angiogenesis) and decrease pathological angiogenesis (pathological neovascularization), thus promoting tissue repair.

[0101] IRE1α is an enzyme encoded by the ERN1 gene in humans (Entrez:2081, Ensembl ENSG00000178607, Uniprot:O75460, Refseq mRNA:NM_152461, NM_001433, Refseq(protein):NP_001424.3). The protein has intrinsic kinase and endoribonuclease activities and is important in altering gene expression in response to endoplasmic reticulum-based stress signals (mainly the unfolded protein response (UPR)). Two alternatively spliced ​​transcript variants were found for this gene that encode distinct isoforms. IRE1α has two functional enzymatic domains, an endonuclease, and a trans-autophosphorylation kinase domain. When activated, IRE1α oligomerizes and performs non-conventional RNA splicing activity to remove introns from X-box binding protein 1 (XBP1) mRNA, which is translated into functional transcription factor XBP1. XBP1 upregulates ER chaperone and endoplasmic reticulum-associated degradation (ERAD) genes that promote recovery from ER stress. Compounds that inhibit IRE1α (i.e., inhibitors) are also known in the art.

[0102] The term "IRE1α inhibitor" or "IRE1α antagonist" as used herein refers to an agent that can reduce or block IRE1α-mediated cell signaling associated with cellular senescence and induction of SASP (i.e., IRE1α ribonuclease activity and XBP1 processing). Non-limiting examples include agents that reduce or block the expression (transcription or translation) of IRE1α, agents that can reduce or block IRE1α activation (e.g., phosphorylation of S724 and / or dimerization of IRE1α). Without being limited in this way, the agent may be natural or synthetic, a small molecule or a protein / polypeptide / nucleic acid, such as, but not limited to, an antisense or shRNA specific to the IRE1α nucleic acid sequence encoding the IRE1α protein or any pharmacological inhibitor described herein. The IRE1α inhibitor or IRE1α antagonist of the present invention binds to the IRE1α nucleic acid or IRE1α protein, reducing the expression, activation or activity of IRE1α, ultimately resulting in a reduction in IRE1α RNAse activity in the cell.

[0103] Inhibitors that target the catalytic core of the RNase domain and / or the ATP-binding pocket of the kinase domain have been described. Non-limiting examples of inhibitors that target the RNAse-binding pocket include salicylaldehyde (e.g., 3-methoxy-6-bromosalicylaldehyde-Volkman et al., 2011, JBC 286(14):12743-12755, PMCID:PMC3069474), 4μ8C, MKC-3946, STF-083010, and toyocamycin. Compounds that inhibit the RNase activity of IRE1α through the kinase domain have also been identified, which are named "kinase-inhibiting RNase attenuators" (KIRAs), including KIRA3 and KIRA6 (Cas#1589527-65-0), and inhibit both the kinase and RNAse activities of IRE1α. Sunitinib and APY29 are examples of compounds that inhibit the ATP-binding pocket but allosterically activate the IRE1α RNase domain (Wang et al., 2012, Nat. Chem. Bio. 8(12):982-989). Additional kinase and / or RNAse inhibitors and activators of IRE1α are described in Wang (supra). In certain embodiments, IRE1α inhibitors used in accordance with the present invention inhibit the RNAse activity of IRE1α but do not inhibit its kinase activity.

[0104] Biguanides have the formula HN(C(NH)NH 2 ) 2These compounds were originally discovered in extracts of the French lilac (Galega officinalis) and shown to lower blood glucose levels. Thus, they were originally used to treat type 2 diabetes. Derivatives of various biguanides are used as pharmaceutical agents for the treatment of diabetes, but also for other diseases and conditions, such as polycystic ovarian syndrome and cancer. Non-limiting examples include metformin (N,N-dimethylimidodicarboxylic acid diamide (IUPAC name), CAS 657-24-9; DrugBank DB00331; ChemSpider 3949; Glucophage XR™; Carbophage SR(TM); Riomet(TM); Fortamet(TM); Glumetza(TM); Obimet(TM); gluformin(TM); Dianben(TM); Diabex(TM); Diaformin(TM); Siofor(TM); and Metfogamma(TM), buformin (1-butylbiguanide, CAS#692-13-7), phenformin (2-(N-phenethylcarbamimidoyl)guanide, Examples of biguanides include biguanide, CAS#114-86-3), proguanil, (1-[amino-(4-chloroanilino)methylidene]-2-propan-2-ylguanidine, also known as chlorproguanil), scintalin A, (1,1'-decane-1,10-diyldiguanidine, Cas#111-23-9) and scintalin B, (1,1'-dodecamethylenediguanidinium dichloride, Cas#61167-43-9). Figure 15 shows the structures of biguanides and some functional derivatives that may be used in accordance with the present invention.

[0105] The SASP inhibitors of the present invention may be administered in combination with other drugs used to treat vascular eye diseases and disorders, such as angiopoietin-2 inhibitors (e.g., those described in WO 2016 / 085750), VEGF antagonists (e.g., anti-VEGF antibodies (e.g., ranibizumab / LUCENTIS™)), small molecule VEGF inhibitors (e.g., snetinib), VEGF-inhibitory fusion proteins (e.g., aflibercept / EYELEA™), and / or SEMA3A antagonists (e.g., SEMA3a antibodies or NRP1 traps, such as those described below (see Table 2 and Figure 18), or e.g., as described in WO 2016 / 033699).

[0106] 3. Reducing or preventing cellular senescence and SASP by inhibiting IRE1α The data presented herein further establish a role for IRE1α in regulating cellular senescence and the SASP. Cellular senescence (such as autocrine and / or paracrine), paracrine senescence can be inhibited or prevented by reducing IRE1α activity (i.e., IRE1α activation and cell signaling).

[0107] IRE1α activity can be inhibited by several approaches. Inhibition of IRE1α cellular activity can be performed directly by reducing IRE1α (i) nucleic acid or protein expression, (ii) activation (serine 724 phosphorylation), and / or (iii) RNAse activity (and optionally its kinase activity) in cells. As mentioned above, IRE1α inhibitors are known in the art, and include agents that inhibit IRE1α expression (e.g., sh_RNA IRE1α antisense), IRE1α activation (e.g., KIRA3, KIRA6), and / or IRE1α ribonuclease (and optionally kinase) activity (e.g., salicylaldehyde, 4μ8C, MKC-3946, STF-083010, KIRA3, KIRA6, and toyocamycin).

[0108] Thus, the present invention provides a method of inhibiting or preventing cellular senescence in a cell or the induction of a senescence-associated secretory phenotype (SASP) in a cell, comprising reducing the level or activity of IRE1α.

[0109] The present invention also relates to a method of inhibiting or preventing cellular senescence in a cell or the induction of a senescence-associated secretory phenotype (SASP) in a cell, comprising contacting the cell with an inhibitor of IRE1α.

[0110] Also provided is a method of inhibiting or preventing cellular senescence of a cell, or the induction of a senescence-associated secretory phenotype in a cell of a subject, comprising administering to the subject an inhibitor of IRE1α.

[0111] The above methods can be used to treat diseases or conditions in which cellular senescence is detrimental, such as various age-related aging conditions (e.g., sarcopenia, neurodegeneration, thinning of the epidermis, skin wrinkling, hair loss and graying, cataracts, obesity, metabolic syndrome, and other diseases of old age), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), atherosclerosis, osteoarthritis, osteoporosis, glaucoma, Parkinson's disease, intestinal diseases, intervertebral disc degeneration, cerebral aneurysms, aortic aneurysms, pancreatic fibrosis, vascular eye diseases (e.g., retinal vascular diseases (proliferative retinopathy, diabetic retinopathy, The aging-related diseases or conditions encompassed by the present invention are intended to exclude one or more vascular eye diseases, such as retinal vascular diseases (proliferative retinopathy, diabetic retinopathy, ischemic retinopathy, macular degeneration, glaucoma) and cystic fibrosis. Inhibition or prevention of cellular senescence may also be useful during and / or after cancer treatment, for example, to reduce the side effects of chemotherapy / radiotherapy, such as metabolic dysfunction, accelerated aging, and increased risk of cancer later in life. In an embodiment, the aging-related diseases or conditions encompassed by the present invention are intended to exclude one or more vascular eye diseases, such as retinal vascular diseases (proliferative retinopathy, diabetic retinopathy, ischemic retinopathy, macular degeneration, glaucoma).

[0112] Various approaches are available to reduce the expression of IRE1α and thus IRE1α-mediated cell senescence.Non-limiting examples include the use of small hairpin shRNA (RNAi), antisense, ribozyme, TAL effector targeting IRE1α promoter, etc.The expression of shRNA or similar inhibitory RNA in cells can be obtained by delivery of plasmid, or delivery via virus (e.g., lentivirus vector, adenovirus vector, etc.) or bacterial vector.

[0113] Therefore, in an alternative embodiment, the present invention provides antisense, shRNA molecules and ribozymes for exogenous administration to cause degradation and / or translation inhibition of target mRNA.The present invention also provides vectors and host cells for delivering and / or expressing the antisense, shRNA molecules, ribozymes, etc. disclosed herein.The antisense, shRNA molecules and ribozymes preferably target IRE1α in mammals, preferably humans. Examples of therapeutic antisense oligonucleotide applications include U.S. Pat. No. 5,135,917, issued Aug. 4, 1992; U.S. Pat. No. 5,098,890, issued Mar. 24, 1992; U.S. Pat. No. 5,087,617, issued Feb. 11, 1992; U.S. Pat. No. 5,166,195, issued Nov. 24, 1992; U.S. Pat. No. 5,004,810, issued Apr. 2, 1991; U.S. Pat. No. 5,194,428, issued Mar. 16, 1993; U.S. Pat. No. 4,806,463, issued Feb. 21, 1989; U.S. Pat. No. 5,286,717, U.S. Pat. No. 5,276,019, and U.S. Pat. No. 5,264,423, issued Feb. 15, 1994; BioWorld Today, April 29, 1994, p.3.

[0114] Preferably, antisense molecules have sufficient complementarity with the target mRNA to avoid non-specific binding of antisense molecules to non-target sequences under the conditions where specific binding is desired, for example, in this case, under physiological conditions, in the case of in vivo assay or therapeutic treatment, or in the case of in vitro assay, under the conditions where assay is performed.Target mRNA for antisense binding can include not only the information for coding protein, but also the relevant ribonucleotides that form, for example, 5' untranslated region, 3' untranslated region, 5' cap region, and intron / exon junction ribonucleotides.The screening method of antisense and ribozyme nucleic acid that can be used to provide such molecules as IRE1α inhibitor of the present invention is disclosed in US Patent No. 5,932,435.

[0115] In some embodiments, antisense oligonucleotides according to the invention may contain from about 5 to about 100 nucleotide units. As will be understood, a nucleotide unit is a base-sugar combination (or a combination of similar structure) suitably linked to adjacent nucleotide units via a phosphodiester or other bond that forms a backbone structure.

[0116] In further embodiments, the expression of the nucleic acid (IRE1α) encoding the polypeptide of interest, or a fragment thereof, can be inhibited or prevented using RNA interference (RNAi) technology, which is a type of post-transcriptional gene silencing. RNAi can be used to create a pseudo "knockout", i.e., a system in which the expression of the product encoded by the gene or coding region of interest is reduced, resulting in a global reduction in the activity of the encoded product in the system. Thus, RNAi can be performed to target the nucleic acid of interest, or a fragment or variant thereof, and reduce the expression and activity level of the product it encodes. Such a system can be used not only to treat disorders related to the activity of such products, but also for functional studies of the products. RNAi is described, for example, in published U.S. Patent Application Nos. 20020173478 (Gewirtz; published November 21, 2002) and 20020132788 (Lewis et al.; published November 7, 2002). Reagents and kits for performing RNAi are commercially available, for example, from Ambion Inc. (Austin, TX, USA) and New England Biolabs Inc. (Beverly, MA, USA).

[0117] The initial agent for RNAi in some systems is a dsRNA molecule corresponding to the target nucleic acid. The dsRNA (e.g., shRNA) is then thought to be cleaved into short interfering RNAs (siRNAs) that are 21-23 nucleotides long (19-21 bp duplexes, each with a 2-nucleotide 3' overhang). The enzyme thought to affect this first cleavage step is called "Dicer" and is classified as a member of the RNase III family of dsRNA-specific ribonucleases. Alternatively, RNAi can be brought about by direct introduction into the cell or by generation within the cell of suitable precursors of such siRNAs or siRNA-like molecules (e.g., a vector (viral vector, such as an adenoviral vector) encoding the precursor(s)). The siRNAs can then associate with other intracellular components to form an RNA-induced silencing complex (RISC). The RISC thus formed then targets the transcript of interest by homology through base pairing interactions between its siRNA component and the target transcript, resulting in cleavage of the target transcript approximately 12 nucleotides from the 3' end of the siRNA. Thus, the target mRNA is cleaved and the levels of the encoded protein product are reduced.

[0118] RNAi can be effected by introducing suitable in vitro synthesized siRNA (shRNA) or siRNA-like molecules into cells. RNAi can be performed, for example, using chemically synthesized RNA. Alternatively, such RNA may be transcribed either in vitro or in vivo using a suitable expression vector. In vitro transcription of the sense and antisense strands (encoded by sequences present on the same vector or on separate vectors) can be effected, for example, using T7 RNA polymerase. In this case, the vector may contain a suitable coding sequence operably linked to a T7 promoter. In embodiments, the in vitro-transcribed RNA may be processed in vitro (e.g., using E. coli RNaseIII) to a size conducive to RNAi. The sense and antisense transcripts combine to form an RNA duplex that is introduced into the target cell of interest. Other vectors may be used that express small hairpin RNA (shRNA) that can be processed into siRNA-like molecules. Methods using a variety of vectors, either in vitro or in vivo, and methods for introducing such vectors into cells (e.g., gene therapy) are known in the art.

[0119] Thus, in one embodiment, expression of a nucleic acid encoding a polypeptide of interest (IRE1α), or a fragment thereof, can be inhibited by introducing into a cell or producing in the cell an siRNA or siRNA-like molecule corresponding to the nucleic acid encoding the polypeptide of interest (e.g., IRE1α), or a fragment thereof, or a nucleic acid homologous thereto. An "siRNA-like molecule" refers to a nucleic acid molecule that is similar to an siRNA (e.g., in terms of size and structure) and can elicit siRNA activity, i.e., can result in RNAi-mediated inhibition of expression. In various embodiments, such methods may involve the direct administration of an siRNA or siRNA-like molecule into a cell or the use of vector-based methods described above. In one embodiment, the siRNA or siRNA-like molecule is less than about 30 nucleotides in length. In a further embodiment, the siRNA or siRNA-like molecule is about 21-23 nucleotides in length. In one embodiment, the siRNA or siRNA-like molecule comprises a 19-21 bp duplex portion, with each strand having a 2-nucleotide 3' overhang. In an embodiment, the siRNA or siRNA-like molecule is substantially identical to a nucleic acid encoding a polypeptide of interest, or a fragment or variant thereof (or a fragment of a variant). Such variants can encode proteins having similar activity to the polypeptide of interest.

[0120] 4. Method for promoting cellular senescence by increasing IRE1α ribonuclease activity Under certain conditions, stimulating cellular senescence may be beneficial. Cellular senescence, such as autocrine and paracrine senescence, can be promoted or induced by stimulating or increasing IRE1α activity (i.e., IRE1α RNAse activity and cell signaling). IRE1α activity can be increased by several approaches, such as increasing the expression of IRE1α in cells or contacting cells with compounds that activate IRE1α RNAse activity (e.g., APY29, sunitinib, or compound 3 (described in Joshi et al., 2015, 6(15):1309-1335)).

[0121] Methods of promoting cellular senescence may be useful in diseases and conditions in which senescence has beneficial effects, such as tissue repair, wound healing, liver fibrosis, renal fibrosis, myocardial infarction, cardiac fibrosis, atherosclerosis, pulmonary hypertension and cancer.

[0122] 5. Compositions and methods for modulating cellular senescence comprising SEMA3A modulators. Class 3 semaphorins (Sema3) are a subfamily of proteins whose known biological roles are diverse and growing. The mechanism of action of Sema3 requires binding to transmembrane receptors that include heteromeric complexes of neuropilins, plexins, and cell adhesion molecules (CAMs). The SEMA3A gene (GeneCard ID: GC07M083955; Entrez Gene ID: 10371; Ensembl: ENSG00000075213) encodes a 771 amino acid protein (NP_006071.1; UniprotKB: Q14563, SEQ ID NO: 50) that contains a signal peptide, an Ig-like C2-type (immunoglobulin-like) domain, a PSI domain, and a Sema domain, which are required for signal transduction. This secreted protein was first described as an axon guidance cue, but is now involved in a variety of physiological and pathological processes, including organ development, bone metabolism, angiogenesis, vascular permeability, growth cone collapse, myogenic regeneration, and neuromuscular junction formation, as well as in the immune system, inflammation, schizophrenia, and retinal diseases such as diabetic retinopathy.

[0123] Sema3a generally signals through a receptor complex that includes neuropilin-1 (NRP1) and co-receptors (e.g., class A plexins (e.g., PLXna1-Plxna4, Plxnd1), L1cam, chL1, Robo1). NRP1 (Ensemble; ENSG00000099250; ENST00000265371; Uniprot:O14786; OMIM:602069; HGNC:8004; GeneCard ID:GC10M033216, SEQ ID NOs:44-47, 95 and 96) is a single-pass transmembrane receptor with a large intracellular domain. The basic structure of neuropilin-1 contains five domains: three extracellular domains (a1a2 (CUB), b1b2 (FV / FVIII), and c (MAM)), a transmembrane domain, and a cytoplasmic domain. The a1a2 domain is homologous to complement components C1r and C1s (CUB), which generally contain four cysteine ​​residues that form disulfide bridges. This domain binds to SEMA3A. Domain b1b2 (FV / FVIII) binds to VEGF. Amino acid Y297 in subdomain b1 is important for binding to VEGF, as substituting Y297 with alanine significantly reduces the binding of VEGF to NRP1. Subdomain b1 also contributes to ligand binding of SEMA3A. Indeed, the applicants surprisingly found that substituting Y297 (Y297A) also significantly reduces the binding of SEMA3A to NRP1. Crystallographic evidence revealed that VEGF165 and Sema3A can simultaneously bind to NRP1 at separate, non-overlapping sites, rather than competing directly for NRP1.

[0124] In addition to the transmembrane form (isoform 1, 923 aa, FIG. 17, SEQ ID NOs: 47, 95, and 96, NM003873, Uniprot: O14786-1), a naturally occurring soluble NRP1 protein (sNRP1) containing only a portion of the extracellular domain can be secreted by cells. 551 to 704 amino acids (isoforms b / s12 NRP1 (644 amino acids; RefSeq: NP_001019799.1; NM_001024628.2, SEQ ID NO: 44), s11 NRP1 (704 amino acids; ENSP00000363956, SEQ ID NO: 46), sIII NRP1 (551 aa) and c / sIV NRP1 (609 amino acids; RefSeq: NP_001019800.1; NM001024629.2, SEQ ID NO: 45) (Cackowski et al., 2004, Genomics, 84(1):82-94; Rossignol M et al., Genomics 2000;70(2):211-222; and Gagnon Different soluble forms ranging from 1 to 100 have been described (ML et al., 2000, Proc. Natl. Acad. Sci. USA; 97(6):2573-2578). The full-length form of the protein contains all 17 exons, whereas the soluble isoforms are generated by alternative splicing or intron read-through of the NRP1 gene. The b / s12 and c / sIV NRP1 isoforms contain the a1a2 and b1b2 domains and most of the b / c linker, but not the c domain. Isoform sIII contains the a1a2 domain, the b1 subdomain, and only a portion of the b2 subdomain. The s11 NRP1 isoform contains the a1a2 and b1b2 domains followed by a portion of the b / c linker encoded by exon 11 and 83 novel amino acids.

[0125] In a second aspect of the present invention, in the following study in a model of ischemic retinopathy, SEMA3A was surprisingly identified as a modulator of cellular senescence. Indeed, an unexpected mechanism was identified triggered by neurons in the avascularized retinal zone, where they enter an early cellular senescence state and adopt a senescence-associated secretory phenotype (SASP). The data described herein show that secretion of SEMA3A by senescent cells drives paracrine senescence via IRE1a, propagating senescence to various cell types, such as neurons, microglia, and the overlying vasculature, in ischemic tissues (paracrine senescence). Furthermore, sustained exposure to SEMA3A was shown to activate IRE1α, inducing senescence, and driving the expression of a group of genes known to be important for promoting and enhancing the senescent state, such as Pai1, I16, Il1β, TGF-β, and Tp53. SEMA3A was also shown to promote senescence-associated DNA damage foci expressing γH2AX, a hallmark of cellular senescence. In particular, and as shown herein, genetic interference with SEMA3A limits aging and stimulates tissue repair.

[0126] The present inventors have found that regulating the level or activity of SEMA3A can control cell senescence and the secretion of proteins released during cell senescence (typically the proinflammatory cytokines of SASP).The present inventors have found that inhibiting the expression or activity of SEMA3A can prevent, limit or reduce cell senescence, and can prevent or reduce the induction of SASP.Similarly, increasing SEMA3A activity (e.g., by increasing its expression or by contacting cells with SEMA3A polypeptide) promotes senescence and induces SASP.

[0127] These data provide evidence for a previously unanticipated role for SEMA3A in regulating SASP-mediated autocrine and paracrine senescence in pathological processes and highlight the therapeutic benefit of modulating SEMA3A activity in aging-associated diseases and conditions.

[0128] (i) A method for inhibiting or preventing cellular senescence by inhibiting SEMA3A signaling Cellular senescence, such as autocrine and paracrine senescence, can be inhibited or prevented by reducing SEMA3A activity (i.e., SEMA3A cell signaling). SEMA3A activity can be inhibited by several approaches. Inhibition of SEMA3A cell activity can be performed directly by (i) reducing the expression of SEMA3A nucleic acid or protein, (ii) inhibiting its secretion by cells, or (III) sequestering secreted SEMA3A to inhibit binding to its receptor on the cell surface, thereby preventing intracellular signaling, activation of IRE1α, and the initiation and / or propagation of cellular senescence. Non-limiting examples of agents and approaches for inhibiting the activity of SEMA3A include: (i) antibodies against SEMA3A, (ii) antibodies against one of its receptors (i.e., those that compete with SEMA3A binding to its receptor), (iii) antisense and RNAi methods to reduce the expression of SEMA3A, and / or (iv) use of soluble receptors or fragments thereof that act as functional SEMA3A traps.

[0129] Thus, the present invention provides a method for inhibiting or preventing cellular senescence in a cell or the induction of a senescence-associated secretory phenotype (SASP) in a cell, comprising reducing the level or activity of SEMA3A.

[0130] The invention also relates to a method of inhibiting or preventing cellular senescence in a cell or the induction of a senescence-associated secretory phenotype (SASP) in a cell, comprising contacting the cell with a SEMA3A antagonist.

[0131] Also provided is a method for inhibiting or preventing cellular senescence or the induction of a senescence-associated secretory phenotype in a cell of a subject, comprising administering to the subject an effective amount of a SEMA3A antagonist.

[0132] The term "SEMA3A inhibitor" or "SEMA3A antagonist" as used herein refers to an agent that can reduce or block SEMA3A-mediated cell signaling associated with SEMA3A induction of SASP and SEMA3A-induced cellular senescence. The "SEMA3A inhibitor" or "SEMA3A antagonist" of the present invention binds to or interacts with SEMA3A polypeptide or SEMA3A nucleic acid (SEMA3A gene or mRNA) to reduce the expression of SEMA3A polypeptide or its interaction with its cognate receptor, so that SEMA3A-mediated cell signaling is reduced or abolished. Non-limiting examples include agents that reduce or block the expression (transcription or translation) of SEMA3A, agents that can reduce or block SEMA3A secretion, or agents that can reduce or block the binding of SEMA3A to its receptor NRP1. Without being limited thereto, the agent may be natural or synthetic and may be, without being limited thereto, a protein / polypeptide such as an antibody that specifically binds to SEMA3A or the NRP1 receptor, a soluble NRP1 polypeptide or a fragment thereof (e.g., an NRP1 trap that binds to SEMA3A), a peptide, a small molecule, a polynucleotide such as, without being limited thereto, an antisense or shRNA specific to the SEMA3A nucleic acid sequence encoding the SEMA3A protein, or a functional variant or fragment thereof.

[0133] The above method may be useful for treating or preventing diseases or conditions in which cellular senescence is detrimental, such as various aging conditions (e.g., sarcopenia, neurodegeneration, thinning of the epidermis, skin wrinkles, hair loss and graying, cataracts, and other senile diseases), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), atherosclerosis, osteoarthritis, osteoporosis and Parkinson's disease, glaucoma, intestinal diseases, intervertebral disc degeneration, cerebral aneurysm, aortic aneurysm, pancreatic fibrosis, cystic fibrosis. Inhibition or prevention of cellular senescence may also be useful during and / or after cancer treatment, for example, to reduce the side effects of chemotherapy / radiotherapy, such as metabolic dysfunction, accelerated aging, and increased risk of cancer later in life. In an embodiment, the aging-related diseases or conditions encompassed by the present invention do not include ocular diseases (e.g., retinal vascular diseases (ischemic retinopathy, macular edema)), inflammation, cerebral ischemia, stroke, or cancer.

[0134] a.Antibodies In certain embodiments of the invention, SEMA3A activity (e.g., SEMA3A-induced IRE1α activation) can be inhibited by using SEMA3A antibodies. These antibodies bind to SEMA3A in a way that inhibits its binding to its cognate receptor, NRP1, thereby preventing SEMA3A-mediated cell signaling (79, 80).

[0135] Alternatively, an antibody that directly targets the NRP1 receptor may be used, blocking the binding of SEMA3A to NRP1. In certain aspects of the present invention, the antibody that targets NRP1 blocks the binding of SEMA3A to its receptor, but does not substantially interfere with the binding of VEGF to NRP1. In one embodiment, the NRP1 antibody binds to the a1a2(A) domain of the NRP1 polypeptide.

[0136] The term "SEMA3A antibody" as used herein refers to an antibody that specifically binds (interacts) with SEMA3A protein and does not show substantial binding to other natural proteins other than those that share the same antigenic determinant with SEMA3A protein. Similarly, the term "NRP1 antibody" refers to an antibody that specifically binds (interacts) with NRP1 protein and does not show substantial binding to other natural proteins other than those that share the same antigenic determinant with NRP1 protein. SEMA3A / NRP1 antibodies include polyclonal antibodies, monoclonal antibodies, humanized antibodies, and chimeric antibodies. The term antibody or immunoglobulin is used in the broadest sense and covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, and antibody fragments, so long as they exhibit the desired biological activity. An antibody fragment includes a portion of a full-length antibody, generally its antigen-binding region or its variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single chain antibody molecules, single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments.Antibody fragments can also refer to binding moieties that include CDRs or antigen-binding domains, such as, but not limited to, VH regions (VH, VH-VH), anticalins, PepBody™, antibody-T cell epitope fusions (Troybodies), or Peptibodies.

[0137] Anti-human SEMA3A / NRP1 antibodies have been previously prepared (80) and are commercially available from various sources, such as Santa Cruz. In general, techniques for preparing antibodies (such as monoclonal antibodies and hybridomas) and for detecting antigens using antibodies are well known in the art, and a variety of protocols are well known and readily available.

[0138] B. A soluble receptor or a fragment thereof Modulation of Sema3-mediated cellular senescence can be achieved by using naturally occurring soluble NRP1 polypeptides or synthetic NRP1 polypeptides (e.g., produced (recombinantly) in cell lines in vitro or chemically synthesized). As used herein, the term "NRP1 trap" or "NRP1 polypeptide trap" encompasses naturally occurring soluble NRP1 polypeptides (e.g., the NRP1 secreted isoforms shown in Figures 17, 19, Table 2, and SEQ ID NOs: 44-47, 95 and 96), non-naturally occurring soluble NRP1 polypeptides, such as any functional soluble fragment of NRP1 or any functional variant of NRP1 that competes with endogenous (cellular, membrane-bound) NRP1 for SEMA3A ligand binding, and synthetic soluble NRP1 polypeptides. In one embodiment, the NRP1 traps of the invention are not naturally occurring (i.e., not naturally occurring), but are "derived" from a naturally occurring NRP1 polypeptide (i.e., they are synthetic, e.g., an NRP1 trap comprising the extracellular domain of NRP1 isoform 1 (e.g., amino acids 1-857 of transmembrane (cellular) NRP1) or a fragment or variant thereof). Generally, the NRP1 traps of the invention initially comprise a signal peptide at their N-terminus (e.g., about amino acids 1-21 of the NRP1 amino acid sequence shown in FIG. 17), which is cleaved upon maturation and secretion by the cell. Thus, the NRP1 polypeptide traps of the invention lack amino acids 1-21 when administered as a purified polypeptide or when prepared as a pharmaceutical composition comprising a purified or substantially pure form. Similarly, the nucleic acid encoding the NRP1 trap of the invention comprises a polynucleotide sequence within 5' that encodes a signal peptide (e.g., the first 63 nucleotides encoding the first 21 amino acids at the N-terminus) that causes the NRP1 trap to be synthesized and secreted by the cell. Depending on the conditions and cell type, the length of the signal peptide that is removed for secretion may vary. Deletion of amino acids 1-20, 1-21 and 1-27 (see Figures 17-18, e.g., SEQ ID NOs: 47, 95 and 96) are described.In certain embodiments, the signal peptide corresponds to the first 21 amino acids of the NRP1 polypeptide set forth in FIG. [Table 1]

[0139] Non-limiting examples of NRP1 traps that may be used in accordance with the present invention include the native soluble NRP1s set forth in SEQ ID NOs: 44-47, 95 and 96, the NRP1 traps described in Table 2 below, FIG. 18 and WO 2016 / 03699 (hereby incorporated by reference). Of course, the mature, active soluble NRP1 trap administered to a subject or contacted with a cell lacks the signal peptide and portions of the amino acid sequence corresponding to the transmembrane and cytoplasmic domains normally present in the cell membrane-bound NRP1. [Table 2] TIFF2025061122000004.tif235160TIFF2025061122000005.tif237160

[0140] In one embodiment, the NRP1 trap of the invention comprises: (i) amino acids 1 to 856 of a human NRP1 polypeptide (preferably in its mature form, from the amino acid following the signal peptide (e.g., amino acid 21, 22 or 28) to amino acid 856), (ii) amino acids 1 to 583 of a human NRP1 polypeptide (preferably in its mature form, from the amino acid following the signal peptide (e.g., amino acid 21, 22 or 28) to amino acid 583), (iii) amino acids 1 to 424 of a human NRP1 polypeptide (preferably in its mature form, from the amino acid following the signal peptide (e.g., amino acid 21, 22 or 28) to amino acid 424), (iv) amino acids 1 to 265 of a human NRP1 polypeptide (preferably in its mature form, from the amino acid following the signal peptide (e.g., amino acid 21, 22 or 28) to amino acid 265), (v) amino acids 1 to 430 and 584 to 856 of the human NRP1 polypeptide (preferably in its mature form, from the amino acid following the signal peptide (e.g., amino acid 21, 22 or 28) to amino acid 430, and from amino acid 584 to amino acid 856), (vi) amino acids 1 to 274 and 584 to 856 of the human NRP1 polypeptide (preferably in its mature form, from the amino acid following the signal peptide (e.g., amino acid 21, 22 or 28) to amino acid 274, and from amino acid 584 to amino acid 856), (vii) amino acids 1 to 430 and 584 to 856 of the human NRP1 polypeptide (preferably in its mature form, from the amino acid following the signal peptide (e.g., amino acid 21, 22 or 28) to amino acid 430, and from amino acid 584 to amino acid 856). In embodiments, the NRP1 polypeptide comprises or consists of the amino acid sequence set forth in Figure 17, SEQ ID NO: 44, 45, 46, 47, 95, or 96, or an allelic or functional variant thereof.

[0141] Given that NRP1 specifically regulates the effect of its ligand on signal transduction and cellular response, it may be advantageous to specifically inhibit the activity of SEMA3A rather than others.In certain embodiments, the NRP1 trap of the present invention may comprise one or more mutations that reduce the ability of NRP1 to bind, for example, to VEGF.Such mutations can be used to more specifically regulate the activity of NRP1 associated with SEMA3A binding, while less affecting the endogenous NRP1 activity associated with other ligands.

[0142] Thus, in one embodiment, the NRP1 trap of the present invention is a polypeptide that binds to SEMA3A but not to VEGF. For example, the NRP1 trap may contain a1 and / or a2 subdomain(s) that bind to SEMA3A but not the b1 and / or b2 subdomain(s) required for VEGF binding (e.g., 1.1, Trap M, Trap N, Trap Y - see Table 2). In one embodiment, the NRP1-derived trap contains domains a1 and a2 corresponding to amino acids 22-275 of the human NRP1 amino acid sequence set forth in FIG. 17. The NRP1 trap may also contain mutations (e.g., deletions or substitutions) that abolish or significantly reduce the binding of VEGF to NRP1 but not SEMA3A to NRP1, or may preferentially bind to SEMA3A compared to VEGF (e.g., Trap Z, see also Tables 2 and 6). Non-limiting examples of such mutations include substitutions of glutamic acid at position 319 and aspartic acid at position 320 of NRP1 (eg, E319K and D320K, as in traps AC and Z).

[0143] In one embodiment, the soluble NRP1 polypeptide or functional variant or fragment thereof (i.e., NRP1 trap) comprises or consists of the trap described in FIG. 17, FIG. 18, SEQ ID NO: 44, 45, or 46, or Table 2, or any functional variant thereof that binds to SEMA3A. In an embodiment, the soluble NRP1 polypeptide trap comprises or consists of the extracellular domain of the polypeptide described in SEQ ID NO: 47, 95, or 96. In an embodiment, the NRP1 trap used according to the invention has a binding affinity for SEMA3A that is at least three times its binding affinity for VEGF165. In an embodiment, the NRP1 trap used according to the invention has a binding affinity for SEMA3A that is at least four times its binding affinity for VEGF165. In an embodiment, the NRP1 trap used according to the invention has a binding affinity for SEMA3A that is at least five times its binding affinity for VEGF165. In an embodiment, the NRP1 trap used according to the invention has a binding affinity for SEMA3A that is at least ten times its binding affinity for VEGF165. In an embodiment, the NRP1 trap used according to the present invention has a binding affinity to SEMA3A that is at least 15 times higher than that to VEGF165. In an embodiment, the NRP1 trap used according to the present invention has a binding affinity to SEMA3A that is at least 18 times higher than that to VEGF165. In an embodiment, the NRP1 trap used according to the present invention has a binding affinity to SEMA3A that is at least 10 times higher than that to VEGF165. In an embodiment, the NRP1 trap used according to the present invention has a binding affinity to SEMA3A that is at least 20 times higher than that to VEGF165 (see, for example, Table 6).

[0144] Because the NRP1 traps of the invention are secreted, they generally lack the transmembrane domain (e.g., corresponding to amino acid residues 860-883 of the NRP1 polypeptide sequence shown in FIG. 17) and the cytoplasmic domain (e.g., corresponding to amino acid residues 884-923 of the NRP1 polypeptide isoform 1 sequence shown in FIG. 17) found, for example, in human NRP1 isoform 1 (SEQ ID NOs: 95, 96, and FIG. 17). In an embodiment, the NRP1 traps of the invention completely or partially lack domain c of NRP1. NRP1 isoform 1 contains a larger c domain (see FIG. 17), whereas the domains of isoforms b and c are shorter.

[0145] As mentioned above, the present invention also encompasses the use of functional variants and functional fragments of the NRP1 polypeptide trap described herein in the methods described herein. Functional variants are derived from the "wild-type" (natural) human NRP1 polypeptide sequence (including any allelic variants found naturally in a population, i.e., allelic variants). Thus, as used herein, "functional variants" or "functional fragments" refer to any NRP1 derivative that has substantially the same biological activity as the NRP1 trap of the present invention with respect to cellular senescence (i.e., can reduce or prevent the induction of SASP and cellular senescence). Thus, functional derivatives include, but are not limited to, proteins that differ from the NRP1 polypeptide trap disclosed herein by any modification, and / or amino acid substitution, deletion, addition (e.g., intrasequence insertion), or carboxyl-terminal fusion. These do not significantly reduce the intended biological effect of the NRP1 trap of the present invention (e.g., inhibiting or preventing SEMA3A-mediated cellular senescence, or inhibiting or preventing SEMA3A-dependent propagation of cellular senescence by SASP, and ultimately inhibiting IRE1α activation, RNAse activity, etc.). Modifications can occur anywhere, including in the polypeptide backbone (i.e., amino acid sequence), the amino acid side chains, and the amino or carboxy termini, so long as they do not substantially adversely affect the intended function of the NRP1 trap of the present invention (i.e., the variant is a functional variant capable of binding and sequestering a SEMA3A polypeptide (e.g., an NRP1 trap corresponding to a naturally occurring human soluble NRP1 isoform or a polypeptide fragment of the extracellular domain of "wild-type" human NRP1, such as those exemplified in Table 2 and Figure 18).

[0146] Table 3 provides examples of amino acids that can be modified (changed or altered) in the NRP1 trap of the present invention. Preferably, the modification(s) in the functional variant (i) are conservative substitutions made according to Table 3 below, (ii) correspond to functional allelic or polymorphic variants found in the population, or (iii) correspond to amino acid variants found in orthologs of human NRP1 polypeptide. Several orthologs of NRP1 protein are known in the art. For example, by comparing the human NRP1 polypeptide sequence with the NRP1 polypeptide sequences of other known orthologs (e.g., mouse and rat - see Figure 17), the skilled artisan can easily identify conserved residues and residues that differ between species. Thus, amino acids that can be modified without substantially affecting the desired biological activity (e.g., inhibiting or preventing cellular senescence, or inhibiting or preventing the propagation of cellular senescence via SASP) can be identified. Non-limiting examples of such amino acids are provided in Table 4. [Table 3] TIFF2025061122000007.tif19160 [Table 4]

[0147] Other functional variants of the NRP1 trap of the present invention can be generated by introducing one or more mutations that correspond to natural (allelic) variants detected in the population. These natural variants can be easily identified using well-known publicly available databases such as the NCBI, GeneCard, HOMIM and Ensembl websites.

[0148] In an embodiment, a functional variant of the NRP1 trap of the invention comprises or consists of amino acids 1-857 of SEQ ID NO: 47 or a functional fragment thereof. In an embodiment, the functional variant comprises one or more conservative amino acid substitutions located at one or more amino acid positions set forth in Table 4. In an embodiment, the amino acid substitutions are as set forth in Table 4. In an embodiment, the soluble NRP1 polypeptide trap of the invention or a functional fragment or variant thereof (allelic variant) may comprise one or more additional polypeptide domain(s) for increasing synthesis, purification, stability and / or bioavailability. For example, the NRP1 trap of the invention may comprise an FC domain (or a portion thereof, such as a human FC domain) or a purification tag (e.g., a 6x-histidine tag). Such additional polypeptide domain(s) may be linked directly or indirectly (via a linker) to the soluble NRP1 polypeptide or a functional fragment or variant thereof. In an embodiment, the one or more additional domains are at the C-terminus of the NRP1 polypeptide trap. In an embodiment, the one or more additional domains are at the N-terminus of the NRP1 polypeptide trap.

[0149] The soluble NRP1 polypeptides of the present invention or functional variants or fragments thereof may optionally comprise one or more polypeptide linkers. Such linkers may be used to link one or more additional polypeptide domain(s) to the soluble polypeptides of the present invention (e.g., a polypeptide domain that increases the stability of the polypeptide in vivo and / or a domain that facilitates purification of the polypeptide). The linker sequence may optionally comprise a peptidase or protease cleavage site that can be used to remove one or more polypeptide fragments or domains (e.g., to remove a purification tag prior to in vivo administration of the soluble NRP1 polypeptide or functional variant or fragment thereof). One non-limiting example of a linker or domain that allows for cleavage of a polypeptide and removal of, for example, a polypeptide domain(s) (e.g., a 6xHis tag purification domain) includes a polypeptide that includes a TEV protease cleavage site (e.g., EXXYXQ\G or S (where \ represents the cleavage site, SEQ ID NOs: 97 and 98). Many other TEV cleavage sites are known, and many other protease / peptidase cleavage sites are known to those of skill in the art, and can be introduced into a polypeptide of the invention to optionally remove one or more polypeptide domains or fragments.

[0150] Polypeptide linkers may also be used to fully or partially replace domains that are normally found in wild-type NRP1 polypeptides but are not present in the soluble NRP1 polypeptides of the present invention or their functional variants or fragments. For example, in the NRP1 traps of the present invention, synthetic linkers may fully or partially replace subdomains a1, a2, b1, b2 and c. The length of the linker may correspond to the full length of the domains that are removed or a portion thereof. Such linkers may increase protein folding, stability, or binding to NRP1 ligands. Non-limiting examples of NRP1 traps that include linkers are described in WO2016 / 033699, which is incorporated herein by reference. One non-limiting example of a useful polypeptide linker is a polyarginine polypeptide. Other linkers are known in the art and may be used according to the present invention.

[0151] Accordingly, the present invention further provides a soluble NRP1 polypeptide or a functional variant or fragment thereof, a nucleic acid encoding a soluble NRP1 polypeptide or a functional variant or fragment thereof, a vector comprising the nucleic acid, and a host cell comprising the nucleic acid or vector.

[0152] c. Inhibition of SEMA3A expression Various approaches are available for reducing the expression of SEMA3A and thus SEMA3A-mediated cellular senescence. Non-limiting examples include the use of small hairpin shRNA (RNAi), antisense, ribozyme, TAL effectors targeting the SEMA3A promoter, CRISPR / Cas9 / Cpf1 systems, etc.

[0153] The expression of shRNA or similar inhibitory RNA in cells can be obtained by plasmid delivery, or by viral (e.g., lentiviral vector) or bacterial vector delivery.Non-limiting examples of shRNA that can be used to inhibit the expression of SEMA3A are provided in Table 9 (see Example 11).

[0154] Therefore, in an alternative embodiment, the present invention provides antisense, shRNA molecule (iRNA) and ribozyme for exogenous administration to cause the degradation and / or translation inhibition of target mRNA.Preferably, antisense, shRNA molecule and ribozyme target mammalian (preferably human) SEMA3A.The exemplary screening method of antisense and ribozyme nucleic acid that can be used to provide such molecule as the SEMA3A inhibitor of the present invention is disclosed in US Patent No. 5,932,435.

[0155] In a further embodiment, the expression of the nucleic acid encoding the polypeptide of interest (SEMA3A or NRP1), or a fragment thereof, can be inhibited or prevented using RNA interference (RNAi) technology, which is a type of post-transcriptional gene silencing.The examples of therapeutic antisense oligonucleotide applications, as well as additional information on antisense molecules, shRNA and RNAi technology, are provided above for the inhibition of IRE1α, and also apply to the inhibition of the expression of SEMA3A to the same extent.

[0156] Thus, in one embodiment, expression of a nucleic acid encoding a polypeptide of interest (SEMA3A or NRP1), or a fragment thereof, can be inhibited by introducing into a cell or producing within a cell an siRNA or siRNA-like molecule corresponding to a nucleic acid encoding a polypeptide of interest (e.g., SEMA3A), or a fragment thereof, or a nucleic acid homologous thereto.

[0157] (ii) A method for promoting cellular senescence by increasing SEMA3A activity Cellular senescence, such as autocrine and paracrine senescence, can be promoted or induced by stimulating or increasing SEMA3A activity (i.e., SEMA3A cell signaling). SEMA3A activity can be increased by several approaches, such as by increasing the expression of SEMA3A in cells or by contacting cells with a SEMA3A polypeptide or a functional fragment or variant thereof.

[0158] Methods of promoting cellular senescence may be useful in diseases and conditions in which senescence has beneficial effects, such as tissue repair, cancer, renal fibrosis, wound healing, liver fibrosis, myocardial infarction, cardiac fibrosis, atherosclerosis and pulmonary hypertension.

[0159] 6. Regulation of lipid parameters The applicant has discovered that the NRP1 gene is involved in the control of lipid metabolism (fat intake / storage / accumulation) and that administration of a soluble NRP1 polypeptide or a fragment thereof (e.g., NRP1 Trap) significantly reduces diet-induced weight gain and improves lipid parameters, with accompanying benefits (or concomitant favorable outcomes) on blood glucose levels and insulin sensitivity.

[0160] Thus, in a further aspect, the invention provides a method of altering lipid parameters in a subject, comprising modulating expression and / or activity of the NRP1 gene and / or its associated NRP1 protein (e.g., transmembrane isoform 1). In certain aspects, the method comprises administering to the subject a compound or composition that reduces or inhibits expression and / or activity of the NRP1 protein. In embodiments, the method comprises administering to the subject (a) a soluble NRP1 polypeptide or fragment thereof (e.g., NRP1 trap), (b) an NRP1 antibody, or (c) a composition comprising (a) and / or (b) together with a pharma- ceutically acceptable carrier.

[0161] As used herein, the expression "disease or condition associated with adiposity" includes any disease or condition that is caused by adiposity or is considered a comorbidity with adiposity (e.g., diet-induced overweight or obesity). A comorbidity is a medical condition whose prevalence is greatly increased (i.e., the risk of suffering from such additional disease or condition is increased) in the presence of an original condition (e.g., adiposity, overweight or obesity). The term can refer to either a condition that exists simultaneously with an original metabolic condition (e.g., adiposity) or the risk of developing such a comorbid condition. Adiposity-related disease or condition is said to be caused by or otherwise associated with adiposity in a subject. Diseases and conditions associated with adiposity include: high BMI, obesity, metabolic syndrome, NAFLD, cardiovascular disease (CVD; heart disease (e.g., congestive heart failure), coronary artery disease (hypercholesterolemia and atherosclerosis), pulmonary embolism, dyslipidemia and stroke), hypertension, and type II diabetes mellitus (TIIDM). In an embodiment, the fat deposit is greater than or equal to 25 kg / m 2 In another embodiment, the adiposity corresponds to a BMI of 30 kg / m 2 This corresponds to a BMI of 100 or more.

[0162] Body composition parameters related to fat accumulation are well known in the art. These body composition parameters include visceral fat area (VFA), body mass index (BMI), waist-to-hip ratio (WHR), waist-to-height ratio, waist circumference (WC), upper arm circumference (AC), conicity index, body fat percentage (PBF), triceps skinfold, subscapular skinfold, white adipose tissue (WAT) level, or brown adipose tissue (BAT) level.

[0163] Modulation of NRP1-mediated lipid metabolism can be achieved using naturally occurring soluble NRP1 polypeptides or synthetic (e.g., recombinantly produced or chemically synthesized) NRP1 polypeptides described herein.

[0164] 7. Compositions / Formulations The active ingredient(s) (e.g., one or more SASP inhibitors, such as one or more IRE1α inhibitors, inhibitors of SEMA3A (e.g., NRP1 trap), etc.) may be provided in a pharmaceutical composition. Pharmaceutical compositions for use according to the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliaries that facilitate processing of the active compounds into pharma-ceutically usable preparations. Pharmaceutical compositions may include other medicinal or pharmaceutical agents, carriers, adjuvants, such as preservatives, stabilizing agents, wetting agents or emulsifying agents, dissolution enhancers, salts for adjusting osmotic pressure, and / or buffers. Appropriate formulations depend on the route of administration selected. For injection, the agents of the present invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers, such as Hank's solution, Ringer's solution, or physiological saline buffer. Methods well known in the art for preparing formulations can be found, for example, in Remington: The Science and Practice of Pharmacy, (20th Edition), Ed., AR Gennaro A R., 2000, Lippencott Williams & Wilkins.

[0165] In an embodiment, the composition of the present invention is formulated for ocular delivery, for example, eye drops or ocular injection.For ocular administration, the compound can be easily formulated by combining the active compound with a pharma- ceutically acceptable carrier suitable for ocular administration, as is well known in the art.In an embodiment, the carrier is a carrier that is not naturally found in the mixture with the compound / agent / inhibitor of the present invention (i.e., a non-naturally occurring carrier).

[0166] For example, the pharmaceutical composition may be formulated for topical administration, intravitreal administration, intracapsular administration, subconjunctival administration, subtenon administration, retrobulbar administration, posterior juxtascleral administration, or a combination thereof. In some embodiments, the pharmaceutical composition is formulated for topical administration. In some embodiments, the pharmaceutical composition is formulated for intravitreal administration.

[0167] Preparations for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative.The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents, such as suspending, stabilizing and / or dispersing agents.

[0168] Alternatively, other delivery systems for pharmaceutical compounds can be used.Liposome and emulsion are well-known examples of delivery vehicles.The delivery system that is particularly useful for periocular drug delivery (for example, in the prevention and / or treatment, or eye disease such as retinal disease) includes the transscleral absorption route, which is considered to be one of the safest means of achieving consistent therapeutic drug concentration in the posterior intima.

[0169] Effective dosage. Pharmaceutical compositions suitable for use in the present invention include compositions in which the active ingredient(s) are contained in an effective amount to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent or reduce the onset of at least one of the existing symptoms of the subject being treated. The determination of an effective amount is well within the capabilities of those skilled in the art.

[0170] In embodiments, an effective amount of a compound(s) used in accordance with the present invention inhibits cellular senescence or the propagation of cellular senescence (via the SASP) sufficiently to reduce or prevent at least one symptom or physiological effect associated with cellular senescence in the diseases and conditions described herein (e.g., ocular vascular disease and other diseases and conditions described herein). Particular compounds having such activity can be identified by in vitro assays that determine dose-dependent inhibition of the SASP and / or IRE1α.

[0171] Alternatively, in other embodiments, the effective amount of the compound(s) used in accordance with the present invention is sufficient to induce or increase SASP and cause cellular senescence.

[0172] For any compound used in the method of the present invention, the therapeutically effective dose can be estimated initially from cell assay. For example, the dose can be formulated in cell and animal models to achieve a circulating concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of the intracellular signaling function of SASP and / or IRE1α) determined in cell assay (usually in response to inflammatory mediators such as Il-1β or other activating stimuli such as hypoxia, ischemia, cell stress, ER stress, etc.).

[0173] A therapeutically effective amount refers to the amount of compound that results in the improvement of symptoms in a subject. Similarly, a prophylactically effective amount refers to the amount necessary to prevent or delay symptoms in a patient (e.g., vascular hyperpermeability, blemished and / or blurred vision, pericytes loss, macular edema, retinal swelling, blood-retinal barrier leakage, pathological neovascularization, reduced vascular repair, etc.). The toxicity and therapeutic effect of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as by determining the maximum tolerated dose (MTD) and ED (effective dose for 50% maximum response). The dose ratio between toxic effect and therapeutic effect is the therapeutic index, which can be expressed as the ratio of MTD to ED50. Compounds that exhibit a high therapeutic index are preferred. The exact formulation, route of administration and dosage can be selected by the individual physician in consideration of the patient's condition.

[0174] Dosage and administration intervals can be individually adjusted to provide a sufficient level of active compound to maintain the desired regulatory effect or minimum effective concentration (MEC). MEC varies from compound to compound, but can be estimated from in vitro data. For example, the concentration required to achieve substantial inhibition of SASP and / or IRE1α expression or activity (e.g., secretion of SASP-associated cytokines, proteases and growth factors, activation of ribonuclease activity and processing of XBP1). The dosage required to achieve MEC depends on individual characteristics and route of administration.

[0175] definition In order to provide a clear and consistent understanding of terms in this application, the following additional definitions are provided.

[0176] The articles "a", "an" and "the" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article.

[0177] As used in this specification and the claims, the words "comprising" (and any form of "comprise", such as "comprise" (comprises")), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" (include")), or "containing" (and any form of containing, such as "contains" (contain)) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps, and are used synonymously with the phrases "including but not limited to" and "comprising but not limited to."

[0178] For the recitation of numerical ranges herein, each intervening number with the same degree of precision therebetween is expressly contemplated.For example, for a range of 18-20, the numbers 18, 19 and 20 are expressly contemplated, and for a range of 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are expressly contemplated.The term "such as" is used herein to mean and is used synonymously with the phrase "such as, but not limited to."

[0179] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. For example, any nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry, and hybridization described herein are well known and commonly used in the art. The meaning and scope of terms shall be clear, but in case of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include plurals, and plural terms shall include singulars.

[0180] The practice of the methods, and the preparation and use of the products and compositions disclosed herein employ, unless otherwise indicated, conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA and related fields, and are within the skill of the art, which techniques are fully explained in the literature. See, e.g., Green and Sambrook MOLECULAR CLONING: A LABORATORY MANUAL, 4th ed., Cold Spring Harbor Laboratory Press, 2014; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 2003 and regularly updated editions; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, 3rd ed., Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, "Chromatin" (P M Wassarman and A P Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, "Chromatin Protocols" (P B Becker, ed.) Humana Press, Totowa, 1999.

[0181] As used herein, the terms "treat / treating / treatment" and "prevent / preventing / prevention" refer to eliciting a desired biological response, i.e., a therapeutic effect and a prophylactic effect, respectively. According to the present invention, a therapeutic effect includes an improvement in symptoms and / or a reduction in the severity of a disease or condition (e.g., vascular eye disease) following administration of a pharmaceutical composition or compound (e.g., a SASP inhibitor) of the present invention.

[0182] As used herein, the terms "prevent" or "prevention" with respect to a disease or condition associated with aging is meant to refer to a reduction in the progression or delay in the onset of at least one symptom associated with the disease or condition or one hallmark of cellular senescence.

[0183] Described herein is a method for regulating cellular senescence. As used herein, "cellular senescence" refers to a state of cells that are viable and metabolically active but lose the ability to proliferate or remain part of a tissue structure but cannot function / communicate properly with the rest of the tissue (i.e., become dormant). Cellular senescence may increase with age or exposure to factors that induce DNA damage, such as mutations or chromosomal damage, or factors that induce DNA damage response or disruption of chromatin structure, resulting in changes in gene expression, such as genes related to SASP. Senescence is believed to be the result of DNA or chromosomal damage, such as telomere shortening, chromosomal aneuploidy, DNA strand breaks, DNA chemical modification (e.g., alkylation), or induction of DNA damage response (DDR). Cellular senescence can be caused, for example, by ischemia, oncogene activation (by DDR) DNA damaging compounds, such as chemotherapeutic agents, or DNA damaging radiation, such as ionizing and UV radiation. Senescence can be caused by a variety of other therapeutic regimens, such as corticoid therapy, antiretroviral therapy, treatment with PPARγ agonists, treatment with xanthine oxidase inhibitors, treatment with bisphosphonates, treatment with antiprotozoal agents, and treatment with inflammatory agents. Senescence can also be caused by metabolic imbalances, such as increased caloric intake, insulin resistance, type II diabetes, hyperinsulinemia, high-fat diet, high-protein diet, ER stress response (UPR response as demonstrated herein), and alterations in the gut microbiota associated with these diseases. Senescent cells develop a unique secretome, including metalloproteases, growth factors, and inflammatory cytokines, a process termed the senescence-associated secretory phenotype (SASP) (37), which allows them to propagate senescence to surrounding tissues in a cell-autonomous and cell-nonautonomous (paracrine) manner (38-40). Thus, paracrine cellular senescence can be induced in cells as a consequence of the senescence-associated secretory phenotype (SASP). Paracrine senescence refers to a state in which senescent cells increase their secretion of proteins, including proinflammatory cytokines (SASP).

[0184] In various embodiments, cellular senescence is caused by: (a) ischemia, (b) cellular senescence, (c) DNA damage to a cell, (d) contact with a chemotherapeutic agent, (e) irradiation of a cell with DNA damaging radiation, (f) contact of a cell with an antiretroviral agent, (g) contact of a cell with a pro-inflammatory agent, (h) contact of a cell with a DNA damaging agent, (i) contact of a cell with an agent that disrupts chromatin structure, (j) telomere erosion, (k) hypoxia, (1) activation of oncogenes, (m) telomere dysfunction, and (o) any combination of at least two of (a)-(m).

[0185] Cells undergoing cellular senescence may exhibit one or more of the following characteristics: growth arrest, formation of γ-H2AX (phosphorylated form of the histone variant H2AX) nuclear foci, elevated levels of pI6INK4A, elevated expression levels of p21 (Cipl / Waf1), increased activity of senescence-associated β-galactosidase, generation of senescence-associated heterochromatin foci (SAHF), loss of proliferation, trimethylation of histone 3 lysine 9 (H3K9me3), induction of endoplasmic reticulum stress and unfolded protein response (UPR), increased levels and / or activation of tp53, increased number and size of PML nuclear bodies, activation of IRE1α, increased glucose consumption, increased expression and / or secretion of proinflammatory cytokines, proteases of the "senescence-associated secretory phenotype" (SASP) and growth factors, among others. These include, but are not limited to; Pai1, IL-6, IL-7, IL-lα, IL-lβ, IL-8, TGF-β1, MCP-2, MCP4, MIP-la, MIP-3a, Eotaxin-3, GM-CSF, MIF, EGF, FGF, HGF, VEGF, KGF, PIGH, NGF, MMP1, MMP3, MMP12, MMP13, MMP14, IGFBP2, IGFBP3, IGFBP4, IGFBP6, IGFBP7, fibronectin, cathepsin B, TIMP-2; lack of expression of Ki67, enlarged and flattened cell morphology, persistent DNA damage response (DDR) signaling, and formation of DNA segments with chromatin changes that enhance senescence (DNA-SCARS), which are nuclear foci that may contain DDR proteins such as phospho-ATM and ATR substrates. Cells undergoing cellular senescence typically have an increased expression level of p16INK4a compared to the expression level of P16INK4a in cells not undergoing cellular senescence. Also, cells undergoing cellular senescence typically have an increased level of SA-β-Gal activity compared to cells not undergoing cellular senescence.

[0186] As used herein, a "senescent cell" or a "cell having a senescent phenotype" refers to a cell that has at least one of the following characteristics: (i) growth arrest, (ii) enlarged and flattened cell morphology, (iii) DNA damage foci in the nucleus, (iv) secretion of growth factor proteases, cytokines and other factors defined as the senescence-associated secretory phenotype (SASP) (e.g., PAI1, TNFAAIP2, IGFBP3, VIM, CDKN1A, FN1, CDKN2B, RRAS, IRF7, HSPA2, TES, CTGF, CCND1, ESM1, THBS1, S100A11, RAB31, IGFBP5, IL6, IL1β, TGFβ1, VEGFA, TP53), (v) senescence-associated β-galactosidase (SA-β-gal) activity (partially reflecting an increase in lysosomal mass), (vi) expression of tumor suppressor p16INK4a (which activates pRB and can lead to the formation of senescence-associated heterochromatin foci (SAHF)), (vii) expression of SEMA3A, (viii) IRE1α activation (S724 phosphorylation) and increased splicing of XBP1, and / or (ix) increased expression of γH2AX, PML and / or p53 activation. In embodiments, a "senescent cell" is a cell having at least the characteristics of (i), (ii) and / or (ii), (v), (vi) and (ix). In embodiments, senescence is secondary to cellular ischemia. In embodiments, senescence is paracrine senescence. In an embodiment, the senescence is post-differentiation senescence. In an embodiment, the senescence is premature senescence. In an embodiment, the premature senescence is characterized by increased expression and / or RNAse activity of IRE1α. In an embodiment, the senescence is retinal senescence. In an embodiment, the senescence is microglial senescence. In an embodiment, the senescence is characterized by (i) increased expression and / or activity of P16INK4a, Tp53, IRE1a, Cdkn1a, Cdkn2a, and / or increased senescence-associated β-gal activity, (ii) expression of γH2Ax and / or PML, and / or (iii) expression of a senescence-associated secretory phenotype (SASP). In an embodiment, the SASP comprises secretion of IL-1β, IL-6, Pai1, TGFβ1, IRE1α and / or VEGF-α. In an embodiment, the above-mentioned SASP is secondary to cellular ischemia.

[0187] In embodiments, the cell is a terminally differentiated cell. In embodiments, the cell is a neuron, a microglial cell, a bone marrow cell, a monocyte, a macrophage, an endothelial cell, a hepatocyte, an adipocyte, a fibroblast, and / or a retinal cell. In embodiments, the cell is suffering from cellular ischemia. In embodiments, the cell is a retinal ganglion cell. In embodiments, the cell is a retinal ganglion neuron. In embodiments, the cell is a vascular cell. In embodiments, the cell is a vascular endothelial cell. In embodiments, the cell is an avascular cell (i.e., located in an avascular region / area). In embodiments, the cell is a hepatic stellate cell. In embodiments, the cell is a microvascular endothelial cell. In certain embodiments, the cell is not an ocular cell. In certain embodiments, the cell is not a retinal cell. In embodiments, the cell is a mammalian cell. In embodiments, the cell is a human cell.

[0188] As used herein, "cellular ischemia" refers to the limited supply of oxygen and / or nutrients (e.g., glucose) necessary for cellular metabolism (to maintain tissue viability) and the inadequate removal of metabolic waste products. Cellular ischemia includes local anemia in a part of the body, which may result from blood congestion (vasoconstriction, thrombosis, or embolism, etc.). Ischemia can be partial (underperfusion) or global. Ischemia is generally caused by vascular problems (e.g., embolism, thrombosis (e.g., atherosclerosis), trauma, aneurysm, cardiomyopathy, hypoglycemia, radiation therapy, hypotension, anemia, etc.), resulting in tissue damage or dysfunction.

[0189] The term "effective amount" as applied to the compound(s), biologics and pharmaceutical compositions described herein means the amount necessary to obtain the desired therapeutic result. For example, an effective amount is a level effective to treat, cure or alleviate the symptoms of the disorder for which the therapeutic compound, biologic or composition is administered. The amount effective for the particular therapeutic purpose sought will depend on a variety of factors, such as the disorder being treated and its severity and / or stage of onset / progression, the bioavailability and activity of the particular compound, biologic or pharmaceutical composition used, the route or method of administration and the site of introduction in the subject, the clearance rate or biological and other pharmacokinetic properties of the particular compound, the duration of treatment, the inoculation regimen, drugs used in combination or simultaneously with the particular compound, biologic or composition, the age, weight, sex, diet, physiology and general health of the subject being treated, and similar factors well known to those skilled in the relevant science and art. Some variation in dosage may occur depending on the condition of the subject being treated, and the physician or other individual administering the treatment will in any event determine the appropriate dose for the individual patient.

[0190] "Homology" and "homologous" refer to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing each position in the aligned sequences. The degree of homology between nucleic acids or amino acid sequences corresponds to the number of identical or matching nucleotides or amino acids at positions shared by the sequences. As the term is used herein, a nucleic acid / polynucleotide sequence is "homologous" to another sequence if the two sequences are substantially identical and the functional activity of the sequence is preserved (the term "homology" as used herein does not presume an evolutionary relationship). Two nucleic acid sequences are considered substantially identical if, when optimally aligned (with gaps allowed), they share at least about 50% sequence similarity or identity, or if the sequences share a defined functional motif. In alternative embodiments, the sequence similarity in optimally aligned substantially identical sequences can be at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical. As used herein, a given percentage of homology between sequences represents the degree of sequence identity in optimally aligned sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, preferably less than about 25% identity, with any of the nucleic acids and polypeptides disclosed herein.

[0191] Substantially complementary nucleic acids are those in which the complement of one molecule is substantially identical to the other molecule.Two nucleic acid or protein sequences are considered to be substantially identical if they share at least about 70% sequence identity when optimally aligned.In alternative embodiments, the sequence identity may be, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%. Optimal alignment of sequences for identity comparison may be performed using a variety of algorithms, including local homology algorithms (Smith and Waterman, 1981, Adv. Appl. Math 2:482), homology alignment algorithms (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443), similarity search methods (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444), and computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI, USA). Sequence identity may also be determined using the BLAST algorithm (Altschul et al., 1990, J. Mol. Biol. 215:403-10 (using published default settings)). Software for performing BLAST analyses is available from the National Center for Biotechnology Information. Informationk (Internet http: / / www.ncbi.nlm.nih.gov / ). The BLAST algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in the database sequences. T is called the neighborhood word score threshold. The initial neighborhood word hits act as seeds to initiate searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased.Extension of word hits in each direction is stopped when the following parameters are met: the cumulative alignment score is decreased by an amount X from its maximum achieved value, the accumulation of one or more negatively scoring residue alignments causes the cumulative score to fall below zero, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program may use as defaults a word length (W) of 11, a BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919), a BLOSUM62 scoring matrix alignment (B) of 50, an expectation (E) of 10 (or 1 or 0.1, or 0.01, or 0.001, or 0.0001), M=5, N=4, and a comparison of both strands. One measure of statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which is an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. In an alternative embodiment of the invention, nucleotide or amino acid sequences are considered to be substantially identical if the smallest sum probability in the comparison of test sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0192] Another indication that two nucleic acid sequences are substantially complementary is that the two sequences hybridize to each other under moderately stringent or preferably stringent conditions. Hybridization of filter-bound sequences under moderately stringent conditions can be, for example, hybridization under conditions of 0.5 M NaHPO at 65° C. 4Hybridization to filter-bound sequences under stringent conditions may be performed, for example, in 0.5 M NaHPO at 65° C., 7% sodium dodecyl sulfate (SDS), 1 mM EDTA, and washed in 0.2×SSC / 0.1% SDS at 42° C. (see Ausubel et al., (eds.), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, p. 2.10.3). Alternatively, hybridization to filter-bound sequences under stringent conditions may be performed, for example, in 0.5 M NaHPO at 65° C. 4 Hybridization may be performed in 500 ng / mL, 7% SDS, 1 mM EDTA, and washed at 68° C. with 0.1×SSC / 0.1% SDS (see Ausubel et al., (eds.), 1989, supra). Hybridization conditions may vary according to known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York). In general, stringent conditions are selected to be about 5° C. lower than the thermal melting point of the specific sequence at a defined ionic strength and pH. For example, in an embodiment, the compound of the present invention is an antisense / RNAi or shRNA that hybridizes to NRP1 or SEMA3A nucleic acid sequence (preferably a human sequence).

[0193] The invention is further illustrated by the following non-limiting examples.

[0194] Example 1 Retinal ischemia-induced cellular senescence To elucidate the cellular processes triggered following vascular degeneration in ischemic retinopathy, mouse pups were subjected to a model of oxygen-induced retinopathy (OIR) that results in an avascular neural band similar to that observed in DR and ROP (27). Mouse pups were exposed to 75% oxygen from postnatal day (P) 7 to 12 to induce vascular occlusion and returned to ambient air, with maximal preretinal neovascularization achieved at P17 (27) (Figure 1A). We then performed unbiased transcriptome analysis by high-throughput RNA sequencing of retinas at P14 OIR (when pathological preretinal vascularization begins) and gene set enrichment analysis (GSEA) to identify regulated, defined gene expression patterns. As expected, we found strong positive correlations in clusters of inflammation (NES (normalized enrichment score) = 1.58; FDRq (false discovery rate) = 0.004) and apoptosis (NES = 1.63; FDRq = 0.002) (Figure 1B). Given the highly postmitotic nature of the neural retina, we unexpectedly noticed a significant enrichment in the Fridman senescence hallmark (28) cluster (NES=1.81; FDRq=0.0) (Fig. 1C).

[0195] Cellular senescence is a state of permanent cell cycle arrest in which cells remain viable and metabolically active (29). In primarily postmitotic tissues such as the retina, senescence can be triggered by stimulation of the DNA damage response or tumor suppressor networks, which have been reported to be activated in the ischemic retina (30). Thus, the senescent state may help protect retinal cells from the low metabolic supply associated with ischemia and avoid hypoxia-associated cell death. Induction of senescence during OIR involves the regulation of p53, p16 INK4a This was further supported by the upregulation of classical senescence-associated proteins, such as Pai1, PML, and γH2AX, and activation of the ER stress effector inositol-requiring enzyme 1α (IRE1α), which has been suggested to promote cellular senescence ( 31 ) ( Figure 1 D), and the transcript levels of the cyclin-dependent kinase inhibitors (CDKi) Cdkn1a and Cdkn2a were significantly increased in OIR retinas ( Figure 1 E).

[0196] To determine which cells initiated the senescence program during OIR, senescence-associated β-galactosidase (SA-β-gal) staining was performed on retinal flat mounts at P14. Counterstaining with isolectin B4 (IB4) revealed that senescent cells were predominantly located in the avascular zone (36.99% of cells were SA-β-gal) compared with vascularized areas (18.79%; P < 0.0001). + A small number of SA-β-gal + cells were also found in control normoxic retinas (Figure 1F and Figure 1G). Consistent with that, analysis of sagittal retinal sections revealed significantly increased SA-β-gal staining in avascularized areas of the retinal ganglion cell layer (GCL) (10.77% vs. 0.3%; P = 0.0167) and to a lesser extent in the inner nuclear layer (INL) (2.61% vs. 0.45%; P = 0.0342) (Figure 1H and Figure 1I). Both layers are closely associated with the inner retinal vasculature that degenerates in ischemic retinopathy. Due to the hypoxic / oxidative and inflammatory nature of the ischemic retina in Figure 1B (32), as well as GSEA of apoptotic genes, we further aimed to ascertain which cells were undergoing apoptotic death during OIR. Terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling (TUNEL) revealed a predominance of apoptotic cells in the INL (Figure 1J) and in most of the periphery (Figure S1A). Collectively (Figure 1J), these data revealed mutually exclusive patterns of retinal cellular senescence and apoptosis, with cells in the GCL associated with the central zone adopting a predominantly senescent phenotype, and cells in the INL being more susceptible to apoptosis.

[0197] Example 2 Retinopathy triggers a senescence-associated secretory phenotype that propagates cellular senescence SASP typically reinforces senescence in an autocrine and paracrine manner, enhancing inflammation and exerting detrimental effects on the tissue microenvironment (34). We investigated whether the cellular senescence initially observed in the GCL during OIR at P14 (Figure 1J) propagated to other cell populations in the retina. Initial SA-β-gal staining of OIR at P14 was concentrated in avascular regions (Figure 2A and Figure 2B, left panel) and was associated with the expression of Brn3a+RGCs and senescence markers (γH2AX, Pai1, and p-IRE1α). S724 At P17, during maximal neovascularization, cellular senescence is localized to pathological vascular tufts (middle panels in Figures 2A and 2B) and retinal microglia, as evidenced by co-localization of the senescence markers γH2AX or PML with IB4 (vessels) and IBA1 (microglia) (Figure 2D). This vasculature is tortuous and leaky (35), with poor and unstable access to pericytes (Figures 11A and 11B) (36). By P21 after OIR, as the retinal vasculature regenerates, SA-β-gal staining is restricted primarily to vascular cells (right panels in Figures 2A and 2B, Figure 10D). At this later time point, pericyte access is regained (Figures 11A and 11B). In addition, we observed SA-β-gal stained cells in the GCL of mice at 8 weeks of streptozotocin-induced type I diabetes (Figure 10E, Figure 10F). The presence of retinal senescence in the STZ model is particularly important, given that the OIR model was performed in mouse pups and that the angiogenic response spreads with age (37). These findings support the presence of cellular senescence in a wide range of ocular vascular disorders. Considering that the OIR model is a well-known and established model in ocular vascular pathology, further studies were performed in this model. Nevertheless, we observed a transient accumulation of senescent cells in different subcellular populations of the retina in different models of retinopathy. The dynamically evolving pattern of cellular senescence in correspondence with disease progression supports paracrine senescence.

[0198] Senescent cells develop a unique secretome, including metalloproteases, growth factors and inflammatory cytokines, a process termed the senescence-associated secretory phenotype (SASP) (39), which allows them to propagate senescence to surrounding tissues in a cell-autonomous and cell-nonautonomous (paracrine) manner (40-42). Moreover, heatmap and GSEA of OIR retinas identified a positive correlation between retinal ischemia and paracrine senescence-related genes (NES=1.4; FDRq=0.049) (Figure 2E). Elevated expression of SASP-related cytokines in OIR was confirmed by RT-qPCR for Pai1, Il6, Il1β, Tgf-β1, Vegf-a, and IRE1α, as well as the tumor suppressor Tp53 (Figure 2F), suggesting that some cytokines central to pathological angiogenesis may originate from senescent retinal cells.

[0199] Example 3 Secretion of SEMA3A by senescent cells drives paracrine senescence Given the spread of cellular senescence, we sought to identify factors driving this process in OIR. An effector molecule associated with retinal ischemia that has been suggested to disrupt the cell cycle (43) is the classical guidance molecule semaphorin 3A (SEMA3A). SEMA3A is induced throughout the vascular occlusion and vascular proliferation phases of OIR (44) and is secreted by hypoxic neurons, biasing the regenerating vascular and metabolic supply towards less affected areas of the retina (44, 45). Given that SEMA3A expression temporally coincides with markers associated with senescent cells during the progression of retinopathy (Figures 3A, 5A, and 5B), we questioned the contribution of SEMA3A to the propagation of paracrine senescence.

[0200] First, evidence for a potential contribution of SEMA3A to paracrine senescence arose from the observation that senescence-inducing oncogenes such as RasV12 (Fig. 3B) and MEK (Fig. 3C) triggered the production of SEMA3A, as well as stress-induced senescent human hepatic stellate cells (HSCs) (Fig. 3D). Moreover, intravitreal injection of recombinant Sema3A induced a marked increase in p53, p16INK4a, and IRE1α (Fig. 3E), accompanied by a marked increase in SA-β-gal staining (Fig. 3F) in P5 pups.

[0201] Finally, exposure of HRMEC to SEMA3A for 7 days (mimicking the first week of OIR) increased cell cycle arrest in G0 / G1 with a significant decrease in S-phase as measured by electrical cell impedance sensing (ECIS) (Figure 3G), and prevented normal endothelial cell growth (Figure 3H).

[0202] The role of SEMA3A in promoting senescence is further demonstrated by directly exposing cells to recombinant SEMA3A, which induces senescence in macrophage-like J774 cells (P < 0.0001), in a cell line of retinal neurons (661W photoreceptors used to model retinal neurons) (46) (P < 0.001), and in HRMECs (P < 0.001) (Figure 3I and J). Furthermore, H 2 O 2 Driven senescence ( 3 ) triggered secretion of SEMA3A in cell lines that model populations that enter senescence in retinopathy, such as macrophage-like J774 cells (J774) and neuronal 661W cells ( Figure 3K ).

[0203] To verify the potential involvement of neuron-derived SEMA3A in driving paracrine endothelial cell senescence, we exposed human retinal microvascular endothelial cells (HRMECs) to conditioned medium (CM) from senescent 661W cells (Figure 3L and Figure 3M), in which Sema3a was silenced by a lentiviral (Lv) vector carrying small hairpin RNA (shRNA) (44, 47). The efficacy of this approach was demonstrated (Figure 5C-Figure 5E). CM from the senescent retinal neuronal cell line effectively propagated senescence in a paracrine manner resulting in induction of SA-β-gal expression in 68% of HRMECs (P<0.005) (Figure 3N, left panel, O). It is highlighted that factors secreted by senescent cells have a propensity to stimulate senescence in neighboring cells. Conversely, CM from Sema3A-deficient senescent retinal neuronal precursor cells caused significantly less paracrine senescence in HRMEC cells (P<0.005) (Figure 3N, right panel, O). In parallel, incubation of HRMEC with conditioned medium (CM) from senescent neuronal precursors was sufficient to activate p53 in a SEMA3A-dependent manner (Figure 5F and Figure 5G). Interestingly, downregulating SEMA3A by intravitreal administration of Lv.sh_Sema3a at P12 of OIR significantly reduced the number of senescent SA-β-gal-positive retinal cells at P14 by 75%, highlighting the crucial contribution of SEMA3A to senescence during OIR (Figure 3P and Figure 3Q). Taken together, these data provide evidence for the production of SEMA3A during cellular senescence and its contribution in the propagation of paracrine senescence.

[0204] . Example 4 Enrichment of ER stress transcripts in retinopathy The pathway of unfolded protein response (UPR), triggered under ER stress conditions, can provide cells with an adaptive mechanism to survive during metabolic imbalance, such as ischemia (48, 49). As supported by our findings, activation of ER stress may help drive premature aging. Transcriptome analysis of OIR retinas revealed significant GSEA enrichment in transcripts related to the UPR (NES=1.41; FDRq=0.047) (Figure 4A). Given that SEMA3A was shown to activate the IRE1α branch of the UPR (Figures 3E and 5H), we investigated the contribution of IRE1α to premature aging in ischemic retinas.

[0205] During ischemic retinopathy, there is a substantial association of microglia and infiltration of myeloid cells expressing microglial markers. fl / fl We observed SA-β-gal staining in the avascular zone (Figure 4B) enriched for EFYP+ myeloid / microglial cells (Figure 4C). EFYP+ microglia also stained with the senescence-associated DNA damage marker γH2AX and localized preferentially to the vascular / avascular border of P14 OIR retinas, and to sites of pathological angiogenesis (tufts) at P17 (Figure 4C).

[0206] Example 5 SEMA3A activates IRE1α, and the RNAse activity of IRE1α contributes to senescence IRE1α is a type I transmembrane protein that has both a serine / threonine kinase domain and a distinct endoribonuclease domain at its cytosolic tail (54, 55). IRE1α preferentially targets mRNAs encoding proteins that traverse the ER-Golgi secretory pathway through its RNase activity, also called IRE1α-dependent decay (RIDD) (56). Given that SEMA3A drives senescence through IRE1α (Figures 2, 3), we next investigated which catalytic arm could explain this physiological response. Given the established evidence that myeloid cells undergo senescence upon retinal ischemia, we used J774 macrophage / monocyte cells and sustained exposure to SEMA3A to activate IRE1α (Figure 7A), induce senescence (Figure 6B), and result in the expression of a panel of genes known to be important for promoting and enhancing the senescent state, including Pai1, Il6, Il1β, TGF-β, and Tp53 (Figure 7B). SEMA3A also promoted senescence-associated DNA-damage foci expressing γH2AX (Figure 7C), a hallmark of cellular senescence (57). Similarly, shRNA-mediated knockdown of IRE1α in endothelial cells prevented SEMA3A-driven senescence (Figures 6D and 6E).

[0207] To determine whether SEMA3A-driven senescence occurs via the kinase or RNAse activity of IRE1α, we used 4μ8c, a selective cell-permeable coumarin o-hydroxyaldehyde pharmacological inhibitor of IRE1α endoribonuclease activity. Exposure to 4μ8c (Figure 7D) prevented SEMA3A-induced growth arrest and abolished SEMA3A-induced senescence (data not shown). In support, 4μ8c also prevented SEMA3A-mediated splicing and activation of the IRE1α effector X-box binding protein-1 (XBP1) (Figure 7D and Figure 7E). Finally, pharmacological inhibition of the endoribonuclease activity of IRE1α, but without any effect on Tnf-α or IRE1α itself, inhibited the production of certain senescence-associated genes Vegf-a, Tgf-β1, Il-1β, Il-6, Pai-1, and Tp53 (Figure 7F). These data highlight the importance of the endoribonuclease activity of IRE1α in inducing senescence.

[0208] Example 6 Metformin abrogates the SASP and pathological retinal angiogenesis To establish the clinical relevance of therapeutic inhibition of SASP and paracrine aging in ischemic retinopathy, we assessed the levels of key SASP proteins in the vitreous of patients with active proliferative diabetic retinopathy (PDR). Angiography and spectral domain optical coherence tomography (SD-OCT) were performed and three-dimensional (3D) retinal maps were generated to assess the extent of retinal damage (Figure 8A). Undiluted vitreous was obtained from PDR patients and control patients with non-vascular pathology such as epiretinal membranes and macular holes, who only showed non-diabetic related retinal damage. Detailed characteristics of the patients are included in Table 1. Assessment of vitreous SASP proteins by multiplex magnetic bead-based immunoassay revealed significant increases in senescence-associated factors Pai-1 (P=0.0004), IL-6 (P=0.001), IL-8 (P=0.0037), and VEGF-A (P=0.0085) in PDR patients (Figure 8B). Given the association between paracrine aging and retinopathy (Figures 1C and 3E), we sought to therapeutically modulate SASP to assess the consequences for pathological retinal angiogenesis. In this regard, metformin, a widely used biguanide antidiabetic drug, has been reported to reduce SASP without interfering with the growth arrest program (58). A single intravitreal injection of metformin at P12 attenuated NF-κB and IRE1α activation in OIR mouse retinas (Figure 8C). This led to a significant decrease in IL6, Cdkn1a, Cdkn2a, and Sema3A as determined by RT-qPCR (Figure 8D), as well as a significant decrease in SA-β-gal at P14 (P = 0.0086) (Figures 8E and 8G, and Figure 12A) and P17 (P = 0.0036) (Figures 8F and 8G, and Figure 12A). Components of the VEGF signaling pathway were unaffected (Figure 13A). We chose to perform intravitreal injections of metformin, given that systemic administration would prevent weight gain in mice and therefore could be a confounding factor (32). [Table 5]

[0209] We next determined whether treatment with metformin and subsequent inhibition of SASP resulted in increased apoptosis. TUNEL staining revealed that treatment with metformin reduced the number of apoptotic cells in the INL layer when compared to vehicle-treated retinas, without exacerbating apoptosis in cells of the GCL (Figure 12B). These findings were confirmed by Western blotting of the retina to cleave caspase-3 at various stages of retinopathy (Figure 12C). Finally, when assessed at P17, intravitreal injection of metformin increased revascularization by more than two-fold (P<0.0001) (Figures 8H and 8J) and inhibited pathological neovascularization by half (P<0.0001) (Figures 8H, 8K). It is important to note that administration of metformin by systemic route did not show any advantage on pathological retinal angiogenesis, highlighting the need for local intravitreal administration. Taken together, these data support therapeutic inhibition of the SASP by biguanides such as metformin in the treatment of pathological ocular angiogenesis (pathological neovascularization).

[0210] Example 7 The VEGF trap eye reverses pathological retinal angiogenesis but does not increase revascularization or reduce aging. We next determined whether anti-VEGF treatments such as the currently used VEGF trap eye (aflibercept) (59, 60) would affect retinal aging during retinopathy (60). Aflibercept is a recombinant fusion protein composed of the extracellular domains and Fc portion of human VEGF receptors 1 and 2. Aflibercept binds to at least VEGF-A and placental growth factor (PLGF) (59). Intravitreal injection of aflibercept at P12 of OIR did not significantly affect SA-β-gal staining (P=0.3087) at P14 (P=0.3087) (Figure 9A, Figure 9B), or at P17 (P=0.1580) (Figure 9C, Figure 9D). Interestingly, in contrast to treatment with metformin, which both reduces SASP and enhances vascular regeneration, aflibercept does not modulate vascular regeneration rate at P14 (P=0.4897) (Figure 9A, Figure 9C) or P17 (P=0.9502) (Figure 8B, Figure 8D), nor is it a driver of senescence (Figure 13B). As expected, intravitreal injection of aflibercept led to a significant reduction in neovascularization at P17 0IR (P=0.0207) (Figure 9G), (*P<0.001)). Thus, treatment with aflibercept only directly blocks pathological angiogenesis without attenuating retinal ischemia or senescence or enhancing retinal vascular repair. Taken together, these data further strengthen the link between cellular senescence and ischemia-driven pathological angiogenesis.

[0211] Example 8 Preparation of soluble SEMA3A neutralizing trap High affinity traps for inhibiting / neutralizing SEMA3A were generated. These traps were derived from neuropilin 1 (NRP1) and were optionally coupled to a 6X-His tag or FC protein (see FIG. 18 and Table 2). Various variants were generated, containing either the entire NRP1 extracellular domain or functional variants capable of maintaining SEMA3A binding. The traps contained the b1 domain (which binds VEGF) and neutralized VEGF 165Traps containing the mutations were generated. The traps were shown to be highly expressed and secreted in transformed human cells. A simple purification and formulation protocol was developed to produce trap samples for structure-activity relationship (SAR) and in vivo efficacy studies.

[0212] method Cell culture and materials. Human neuropilin 1 (GenBank™ accession number NM_003873, SEQ ID NO: 66) was obtained from Origene Inc. The Origen clone contains a conservative mutation at amino acid 140 that changes leucine to isoleucine. 293T (ATCC) cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The pFUSE-hIgG1-Fc1 vector was purchased from InvivoGen Inc.

[0213] Cloning. The extracellular domain of neuropilin-1 (residues 1-856), or a portion thereof, was PCR amplified from Origene clone RC217035 using Phusion™ high-fidelity polymerase (New England Biolabs) and cloned into EcoR1-BglII of pFUSE-hIgG1-Fc1 in frame with the human FC-1 coding sequence. Constructs encoding soluble forms of trap were generated by inserting a sequence encoding a TEV protease cleavage site, followed by 6xHis residues and a stop codon, upstream of the FC coding portion of the corresponding FC construct. Further deletions (b1, b1b2) or VEGF165-binding mutations (e.g., Y297A) were introduced using the Q5 site directed mutagenesis kit (NEB). All construct sequences were verified by Sanger sequencing (Genome Quebec).

[0214] Evaluation of Trap expression in human cells. Constructs encoding mouse and human Trap were transfected into 293T cells. Cells were grown 48 hours post-transfection in FreeStyle™ 293 medium (Invitrogen). Cell lysates were prepared from 293T cells 48 hours post-transfection. Cells were washed extensively with PBS and lysed in ice-cold lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1% Triton XH-100 and 10% glycerol) supplemented with standard amounts of protease inhibitors (AEBSF, TPCK, TLCK, aprotinin, leupeptin, pepstatin, and E64, Sigma). Cell lysates were cleared by microcentrifugation (12000 g, 20 min). Lysate concentrations were determined by standard microBCA (Sigma). Equal amounts of protein were loaded onto 5–20% PAGE-SDS gradient gels and transferred to PVDF (Amersham). Clarified conditioned media from transfected cells was incubated with either Protein A Sepharose (Pharmacia) or Talon resin (Clontech) for FC or 6xHis tag. Resins were washed with PBS and diluted in 2XPAGE-SDS sample buffer before gel separation and transfer. Antibodies used for immunoblotting were anti-human neuropilin-1 (Cell signaling), mouse monoclonal anti-6X-HIS (In Vitrogen), and reporter HRP-conjugated anti-human, mouse and rabbit IgG (BioRAD). All antibodies were used at 1 / 2000 dilution. After incubation of the membrane with ECL (Amersham), chemiluminescence signals were captured using a Fuji imaging system.

[0215] Expression and purification of traps. 293-T cells were transfected with plasmids encoding various traps by either polyethylamine (PEI) or calcium phosphate precipitation standard transfection methods. The next day, cells were washed twice with serum-free medium and fed with serum-free complete medium (Free style 293 medium, InVitrogen). After 60-72 hours of growth in serum-free medium, conditioned medium was collected and cleared from cell debris by swinging bucket centrifugation (2000 RPM, 20 min). FC traps were purified from conditioned medium of transfected 293T cells by passaging on protein A or G Sepharose (Pharmacia), followed by extensive washing with PBS and elution with 0.1 M glycine (pH 3.0). The eluted fraction was immediately neutralized by adding 1 / 10 volume of 1 M Tris (pH 8) and 1 / 10 volume of 10x PBS (pH 7.4). Soluble 6XHIS-tagged TRAP was purified from conditioned medium of transfected 293T cells by passage through Talon agarose (Clontech), followed by extensive washing with PBS and stepwise imidazole elution (typically ranging from 10 to 150 μM). Samples of purified TRAP fractions were analyzed on 5-15% or 5-20% gradient PAGE-SDS gels. Gels were stained using the Safely Blue staining kit (InVitrogen).

[0216] Sterile formulation of purified traps for in vivo injection. Purified elution fractions from 40 ml of conditioned medium were pooled and diluted to a total volume of 10 ml in PBS. The diluted trap protein was sterilized by filtration through a 0.2 μM low protein binding filter (Progene). The protein solution was concentrated and buffer exchanged with PBS using a sterile PES concentrator (Pierce, nominal MWCO 30KD). Sterile concentrated trap samples (approximately 30-50 μl) were analyzed and stained by PAGE-SDS as described above.

[0217] Example 9 Trapping affinity of SEMA3A AP-VEGF 165The coding sequence of human VEGF165 variant 1 (NM_001025366) was subcloned in frame with the alkaline phosphatase domain (AP-VEGF165) into the pAPtag5 vector (GenHunter). HEK293T cells were transfected with the AP-VEGF165 construct using the polyethylenimine (PEI) transfection method. After an overnight transfection step, cells were cultured for an additional 60 h in serum-free medium (Invitrogen). The cell medium was collected and concentrated with a PES device (Pierce). The concentrated AP-VEGF165 ligand was analyzed by PAGE-SDS and quantified using SimplyBlue safe stain (Life technologies).

[0218] Sema3A and AP-VEGF 165 Binding assay. Saturation curves to determine the KD of binding to SEMA3A or VEGF165 were obtained as follows. Wells of a high protein binding 96-well plate (Maxisorp, Nunc) were coated with purified trap diluted in PBS and then blocked with binding buffer (PBS containing 2% casein and 0.05% Tween 20). SEMA3A-FC (R&D systems) or AP-VEGF165 ligand was diluted in binding buffer over a wide range of concentrations and added to the wells. After overnight incubation, the wells were washed with PBS containing 0.05% tween. Bound SEMA3a-FC was detected using HRP-conjugated anti-human IgG (Biorad) and ECL substrate (Pierce). Alternatively, bound AP-VEGF165 was detected using CPD star substrate (Roche). Chemiluminescence signals were acquired on a TECAN reader. Dissociation constants (KD) were determined by nonlinear curve fitting using Graph Pad prism software.

[0219] The relative affinity of the traps of the invention for SEMA3A and VEGF was assessed. The traps were prepared as described in Example 8. A schematic of the tested traps (without HIS or FC tags) is also shown in FIG. [Table 6]

[0220] The soluble NRP1 traps tested generally bind more efficiently to SEMA3A than to VEGF. This preference for SEMA3A was found to be surprising, since SEMA3A and VEGF are usually thought to have the same general affinity for NRP1. Applicants also surprisingly found that introducing a mutation at position 297 (Y297A) of NRP1 inhibits binding to NRP1 as well as VEGF. Such mutations, previously thought to be associated with increased affinity to SEMA3A, may be advantageous in conditions where SEMA3A inhibition is preferred over VEGF inhibition. Inhibiting VEGF with VEGF inhibitors such as bevacizumab has been suggested to induce cellular senescence in colorectal cancer cells in vitro and in vivo (Hasan et al., 2011 Int. J. Cancer 1; 129(9): 2115-2123), and using an NRP1 trap with low affinity for VEGF may be preferred in the context of aging-related diseases and conditions. Furthermore, an NRP1 trap that preferably interacts with Sema3a rather than VEGF is expected to exhibit reduced side effects associated with inhibition of VEGF cell signaling.

[0221] Example 10 Attenuation of cellular senescence by trapping NRP1 OIR mice were intravitreally injected with NRP1 Trap G or M or vehicle at P12 and the retinas were monitored for cellular senescence. As shown in Figure 20, P17 OIR flat-mounted retinas quantified by SA-β-gal staining showed significant attenuation of cellular senescence when mice received a single injection of Trap M or Trap G, with Trap M proving to be more effective at inhibiting cellular senescence. Interestingly, Trap M has a kd for SEMA3A that is approximately 20-fold greater than for VEGF, whereas Trap G has a less significant preference for SEMA3A (see Table 6).

[0222] Example 11 Experimental procedure Animals. All studies were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision Research and approved by the Animal Care Committee of the University of Montreal in agreement with the guidelines established by the Canadian Council on Animal Care. C57Bl / 6 wild type (WT) mice were purchased from The Jackson Laboratory. LyzM-Cre (Lyz2 tm1(cre)Iflo / J ; no. 004781) were purchased from The Jackson Laboratory. (C57Bl / 6WT, LysM-Cre, and LysM-Cre / ROSA26EYFP) fl / f , we generated mice carrying EYFP-expressing cells of the myeloid lineage ( 71 ).

[0223] O2-induced retinopathy in different strains (C57Bl / 6WT, LyzM-Cre, LysM-Cre / IRE1 fl / fl , LysM-Cre / IRE1 + / + , IRE1 fl / fland LysM-Cre / ROSA26EYFP fl / fl ) and their foster mothers (CD1, Charles River) were maintained in 75% O2 from postnatal day 7 (P7) to day 12. 2 The mice were exposed to hypoxia and then returned to room air. This model serves as a surrogate for human ocular neovascular diseases such as ROP and diabetic retinopathy, which are characterized by a late stage of destructive pathological angiogenesis (72, 73). Upon return to room air, hypoxia-driven neovascularization (NV) occurs from P14 onwards (27). We enucleated the eyes at different time points and dissected the retinas for mRNA, protein assays or flat mounting.

[0224] RNA-Seq sample preparation and sequencing. Total RNA was isolated from the retina using RNeasy Mini Kit (QIAGEN). mRNA was then purified from 1 μg of total RNA using Dynabeads® mRNA DIRECT™ Micro Kit (Thermo Fisher SCIENTIFIC). Whole transcriptome libraries were prepared using Ion Total RNA-seq Kit v2. Amplified library yield and size distribution were assessed on an Agilent bioanalyzer using a DNA1000 kit. Sequencing was performed on an Ion Chef™ Instrument (Ion Torrent™, Thermo Fisher SCIENTIFIC).

[0225] cDNA library construction and sequencing. Analysis was performed using Torrent Suite software v4.4 (Thermo Fisher) and the Whole Transcriptome Analysis Plugin v 4.2-r7 (Thermo Fisher). The Whole Transcriptome Analysis Plugin aligns reads to the mouse reference genome (mm10) using Tophat2, then unmapped reads are aligned using Bowtie2 and merged together. FPKM is calculated using Cufflink.

[0226] Gene set enrichment analysis (GSEA). Gene set enrichment analysis was performed using GSEA v2.2.1 software provided by the Broad Institute of MIT and Harvard University. GSEA was used to validate correlations between molecular signatures in phenotypes of interest. Enrichment analysis was performed using log2-normalized FPKM (Fragment Per Kilobase of transcript per Million) data generated by the ToPhat / Cuffdiff command pipeline. FPKM values ​​were transformed as ratios (FPKMx / [FPKM normoxia]mean), then log2-normalized (log2[ratio]) and median-centered (log2ratio-[log2ratio normoxia]mean).

[0227] Default parameters were changed as follows: Gene sets of interest were found in the catalogue of functionally annotated gene sets from the Molecular Signature Database (MSigDB). Phenotypes were permuted 1000 times. Phenotype labels were "OIR" vs. "normoxia". Gene sets smaller than 15 and larger than 500 were excluded from the analysis. The statistic used to score hits was "weighted p2" and the class separation metric used was "t-test".

[0228] Semi-quantitative and real-time PCR analysis. RNA was isolated using the GenElute™ Mammalian Total RNA Miniprep Kit (Sigma) and digested with DNase I to prevent amplification of genomic DNA. RNA was reverse transcribed using M-MLV reverse transcriptase and gene expression was analyzed using SybrGreen™ on an ABI Biosystems Real-Time PCR instrument. β-actin was used as a reference gene. Primer sequences are listed in Table 7. XBP-1 splicing was examined by incubating XBP-1 semi-quantitative PCR products with 0.4 U / μL PstI enzyme for 5 h at 37° C., followed by separation on a 2.5% agarose gel. [Table 7]

[0229] Flow cytometry analysis. Human retinal microvascular endothelial cell (HRMEC) cell cycle analysis (PI biolegend) was performed according to the manufacturer's instructions and as previously reported (43). Briefly, HRMECs (1x10 6 ) were seeded into 6-well plates and incubated with 100 ng / ml and 500 ng / ml SEMA3A for 7 days. Samples were analyzed by flow cytometry. FACS was performed on an LSRII (BD Biosciences) instrument and data were analyzed using FlowJo software (version 7.6.5).

[0230] Electrical cell-substrate impedance sensing (ECIS) proliferation assay. Real-time analysis of transendothelial electrical resistance was performed by seeding 5000 HRMEC / ml into 8W10E+ standard 8-well arrays (Applied BioPhysics, NY). Cells were grown to a capacitance of less than 10 nF. Cells were starved for 5 h with endothelial basal medium (EBM-2, Lonza) and then treated with 100 ng / ml SEMA3A or vehicle (EBM-2) for 120 h and impedance was measured using an ECIS Zθ impedance instrument (Applied BioPhysics, NY). Measurements were taken 120 h after treatment.

[0231] Human samples. Approval of the human clinical protocol and informed consent form by the Maisonneuve-Rosemont Hospital (HMR) Ethics Committee (Ref. CER:10059) and recruitment of patients for local core vitreous biopsy sampling from patients affected by proliferative retinopathy were received. The entire procedure was performed as an outpatient procedure in a small treatment room within the outpatient clinic of the Ophthalmology Department of Maisonneuve-Rosemont Hospital. All instruments were unpacked and handled in a sterile fashion. The study complies with the principles of the Declaration of Helsinki.

[0232] Vitrectomy. All patients previously diagnosed with PDR were followed and operated on by a single vitreoretinal surgeon (FAR). Control patients were surgically treated for nonvascular pathology (ERM or MH) by the same surgeon. In an operating room setting, patients were operated on under topical retroorbital / periorbital anesthesia. Periocular skin was cleansed using 5% povidone-iodine solution, and topical instillation into the eye and into the cul-de-sac was left in place for 5 minutes. A three-port 25-gauge transciliary pars plana vitrectomy was performed with a 25-gauge valved cannula (Alcon). Undiluted vitreous was collected with a 25-gauge vitrectomy device (vitrector) under microscopic visualization using a wide-angle viewing system (Resight, Zeiss). After vitreous biopsy, the infusion line was opened, and vitrectomy and membrane peeling were performed in the usual fashion to treat diabetic vitreous hemorrhage and tractional retinal detachment. This was followed by panretinal intraocular laser photocoagulation, fluid-air exchange, and intravitreal anti-VEGF injections.

[0233] Multiplex cytokine quantification. Vitreous samples were frozen on dry ice and stored at -80°C immediately after biopsy. Prior to analysis, vitreous samples were centrifuged at 15000xg for 5 min at 4°C. Pai1, VEGF, IL-6, IL-8. The multiplex panel (Cancer Panel 1 (Bio-rad)) was used according to the manufacturer's protocol. Luminex assays were analyzed using a Bio-Plex 200 array reader (Bio-rad). Quantitative measurement of each analyte was achieved by comparing the raw data obtained from patient samples to standard curves. A total of four cytokines (Croa, Grob and IL-1β) had to be excluded due to detection limits.

[0234] Immunofluorescence (IF). To localize protein expression, eyes were enucleated from mice, fixed in 4% paraformaldehyde for 4 h at room temperature, incubated overnight in 30% sucrose, and then frozen in OCT compound. Whole eyes were then embedded in optimal cutting temperature compound at -20 °C and 12 μm sections were made. IF experiments and visualization of sections were performed using an epifluorescence microscope (Zeiss AxioImager) or a confocal microscope (Olympus confocal FV1000).

[0235] To visualize the pan-retinal vasculature, dissected retinas were flat-mounted and stained with 1 mM CaCl in PBS for the retinal vasculature. 2 The extent of avascular or neovascularized areas at P17 was determined using ImageJ and the SWIFT-NV method ( 74 ).

[0236] For protein localization, flat-mounted retinas were incubated with different antibodies as indicated. For in vitro IF, cultured cells were plated on 0.1% gelatin-coated coverslips, serum-starved overnight, and stimulated with SEMA3A (100 ng / ml) for 7 days. Cells were washed briefly with cold PBS and fixed for 20 min in 3.5% paraformaldehyde in PBS. Cells were rinsed with PBS and permeabilized with 0.3% Triton in PBS for 5 min. Fixed cells were blocked with 1% BSA and then incubated with primary antibodies in 0.1% BSA in PBS for 1 h. Bound primary antibodies were visualized after 1 h incubation with Alexa Fluor secondary antibodies. Coverslips were mounted with Fluoromount (Sigma-Aldrich) and analyzed by confocal microscopy (Olympus confocal FV1000). Samples were viewed with a x63 / 1.4NA oil or 30x objective. Images were assembled using Photoshop CS4 (Adobe Systems). For all antibodies used for immunohistochemistry, see Table 8. [Table 8] Senescence-associated β-galactosidase (SA-β-gal) assay. Senescence-associated β-galactosidase assays were performed as previously described (57, 75).

[0237] Quantification of SA-β-gal in vivo. Senescence-associated β-galactosidase staining in flat-mounted retinas or sagittal sections of the eyes was analyzed using Image J software as described in FIG.

[0238] Production of lentivirus. Lentivirus (HIV-derived) was prepared by transfecting HEK293T cells (Invitrogen) with vector plasmids containing Cre, green fluorescent protein GFP, or small hairpin RNA (Sh_RNA) for SEMA3A, IRE1α, or GFP (see Table 9 below) together with third generation packaging plasmids pV-SVG, pMDL, and pREV (Open Biosystems) as previously described by us and others (35, 44, 76). Lentivirus was incubated at 37°C for 1 h in DMEM complete medium (Invitrogen) for 30 min at 4°C for 1 h. 7 Cells were seeded, transfected with the above plasmids, and incubated for 30 h. The supernatant was then replaced with fresh complete DMEM medium and incubated for another 30 h. Secreted viruses were harvested, ultracentrifuged at 50,000 g, resuspended in PBS, aliquoted, and stored at -80°C.

[0239] Intravitreal injection. P2, P10 or P12 C57BL / 6 pups were anesthetized with 3.0% isoflurane and injected with 0.5 μL of lentivirus (see "Lentivirus Production"), recombinant SEMA3A (100 ng / μL), metformin (10 μg / μL), or aflibercept (10 μg / μL) into the vitreous cavity using a 10 μL Hamilton syringe fitted with a 50-gauge glass capillary tip. Approximately 254 ± 11.0 ng / μL of lentivirus Sh_GFP and 323.3 ± 15.3 ng / μL containing Sh_Sema3a, Lv.Cre (15.0 ng / mL), and Lv.GFP (15.0 ng / mL) were injected. Viral titers were assessed using a p24 ELISA kit (ZeptoMetrix). The titers of the lentiviruses used (in ng p24) were: LV.Sh_RNAIRE1α (8.52 ng / mL), and LV.Sh_RNA.GFP (8.47 ng / mL). [Table 9]

[0240] Preparation of conditioned medium (CM). Human retinal microvascular endothelial cells (HRMEC), retinal neuronal 661W photoreceptor cells and mouse macrophages (J774A.1 cell line) were conditioned for 48 h after transfection or as indicated in each experiment with recombinant SEMA3A (100 ng / μL), H 2 O 2 (150 μM, 2 h) for 7 days. The supernatant was centrifuged, filtered, and then frozen for subsequent use. For Western blots, the cells were concentrated with CM using ultracentrifugal Amicon filter units (Millipore).

[0241] Western blotting. We enucleated the eyes at various time points and rapidly dissected and homogenized the retinas to assess retinal protein levels. Protein concentrations from retinal homogenates, cell lysates were assessed by BCA assay (Sigma), and then 30 μg of protein was analyzed for each condition by standard SDS-PAGE method. Antibodies used for Western blotting are listed in Table 8 above.

[0242] Statistical analysis. Student's T-test and ANOVA were used where appropriate. AP<0.005 and P<0.05, respectively, were considered statistically different using Prism, version 5 software (GraphPad).

[0243] Recombinant proteins used: Recombinant human semaphorin 3A (mouse myeloma cell line, NS0) (R&D Systems) concentrations of 100 ng / ml and 500 ng / ml were used in vitro and 100 ng / ml in vivo.

[0244] Materials. Metformin, assay (RIPA) buffer, protease inhibitor cocktail, and phosphatase inhibitors were purchased from Sigma Chemicals. Aflibercept (Eylea™) was purchased from Bayer. 4μ8c inhibitor was obtained from Torcis (Biosciences).

[0245] Plasmids and generation of stable cell lines and transfection. 661W cells and HRMEC (Open Biosystems) cells were stably transfected with 500 ng of Sh_RNA plasmid targeting, Sema3a, IRE1α and unrelated sh_RNA (sh_GFP) respectively using Lipofectamine™ 2000 for 16 hours at 37°C according to the manufacturer's instructions. We generated stable cell lines by selecting with 2 mg of puromycin for 2 weeks. Expression plasmids of GFP, IRE1αWT, dominant negative mutant of IRE1α, RNase dead mutant K907A in J774 cells using Lipofectamine™ 2000. Plasmids for IRE1α were obtained from Addgene (Fumihiko Urano: plasmids #20744 and #20745).

[0246] Example 12 Experimental Procedures for Examples 13-15 Mice. All studies were performed in accordance with the Canadian Council on Animal Care guidelines and approved by the Animal Care Committee of the University of Montreal. C57Bl / 6 wild-type mice, LysM-Cre mice (B6.129P2-Lyz2 tm1(cre)Ifo / J; No. 004781), and neuropilin-1 floxed mice (B6.129(SJL)-NRP1 tm2Ddg / J; No. 005247) were purchased from The Jackson Laboratory and raised in a house. Diet: HFD: 60% fat calories, BioServ F3282. Control diet: 2018 Teklad Global 18% protein rodent diet.

[0247] Fluorescence-activated cell sorting (FACS) of adipose tissue macrophages. Retroperitoneal fat pads were collected, weighed, homogenized in DMEM F12 medium, and then incubated with 1 mg / mL collagenase D (Sigma) for 45 min at 37 °C. EDTA was then added at a concentration of 10 mM, and the mixture was incubated for an additional 5 min. The homogenate was then filtered through a 70 μm cell strainer and centrifuged. The pellet was resuspended and lysed in lysis buffer (10 mM KCHO). 3 ;150mM;NH 4The cells were incubated in 0.1 mM EDTA (Cl; 0.1 mM EDTA) for 5 min at room temperature and centrifuged. The pellet was resuspended in 1xPBS and filtered through a 100 μm cell strainer. The cell suspension was incubated with Zombie Aqua Fixable Viability Kit (BioLegend) for 15 min at room temperature. The cells were then incubated with LEAF purified anti-mouse CD16 / 32 (Biolegend) for 15 min at room temperature to block Fc receptors. The cells were then incubated with the following antibodies for 25 min at 4° C.: Brilliant Violet 785 anti-mouse CD45.2 (BioLegend), Brilliant Violet 711 anti-mouse / human CD11b (BioLegend), APC / CY7 anti-mouse Ly-6G (BioLegend), Pe / Cy7 anti-mouse F4 / 80 (BioLegend), PE anti-mouse CD11c (BioLegend), FITC anti-mouse Ly-6C (BioLegend) and APC anti-mouse CD304 (Neuropilin-1) or APC rat IgG2a, kappa isotype control (BioLegend). To analyze CD206 expression, permeabilization and fixation of the cells was performed with Cytofix / Cytoperm kit (BD Bioscience) for 20 min at 4° C. The cells were then incubated with rat serum (Cedarlane) for 25 min at 4° C. to block intracellular receptors. Cells were finally stained with Brilliant Violet 421 anti-mouse CD206 (MMR) (BioLegend) for 25 min at 4° C. FACS was performed on a Fortessa (BD Biosciences) instrument and data were analyzed using FlowJo software (version 7.6.5).

[0248] In vivo BODIPY uptake. In vivo BODIPY uptake assays were performed on LysM-Cre-NRP1 cells fed a HFD for 10 weeks. + / + and LysM-Cre-NRP1 fl / flThe study was performed on male mice. Mice were starved for 4 hours, followed by intraperitoneal injection of 100 μL of 30 μM BODIPY™ 500 / 510C1, C12 in 1% BSA. Three hours after BODIPY™ injection, mice were euthanized. Blood was collected by cardiac puncture, and plasma was then separated by centrifugation. Heart, liver and white adipose tissue samples were collected and homogenized in 1xRIPA buffer (Cell Signaling). BODIPY™ fluorescence of homogenates and plasma was read at a wavelength emission of 488 nm and excitation of 525 nm using an Infinite M1000 Pro reader (Tecan) and normalized to protein concentration (quantified using the QuantiPro™ BCA assay kit (Sigma)).

[0249] Primary macrophage cultures. 8–12 week old LysM-Cre-NRP1 + / + and LysM-Cre-NRP1 fl / fl Mice were anesthetized with 2% isoflurane in oxygen at 2 L / min and then euthanized by cervical dislocation. After that, a small incision was made in the abdominal skin of the mice. The skin was pulled out for each size mouse, and the abdominal cavity was washed with 5 ml of PBS + 3% FBS for 2 min. The harvested cells were then centrifuged at 1000 rpm for 5 min, resuspended in culture medium (DMEM F12 + 10% FBS and 1% streptomycin / penicillin) and plated. Incubated at 37 °C, 5% CO 2 After 1 h of incubation under ambient atmosphere, the medium was replaced and the cells were cultured under the same conditions for the next 24 h before being used for BODIPY uptake, pHrodo phagocytosis assay, or Oil Red-O staining.

[0250] Quantitative RT-PCR (qPCR) analysis. RNA extraction was performed using 100–500 mg of frozen (-80 °C) RP-WAT according to the Trizol reagent protocol (Invitrogen). Total RNA (1 μg) was reverse transcribed (iScript cDNA synthesis kit, Bio-Rad) according to the manufacturer's instructions. qPCR was performed (7500 Real-Time PCR System, Applied Biosystem) using SYBR Green (Bio-Rad) and 40 ng of cDNA per reaction. Expression levels were normalized to the expression of β-actin. Primer (Integrated DNA Technologies) sequences are listed below: [Table 10]

[0251] ImmGen skyline dataset. Immunological Genome Project data phase 1 (GEO accession code GSE15907) and phase 2 (GSE37448) were extracted and normalized in R by robust multiarray averaging (RMA) and antiLog values ​​were plotted.

[0252] Immunohistochemistry (IHC). RPWAT tissues were fixed in 4% PFA for 48 hours, then incubated in 20% methanol for 10 minutes and rinsed with PBS. After blocking in 3% BSA (Hyclone, GE) + 0.3% Triton™ X-100 (Sigma) for 1 hour, they were incubated with rhodamine-labeled Bandeiraea simplicifolia Receptin I (Vector Laboratories Inc.), anti-rat F4 / 80 (donkey IgG; eBioscience), anti-rabbit perilipin (donkey IgG; Abcam), and anti-rat neuropilin-1 antibodies (donkey IgG; R&D) at 4°C overnight. Alexa-Fluor secondary antibodies were incubated for 2 hours at 20°C. RPWAT was then mounted on a microscope slide and images were taken by confocal microscopy.

[0253] Macrophage BODIPY uptake. LysMCRE-NRP1 + / + and LysMCRE-NRP1 fl / fl 1x10 macrophages extracted from 5 Cells / well were seeded in 48-well plates. BODIPY500 / 510C1, C12 (Life technologies) were added at concentrations of 0.5 and 1 μg / ml and incubated at room temperature for 5 minutes, then placed on ice. Wells were washed with cold PBS and then fixed with 1% paraformaldehyde (Electron Microscopy Science). Fluorescence was read using an Infinite M1000 Pro reader (Tecan) at wavelengths emission 488 nm and excitation 525 nm. Cells were then stained with DAPI (Life Technologies) at a concentration of 1 / 20000 and fluorescence was measured at excitation 358 nm and emission 461 nm.

[0254] pHrodo phagocytosis assay. LysMCRE-NRP1 + / + and LysMCRE-NRP1 fl / fl Macrophages extracted from 1x10 cells were placed in a 96-well plate. 5 Cells were seeded at 1000 x g / well. pHrodo® Green Zymosan Bioparticles Conjugate® (Life Technologies) was resuspended in FluoroBrite™ DMEM medium + 10% FBS + 1% PenStrep at a concentration of 0.5% / mL. 100 μL of the bioparticle resuspension was added to the cells and to empty wells as a negative control. Cells were incubated at 37° C. for 90 min and pH / phagocytosis-dependent fluorescence was detected on a TECAN plate reader at excitation 509 nm and emission 533 nm. Net phagocytosis was calculated by subtracting the fluorescence of the negative control from the fluorescence of the experimental samples.

[0255] Oil Red O staining and quantification. Cultured adipocytes and peritoneal macrophages were washed with PBS, fixed in 10% PFA for 30 min, and rinsed. Cells were then incubated with twice-filtered 0.3% Oil Red O solution for 60 min and rinsed. Photographs were taken under a light microscope at a magnification of 10x for adipocytes and 63x for macrophages. Quantification of lipid droplets was performed using the Limit of threshold method from ImageJ.

[0256] Weight gain in the presence of the AdenoTrapM protocol. 6-8 week old C57B16 / J mice were divided into six groups (normal diet + saline, normal diet + AdenoTrapM, normal diet + AdenoGFP, high fat diet + saline, high fat diet + AdenoTrapM, high fat diet + AdenoGFP). Mice were injected intravenously (tail vein) with saline, AdenoTrapM or AdenoGFP (0.25x10 10 PFU / injection). Half of these mice were fed a high-fat diet and the other half were fed a normal diet and weighed at weekly intervals.

[0257] A drop of blood was taken from the tail at 2 and 8 weeks post-injection, and the presence of Trap M was assessed in the blood by immunoprecipitation with anti-His antibody (see Figures 26, 28 and 29).

[0258] Glucose tolerance test (GTT). Six to eight week old C57B16 / J mice were intravenously injected with saline or Adeno-Trap M (0.25x10 10 PFU / injection). Mice were fed a high fat diet immediately after injection. Glycemia was assessed at baseline and 15, 30, 60, 120, and 240 min after intraperitoneal injection of 2 g D-glucose / kg. Recorded measurements are shown in Figure 25 (N=5, NS means not significant by two-way anova Bonferroni post-hoc test).

[0259] Insulin Tolerance Test (ITT). Mice were fasted for 5.5 h (morning). Blood glucose was measured at baseline, 30, 60 and 120 min after intraperitoneal injection of 0.75 U / kg insulin.

[0260] In vivo BODIPY™ uptake. The in vivo BODIPY™ uptake assay was performed using LysM-Cre-NRP1 + / + and LysM-Cre-NRP1 fl / fl The study was performed in male mice fed a HFD for 10 weeks. Mice were starved for 4 hours, followed by intraperitoneal injection of 100 μL of 30 μM BODIPY™ 500 / 510C1, C12 (Life technologies) in 1% BSA (Hyclone, GE). Mice were euthanized 3 hours after BODIPY injection. Blood was collected by cardiac puncture, and plasma was then separated by centrifugation. Heart, liver and white adipose tissue samples were collected and homogenized in 1x RIPA buffer (Cell Signaling). BODIPY fluorescence of homogenates and plasma was read at a wavelength emission of 488 nm and excitation of 525 nm using an Infinite M1000 Pro reader (TECAN) and normalized to protein concentration (quantified using the QuantiPro™ BCA assay kit (Sigma)).

[0261] Oil Red O staining and quantification. Cultured adipocytes and peritoneal macrophages were washed with PBS, fixed in 10% PFA for 30 min, and rinsed. Cells were then incubated with twice-filtered 0.3% Oil Red O (Sigma) solution for 60 min and rinsed. Photographs were taken under a light microscope at a magnification of 10x for adipocytes and 63x for macrophages. Quantification of lipid droplets was performed using the Limit of threshold method from ImageJ.

[0262] Adenovirus production. Traps AD and AE were derived from traps M and O (described in WO 2016 / 033699, supra) by introducing the VEGF165-binding mutant residue D320K using the Q5 site directed mutagenesis kit (New England Biolabs). Adenovirus trap constructs were generated by first subcloning the coding sequences of traps M, G, A and D into the EcoRI-EcoRV sites of pENTR1A (Life technologies), followed by LR clonase homologous recombination in the destination vector pAd / CMV / V5-DEST (Life technologies). The current set of constructs is referred to as pAdeno-TrapA, C, G or M and pAdeno-GFP. All construct insert sequences were verified by Sanger sequencing (Genome Quebec). All junction regions generated after recombination of trap coding sequences into pAD / CMV / V5-dest were sequenced as well.

[0263] Statistical analysis. Data are expressed as mean ± SEM. Different groups were compared using two-tailed Student's t-test and ANOVA, where appropriate. P<0.05 was considered statistically different.

[0264] Example 13 NRP1-expressing macrophages accumulate in adipose tissue during diet-induced obesity During diet-induced obesity (DIO), necrotic adipocytes release fatty acids (FAs) that are partially taken up by surrounding macrophages forming coronary structures. Given the importance of macrophages in lipid metabolism and obesity, the expression profile of NRP1 in myeloid cells was analyzed using data from the immunological consortium ImmGen (Heng and Painter, 2008). NRP1 expression was most robust in adipose tissue macrophages (ATMs) compared to other steady-state tissue-resident macrophages, monocytes and neutrophils (Figure 21A). This data indicated a potential role for NRP1+ macrophages in adipose tissue homeostasis.

[0265] Therefore, C57BL / 6 mice were placed on a high-fat diet (HFD; 60% fat calories) for 10 weeks starting at 8 weeks of age, and ATM populations were examined by fluorescence-activated cell sorting (FACS). In accordance with other studies, an increased presence of ATMs was detected in adipose tissue of HFD-fed mice when compared to age-matched controls on a normal diet (RD; 18% fat calories) (Figure 21B). This was paralleled by a proportional increase in NRP1+ATMs (Figure 21C). Immunohistochemistry (IHC) of retroperitoneal white adipose tissue (RPWAT) from both 10-week-old HFD-fed and age-matched RD mice confirmed robust expression of NRP1 in macrophages and blood vessels (data not shown). After 22 weeks of HFD, NRP1 was localized to coronary structures, which corresponded to clusters of phagocytic macrophages surrounding dying and dead adipocytes (data not shown). Of the NRP1 ligands examined, only transforming growth factor beta 1 (Tgfb1) was significantly elevated in retroperitoneal white adipose tissue (RPWAT) of HFD-fed mice (Figure 21G), whereas semaphorin 3A (Sema3a), vascular endothelial growth factor-A (Vegfa), or -B (Vegfb) were unaffected (Figure 21D-F). Collectively, these data demonstrate robust expression of NRP1 in ATMs and suggest an increase in NRP1+ macrophages in adipose tissue during HFD-induced weight gain.

[0266] Example 14 NRP1 promotes the uptake and phagocytosis of fatty acids by macrophages. In obesity, long-chain fatty acid (FA) uptake is upregulated in adipocytes (Berk et al., 1999; Petrescu et al., 2005). NRP1 in adipose tissue homeostasis and weight gain + To elucidate the role of macrophages, we used LysM-CRE-NRP1 to specifically ablate NRP1 in cells of the myeloid lineage. fl / fl We generated a mouse line (Dejda et al., 2014) that is able to uptake long-chain FA analogs (C1-BODIPY-C12, an 18-carbon FA) by the LysM-Cre-NRP1 fl / fl and control LysM-Cre-NRP1 + / + In macrophages, NRP1-deficient macrophages ingested significantly less FAs than control macrophages during acute exposure (Figure 22A). In addition, systemic administration of C1-C12 BODIPY reduced the uptake of LysM-Cre-NRP1 when compared to plasma and heart (Figure 22D, E). fl / fl We found significantly elevated levels of tagged FAs in mouse RPWAT and liver (Fig. 22B and Fig. 22C), which implicates NRP1 in lipid uptake. + The role of macrophages is solidified.

[0267] To determine whether NRP1 affected lipid sequestration in macrophages, neutral lipids in macrophages were stained with Oil Red O staining, which was consistent with the results of staining LysM-Cre-NRP1 cells incubated in adipocyte-conditioned medium. fl / flIn macrophages, neutral lipids were significantly reduced (Figures 22F-G). Because adipocyte and macrophage media differ in glucose and insulin concentrations, we assessed whether the reduction in internalized lipids in NRP1-deficient macrophages also occurred in adipocyte media with or without insulin, as well as in non-conditioned media such as macrophage media. In all conditions, NRP1 macrophages sequestered significantly less neutral lipids than controls (Figures 22H-K).

[0268] As adipocyte death increases in obese mice and humans, macrophages are attracted to the necrotic site to phagocytose cellular debris and sequester released lipids (Cinti et al., 2005). Having observed a decrease in lipid uptake in NRP1-deficient macrophages, we wondered whether their phagocytic capacity was also impaired. Phagocytosis was enhanced by the LysM-Cre-NRP1 fl / fl and pHrodo Green Zymosan Bioparticle Conjugate in control macrophages and found that macrophages lacking NRP1 had reduced phagocytic capacity (Figure 23).

[0269] Taken together, the above results demonstrate that NRP1-deficient macrophages have impaired FA uptake and phagocytic capacity.

[0270] Example 15 NRP1 trap reduces weight gain associated with high-fat diet The effect of NRP1 trap on weight gain was assessed. Adenovirus expressing soluble NRP1 trap containing domains a1, a2 and b1 of NRP1 (TrapM, see Table 2); Adeno-GFP; or saline (control) was administered to male mice while they were switched from a normal diet to a high-fat diet (HFD, T0). Weight gain was monitored over a period of 10 weeks. Data are presented as mean ± SEM. Student's unpaired t-test, *p<0.05, **p<0.01, saline vs. Adeno-TrapM, two-way anova, Bonferroni post-hoc test, N=5.

[0271] As shown in Figure 24, administration of NRP1 prevented weight gain in mice. The increase in weight gain observed at weeks 7 and 8 is consistent with the reduction in circulating adenovirus expressing NRP1 trap. Surprisingly, the prevention of weight gain was more significant in male mice than in female mice (data not shown).

[0272] The effect of NRP1-Trap on glucose tolerance was also assessed. 6-8 week-old C57B16 / J mice were intravenously injected with saline, Adeno-GFP or Adeno-Trap-M and fed a high-fat diet immediately after injection. Glycemia was assessed at different time points after intraperitoneal injection of 2 g glucose / kg mouse. As shown in Figure 25B, mice treated with Adeno-Trap-M had a higher tolerance to glucose than mice treated with saline or Adeno-GFP. [Table 11]

[0273] The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the entire description. References [Table 12] TIFF2025061122000017.tif223160TIFF2025061122000018.tif226160TIFF2025061122000019.tif232160TIFF2025061122000020.tif113160

Claims

1. A composition for treating (i) a disease or condition associated with premature cellular aging, and / or (ii) a metabolic disease or condition in a subject, comprising a SEMA3A antagonist and a carrier, wherein the SEMA3A antagonist is (a) a soluble NRP1 polypeptide that binds to SEMA3A and has an amino acid sequence having at least 95% identity to the sequence of SEQ ID NO: 82, or (b) an anti-SEMA3A antibody or an antigen-binding fragment thereof.

2. The composition of claim 1 , wherein the SEMA3A antagonist is a soluble NRP1 polypeptide comprising an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO:

82.

3. The composition of claim 2, wherein the soluble NRP1 polypeptide comprises the amino acid sequence of SEQ ID NO:

82.

4. The composition of claim 2 or 3, wherein the soluble NRP1 polypeptide is fused to a fragment crystallizable (Fc) domain.

5. The composition of claim 4 , wherein the Fc domain is fused to the amino terminus of the soluble NRP1 polypeptide.

6. The composition of claim 4 or 5, further comprising a linker between the soluble NRP1 polypeptide and the Fc domain.

7. The composition of claim 1 , wherein the SEMA3A antagonist is an anti-SEMA3A antibody or an antigen-binding fragment thereof.

8. 8. The composition of any one of claims 1 to 7, wherein the disease or condition associated with premature cellular aging is neurodegeneration, sarcopenia, thinning of the epidermis, skin wrinkling, hair loss, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), atherosclerosis, osteoarthritis, osteoporosis, Parkinson's disease, intestinal disease, glaucoma, intervertebral disc degeneration, cerebral aneurysm, aortic aneurysm, pancreatic fibrosis, or cystic fibrosis.

9. The composition of any one of claims 1 to 7, wherein the metabolic disease or condition is obesity, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), or diabetes.

10. The composition of any one of claims 1 to 9, wherein the carrier is a pharma- ceutically acceptable carrier suitable for systemic administration.

11. The composition of any one of claims 1 to 9, wherein the carrier is a pharma- ceutically acceptable carrier suitable for topical ocular administration.

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