Production and use of ENAMPT contained in extracellular vesicles

Abstract

The present invention relates to various compositions comprising NAMPT and / or variants thereof, processes for preparing these compositions, and various methods of using these compositions to prevent or treat age-related conditions in a subject. The present invention also relates to methods for increasing NMN and / or NAD+ biosynthesis in cells.

Description

Technical Field

[0001] Statement regarding federally sponsored research or development This invention was made with government support under grants AG037457 and AG047902 awarded by the National Institutes of Health. The government has certain rights in this invention. The present invention relates to various compositions comprising NAMPT and / or its variants, processes for preparing these compositions, and various methods of using these compositions for preventing or treating age-related conditions in a subject. The present invention also relates to methods of increasing NMN and / or NAD+ biosynthesis in a subject or cell.

Background Art

[0002] Aging is a major risk factor for tissue dysfunction and chronic diseases. In recent years, nicotinamide adenine dinucleotide (NAD + ) metabolism has emerged as a central topic in the fields of aging and lifespan research due to the apparent age-related decline in systemic NAD + availability across many species (Canto et al., 2015, Rajman et al., 2018, Verdin, 2015, Yoshino et al., 2018). It is now established that NAD + availability declines with age at the whole-body level and causes various age-related pathophysiological changes in diverse model organisms. In mammals, the age-related decline in NAD + availability appears to be caused by two major events: a decrease in NAD+ biosynthesis and an increase in NAD + consumption (Imai, 2016, Imai and Guarente, 2014). The former can be caused by chronic inflammation associated with increased oxidative stress and / or increased inflammatory cytokines, while the latter can be caused by increased DNA damage. As a result, in multiple tissues including adipose tissue, skeletal muscle, liver, pancreas, skin, neurosensory retina, and brain, NAD +Levels decline with age (Canto et al., 2015, Lin et al., 2018, Rajman et al., 2018, Verdin, 2015, Yoshino et al., 2018). Such systemic NAD decline as a fundamental event in age-related pathophysiology + is currently providing strong evidence for developing effective anti-aging interventions using major NAD + intermediates such as nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) (Rajman et al., 2018, Yoshino et al., 2018). Indeed, many studies have already demonstrated the efficacy of NMN and NR in alleviating age-related functional decline and treating age-related disease states in various mouse models (Rajman et al., 2018, Yoshino et al., 2018).

[0003] In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the major NAD + biosynthetic pathway and converts nicotinamide and 5'-phosphoribosyl-pyrophosphate (PRPP) to NMN (Garten et al., 2015, Imai, 2009). Interestingly, mammals have two different forms of NAMPT, intracellular and extracellular NAMPT (iNAMPT and eNAMPT, respectively) (Revollo et al., 2007). Important NAD +The function of iNAMPT as a biosynthetic enzyme is fully established, but the physiological relevance and function of eNAMPT have long been a subject of debate. eNAMPT was previously identified as pre-B cell colony-enhancing factor (PBEF) and insulin-mimetic visfatin, neither of which has been reconfirmed to date (Fukuhara et al., 2007, Garten et al., 2015, Imai, 2009, Samal et al., 1994). In addition, eNAMPT has also been reported to function as a pro-inflammatory cytokine, but this specific function has not yet been confirmed in loss-of-function or gain-of-function Nampt mutants (Dahl et al., 2012). Previously, the physiological relevance of eNAMPT in vivo was demonstrated by adipose tissue-specific genetic manipulation of Nampt (Yoon et al., 201 Yoon et al., title “SIRT1-Mediated eNAMPT Secretion from Adipose Tissue Regulates Hypothalamic NAD(+) and Function in Mice,” (2015) Cell Metab 21, 706-717). Adipose tissue-specific Nampt knockout (ANKO) mice, especially females, show a significant decrease in circulating eNAMPT levels. Surprisingly, ANKO mice exhibit a significant reduction in NAD + levels not only in adipose tissue but also in other remote tissues such as the hypothalamus (Yoon et al., 2015). Subsequent intensive investigations revealed a novel function of eNAMPT that enhances NAD + , SIRT1 activity, and neuronal activation in the hypothalamus in response to fasting. These findings suggest the existence of a novel inter-tissue interaction system between adipose tissue and the hypothalamus mediated by eNAMPT (Imai, 2016).

[0004] However, whether eNAMPT affects NAD in the hypothalamus +It remains difficult to understand how to precisely adjust the levels. Furthermore, there is currently no method to effectively deliver eNAMPT in vitro or in vivo, or to apply it to alleviate any disease or condition. [Overview of the project]

[0005] The present invention relates to various compositions comprising lipids and NAMPT and / or its variants, processes for preparing these compositions, and various methods for using these compositions to prevent or treat age-related conditions in a subject. The present invention also relates to methods for increasing NMN and / or NAD+ biosynthesis in a subject or cells.

[0006] Other subjects and characteristics are partially evident and partially noted below. [Brief explanation of the drawing]

[0007] [Figure 1A] plasma eNAMPT levels in 6-month and 18-month-old female and male mice (n=5). [Figure 1B] Plasma eNAMPT concentrations in 6-month and 18-month-old female and male mice (n=5 per time point for each age) over a 24-hour period. Differences between plasma eNAMPT levels at 6 months and 18 months of age were assessed by two-way repeated measures ANOVA, and the effect of age was significant in both males and females (p<0.05). [Figure 1C] Plasma eNAMPT levels in mice and humans (n=9 for mice, n=13 for humans) across different age groups. [Figure 1D] Relationship between plasma eNAMPT levels and the remaining lifespan of individual mice (n=8). eNAMPT levels were measured at 26–28 months of age. [Figure 2A] Plasma eNAMPT levels in 4-month-old controls (left bar) and ANKI mice (right bar) (n=3-4 per group, sex-specific). [Figure 2B]Plasma eNAMPT levels in 24-month-old controls (left bar) and ANKI mice (right bar) (n=3-4 per group, sex-specific). [Figure 2C] Tissue NAD+ levels in 20-month-old control and ANKI mice (n=3 per group, sex-specific). [Figure 3A] Circle running activity of 4-month and 18-month-old control (CTRL) and ANKI female mice (per group, 4 months old, n=3; 18 months old, n=10-13). [Figure 3B] Total walking activity (top panel) and standing activity (bottom panel) of 18-month-old control and ANKI female mice (n=4-5 per group). [Figure 3C] Levels of sleep fragmentation in 4-month and 20-month-old male mice (n=6 per group), as well as in 20-month-old controls (left bar) and ANKI mice (right bar) (n=7-8 per group; males and females combined). The number of transitions between NREM sleep (NR) and wakefulness (W) cycles is shown. [Figure 3D] mRNA expression levels of Ox2r and Prdm13 in the hypothalamus of 20-month-old control and ANKI female mice (n=3-6 per group). [Figure 4A] Blood glucose (n=8-13) and insulin (n=8-9). [Figure 4B] Blood glucose levels (n=8-13) during IPGTT in 17-20 month old controls (left bar) and ANKI male mice (right bar). [Figure 4C] Total number of pancreatic islets in the spleen of 20-month-old control mice (left bar) and ANKI male mice (right bar) (n=3 per group). [Figure 4D] Representative images of pancreatic islets in the spleen of 20-month-old control and ANKI male mice (n=3 per group). [Figure 4E] Size distribution of pancreatic islets in the spleen of 20-month-old control (left bar) and ANKI male mice (right bar) (n=3 per group). [Figure 4F]Dark adaptation a-wave, dark adaptation b-wave, and light adaptation b-wave from ERG analysis of 18-20 month old control and ANKI mice (n=6-7 per group; males and females combined). [Figure 5A] Kaplan-Meier curves for female and male ANKI mice (female, control 39, ANKI40; male, control 39, ANKI39). [Figure 5B] Age-related mortality rates in control and ANKI female and male mice. [Figure 6A] Comparison of eNAMPT, extracellular vesicle (EV) marker proteins (TSG101, CD63, CD81, and CD9), and non-EV proteins (transferrin and albumin) in total plasma, EV fraction, and supernatant isolated by ultracentrifugation and a total exosome isolation (TEI) kit. When EVs were reconstituted in PBS in a volume equal to the starting volume of plasma, the protein concentrations were typically about 0.4 and 1 μg / μl for EVs purified by ultracentrifugation and the TEI kit, respectively. 40 μg of protein from each fraction was packed. [Figure 6B] Comparison of eNAMPT and EV marker proteins in six fractions (F1-F6) isolated by sucrose density gradient centrifugation. 2 mL of plasma was used for this fraction. [Figure 6C] Comparison of eNAMPT in whole plasma (P), extravasation fraction (E), and supernatant (S) isolated from three 4-month-old male mice and male human donors aged 37, 41, and 45 years. Each fraction was filled after being adjusted to equal volumes. [Figure 6D] Comparison of eNAMPT, TSG101, transferrin, and immunoglobulin light chain (Ig LC) in the treatment of mouse plasma with proteinase K and / or Triton X-100. [Figure 6E] Levels of EV-containing eNAMPT (EV-eNAMPT) and CD63 in plasma from 6 and 22-month-old mice (n=4 per group). [Figure 6F]Levels of EV-containing eNAMPT (EV-eNAMPT) and CD63 in plasma of 24-month-old control (CTRL) and ANKI mice (n=4 per group). [Figure 7A] Fluorescence images of primary hypothalamic neurons after incubation with BODIPY-labeled EV. EV purified from 400 μl of mouse plasma was added to 200 μl of culture medium. Arrows indicate neurons that have internalized BODIPY-labeled EV. [Figure 7B] Cytoplasmic levels of FLAG-tagged recombinant NAMPT (recNAMPT) and cellular NAD+ in primary hypothalamic neurons after incubation with recNAMPT alone or with EV-containing recNAMPT (n=3). [Figure 7C] Relative rate of NAD+ biosynthesis in primary hypothalamic neurons (n=4) after incubation with EV isolated from plasma by ultracentrifugation and TEI kit. [Figure 7D] Relative cellular NAMPT activity in primary hypothalamic neurons (n=3-6) after incubation with control (CTRL) and NAMPT knockdown (NAMPT-KD) EVs generated from OP9 adipocytes. [Figure 7E] Cytoplasmic NAMPT and NAD+ levels in primary hypothalamic neurons after incubation with EVs isolated from 6 and 18-month-old mice (n=4). [Figure 7F] Changes in NAD+ levels after incubation with EVs isolated from 6 and 20-22 month old mice (n=9-13). [Figure 7G] NAD+ levels in primary hypothalamic neurons after incubation with EVs isolated from 20-month-old controls (left bar) and ANKI mice (right bar). [Figure 7H] Total number of circular running activities in 20-month-old female mice during dark and light conditions, before and after injection of EV purified from 4-6 month-old mice (n=5) four times a day. [Figure 7I]Total circular activity counts in 25-month-old female mice (n=6) during dark time, before and after injection of control (CTRL) and Nampt knockdown (NAMPT-KD) EV purified from OP9 adipocytes four times daily. [Figure 7J] Kaplan-Meier curve and representative image. [Figure 7K] Kaplan-Meier curves of aged female mice injected with vehicle or EV isolated from 4-12 month old mice (n=11-12). Mouse images were taken 3 months after treatment. [Figure 8A] NAMPT levels in primary adipocytes isolated from 8-month-old male mice (n=5). [Figure 8B] Western blots for plasma eNAMPT levels over 24 hours in female mice aged 6 months and 18 months (n=5 per age-specific time point). 6-1~5 and 18-1~5 are individual plasma samples from 6 months and 18 months of age, respectively. The order of samples in each blot was intentionally randomized to avoid bias in signal quantification. [Figure 8C] Western blots for plasma eNAMPT levels over 24 hours in male mice aged 6 months and 18 months (n=5 per age-specific time point). 6-1~5 and 18-1~5 are individual plasma samples from 6 months and 18 months of age, respectively. The order of samples in each blot was intentionally randomized to avoid bias in signal quantification. [Figure 9A] Plasma eNAMPT levels in 6-month-old control, 18-month-old ANKI, and 18-month-old control mice (n=3). Plasma eNAMPT shows a double band (right panel), both of which were quantified (left panel). [Figure 9B] Relationship between plasma eNAMPT levels and hypothalamic NAD+ levels in 20-month-old control and ANKI female mice (n=5 per group). [Figure 10A] Circle running activity of 6- and 18-month-old wild-type mice (n=3-9). Differences were assessed by Wilcoxon signed-rank test. [Figure 10B] Walking activity (top) and standing activity (bottom) of 18-month-old control (CTRL) and ANKI male mice (n=4-7). [Figure 11A] Relative blood glucose levels of 17-20 month old control and ANKI male mice (right bar) (n=8 per group) during insulin loading tests. Glucose levels at each time point are normalized to the glucose level at 0 minutes. [Figure 11B] Body weight of 18-20 month old controls (left bar) and ANKI female and male mice (right bar) (males, n=20-36; females, n=19-23). [Figure 11C] Body composition of 17-20 month old controls (left bar) and ANKI male and female mice (right bar). [Figure 11D] Daily food intake of 23-month-old control mice (left bar) and ANKI male and female mice (right bar) (n=6-9). [Figure 11E] Relative plasma cytokine levels of 23-month-old controls (left bar) and ANKI male and female mice (right bar) (n=3). [Figure 11F] Contextual fear conditioning tests in 20-month-old control and ANKI female mice. Graph 1: Percentage of freezing behavior time in control and ANKI mice at baseline and during shock tone training; Graph 2: Contextual fear response on day 2; Graph 3: Baseline and auditory cue responses on day 3 (n=5). [Figure 12A] eNAMPT and EV marker proteins in six fractions isolated by suspension of purified EV onto a sucrose density gradient using ultracentrifugation. [Figure 12B] Comparison of densities in 12 fractions, as well as eNAMPT and the EV marker Alix in each fraction isolated from sucrose density gradient separation of EV purified by the Total Exosome Isolation (TEI) kit. The very small fractions of eNAMPT and Alix co-fractionated in fraction #11 are most likely due to contamination by protein aggregates. [Figure 12C]Electron micrographs and particle size distribution of EVs isolated from mouse and human plasma. [Figure 12D] Electron microscope images and particle size distribution of EVs isolated from mice (n=100 for each analysis). [Figure 12E] Comparison of eNAMPT, EV marker proteins (CD9, C81, Hsp70, and TSG101), and non-EV proteins (adiponectin and adipsin) in conditioned medium for OP9 adipocytes. The EV fraction and supernatant were separated by ultracentrifugation. 40 μg of protein from each fraction was packed into the cells. [Figure 13A] Fluorescence images of primary hypothalamic neurons after incubation with BODIPY-labeled EV from OP9 adipocytes. [Figure 13B] Relative NAMPT enzyme activity in primary hypothalamic neurons after incubation with each fraction (culture medium, EV, and supernatant) isolated from OP9 adipocyte conditioned medium. NMN biosynthesis levels, measured by mass spectrometry using D-4-NAM, were normalized to those in untreated cells. [Figure 13C] eNAMPT levels in EVs isolated from control and NAMPT knockdown (NAMPT-KD) OP9 adipocytes. Protein concentrations of purified EVs from both conditioned media were very similar, suggesting no difference in the amount of EV released from both cell lines. [Figure 13D] Levels of cytoplasmic NAMPT in primary hypothalamic neurons after incubation with plasma from 6- and 18-month-old mice. [Figure 13E] Total 24-hour circling activity counts of 20-month-old female mice before and after injection of EV isolated from 4-6 month-old mice (n=5) four times daily. [Figure 13F] Total circular activity count during light exposure in 25-month-old female mice (n=5-6) before and after injection of EV purified from control (CTRL) and NAMPT-KD OP9 adipocytes four times daily. [Figure 13G]Total number of circular running activities in 20-month-old male mice in darkness and light conditions, before and after injection of EV purified from 4-6 month-old mice (n=5) four times a day. [Figure 14] Cultured primary mouse hippocampal neurons treated with mouse plasma-derived extravasation veneers (****p<0.0001, one-way ANOVA with Sidac correction for multiple comparisons). [Figure 15] Astrocyte concentrated culture treated with Bodipy-TR-ceramide-labeled EV. [Figure 16] Microglia cultures treated with Bodipy-TR-ceramide-labeled EV. [Modes for carrying out the invention]

[0008] The present invention relates to various compositions comprising nicotinamide phosphoribosyltransferase (NAMPT) and / or its variants, processes for preparing these compositions, and various methods for using these compositions to prevent or treat age-related conditions in a subject. The present invention also relates to methods for increasing NMN and / or NAD+ biosynthesis in cells. The methods and compositions enable an improved delivery system of NAMPT and / or its variants to cells and organisms, where NAMPT and / or its variants can be utilized and act as anti-aging modifiers.

[0009] This invention is based on the discovery that circulating levels of extracellular nicotinamide phosphoribosyltransferase (eNAMPT) in mice and humans decline significantly with age. Increasing circulating eNAMPT levels in aging mice through adipose tissue-specific overexpression of NAMPT is linked to NAD levels in multiple tissues. + The goal is to increase the levels of NAMPT, thereby enhancing their functions and extending healthy lifespan in female mice. However, prior to this invention, it was unclear how eNAMPT is delivered in vivo or how NAMPT levels can be increased in aging individuals.

[0010] Extracellular vesicle (EV) delivery of NAMPT has been found to provide an effective method for supplementing NAMPT in cell lines or organisms. eNAMPT is carried in extracellular vesicles (EVs) through systemic circulation in mice and humans. EV-mediated delivery of eNAMPT is linked to intracellular reintegration and NAD + It induces biosynthesis. Supplementing with eNAMPT-containing EV isolated from young mice significantly improves running activity and extends lifespan in aging mice. Therefore, the inventors believe that systemic NAD + This study reveals a novel EV-mediated delivery mechanism of eNAMPT that promotes biosynthesis and combats aging, suggesting a potential means for anti-aging interventions in humans.

[0011] In recent years, numerous studies have reported the crucial role of extracellular proteins (EVs) as novel intercellular or intertissue interaction tools for transporting proteins and microRNAs (Whitham et al., 2018; Ying et al., 2017; Zhang et al., 2017). Indeed, adipose tissue has recently been demonstrated to be a major source of circulating EV-containing microRNAs that regulate gene expression in distant tissues (Thomou et al., 2017). In this regard, it is interesting that eNAMPT in the blood circulation is contained almost exclusively in EVs. In adipose tissue, SIRT1-dependent deacetylation of lysine 53 on iNAMPT facilitates protein secretion, suggesting that this deacetylation may be involved in the process of incorporating the NAMPT protein into EVs (Yoon et al., 2015). However, how eNAMPT-containing EVs are specifically targeted in certain tissues such as the hypothalamus, hippocampus, pancreas, and retina remains unknown. EV-mediated delivery occurs when eNAMPT is properly internalized within the cell, and NMN / NAD + It has been found to be important for enhancing intracellular biosynthesis. When eNAMPT protein is administered alone, the protein is not properly internalized.

[0012] Furthermore, it has been found that eNAMPT-containing extracellular matrix (EV) can be transferred from one individual to another. In particular, supplementation with eNAMPT-containing EV purified from young mice has been found to significantly enhance circulating activity and extend lifespan in aging mice. In human blood, eNAMPT is also found only in EV. Therefore, this model supports the use of EV-containing eNAMPT as an anti-aging biopharmaceutical in humans. These findings present novel possibilities for using EV-mediated systemic delivery of eNAMPT as a biopharmaceutical for effective anti-aging interventions.

[0013] Accordingly, various compositions of the present invention comprise nicotinamide phosphoribosyltransferase (NAMPT) and / or its variants, as well as lipids, the lipids forming a layer that at least partially encapsulates NAMPT and / or its variants. For example, the lipids can aggregate to form micelles or liposomes that encapsulate NAMPT and / or its variants. In some embodiments, the composition comprises exosomes containing NAMPT and / or its variants.

[0014] In some embodiments, the lipids include phospholipids. For example, phospholipids may be selected from the group consisting of phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylinositol phosphate, phosphatidylinositol diphosphate, phosphatidylinositol triphosphate, diphosphatidylglycerol, and combinations thereof. In various embodiments, the lipids include sphingolipids. For example, sphingolipids may be selected from the group consisting of ceramide phosphorylcholine, ceramide phosphorylethanolamine, ceramide phosphoryllipids, and combinations thereof.

[0015] In various embodiments, the concentration of NAMPT and / or its variants in the composition is about 1% by weight to about 20% by weight. In some embodiments, the composition has a weight ratio of NAMPT and / or its variants of about 1:1 to about 100:1.

[0016] Typically, the composition comprises a plurality of vesicles. In various embodiments, the vesicles are characterized by having an average particle size of about 10 nm to about 200 nm, about 10 nm to about 100 nm, or about 20 nm to about 100 nm. In some embodiments, the vesicles further contain water.

[0017] In various embodiments, the composition does not contain or is essentially free of certain biological components (e.g., less than 1% by weight or even less than 0.1% by weight). For example, in some embodiments, it does not contain or is essentially free of adipocytes, blood, and / or plasma (e.g., less than 1% by weight or even less than 0.1% by weight).

[0018] The compositions described herein, though not limited to these, may be administered by routes including oral, intravenous, intramuscular, intraarterial, intramedullary, subarachnoid, intraventricular, percutaneous, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. In various embodiments, administration is selected from the group consisting of oral, intranasal, intraperitoneal, intravenous, intramuscular, rectal, and percutaneous methods. In some embodiments, the compositions may be administered orally. In various embodiments, the compositions may be administered parenterally.

[0019] Compositions for oral administration may be formulated in doses suitable for oral administration using pharmaceutically acceptable carriers and excipients known in the art. Such carriers allow the composition to be formulated as tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, etc., for digestion by the subject. In certain embodiments, the composition is formulated for parenteral administration. Further details of the formulation and administration techniques can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Co., Easton, Pa., which is incorporated herein by reference). After the compositions are prepared, they may be placed in appropriate containers and labeled for the treatment of the indicated condition. Such labeling would include the amount, frequency, and method of administration.

[0020] In addition to the active ingredient (e.g., inhibitor compounds), the composition may contain suitable pharmaceutically acceptable carriers and excipients. In some embodiments, the composition further includes a carrier, such as water. Also in various embodiments, the composition further includes excipients. Various excipients include, for example, various non-toxic, inert solids, semi-solids, or liquid fillers, diluents, encapsulating materials, or any kind of formulation aid. Some examples of materials that can function as pharmaceutically acceptable excipients are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; celluloses and their derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; Tween Detergents such as 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogenic substance removal water; isotonic saline solution; Ringer's solution; ethyl alcohol; artificial cerebrospinal fluid (CSF); and phosphate buffer, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and colorants, release agents, coating agents, sweeteners, flavoring agents, and fragrances, and preservatives and antioxidants may also be present in the composition.

[0021] As described above, the present invention also relates to various methods of using the NAMPT-containing compositions or NAMPT variant-containing compositions described herein. One method relates to increasing NMN and / or NAD+ biosynthesis in cells. This method involves applying the compositions described herein to cells. Without being constrained by theory, it is thought that the lipid membrane of the vesicles of the composition can fuse with the cell's plasma membrane, thereby facilitating the transfer of the vesicle contents (i.e., NAMPT) into the cell. Once internalized, NAMPT and / or its variants can be used in cellular biosynthetic pathways to produce, for example, NMN and / or NAD+. Other methods relate to increasing NMN and / or NAD+ biosynthesis in subjects. These methods involve administering the compositions described herein to subjects.

[0022] Another method is intended to prevent or treat age-related conditions in a subject (e.g., a subject requiring prevention or treatment). The method involves administering an effective amount of the composition described herein to the subject. The methods described herein can increase NMN and / or NAD+ biosynthesis above physiological levels. Physiological levels correspond to the amount of product expected to be produced by a cell or organism at a particular point in time. Production can naturally vary throughout the organism's lifespan. Therefore, in various embodiments, the increase in NMN and / or NAD+ may be determined relative to the amount reasonably synthesized by the subject at that time.

[0023] In various embodiments, age-related conditions include physiological conditions selected from the group consisting of reduced physical activity, reduced sleep quality, reduced cognitive function, reduced glucose metabolism, reduced vision, and combinations thereof. In various embodiments, the methods disclosed herein, though not limited to these, may be used to treat, improve, alleviate, or reverse any age-related disease or condition involving NMN metabolism, such as type II diabetes, obesity, age-related obesity, increased age-related blood lipid levels, reduced age-related insulin sensitivity, loss or reduction of age-related memory function, loss or reduction of age-related ocular function, age-related physiological decline, impaired glucose-stimulated insulin secretion, diabetes, improved mitochondrial function, neuronal death, and / or cognitive function in Alzheimer's disease, protection of the heart from ischemia / reperfusion injury, maintenance of neural stem cell / progenitor cell populations, recovery of skeletal muscle mitochondrial and arterial function after injury, and age-related decline in function.

[0024] In various embodiments, age-related conditions may include age-related loss of insulin sensitivity and / or insulin secretion in subjects requiring it. In some embodiments, age-related conditions include age-related memory impairment. In various embodiments, age-related conditions include decreased ocular function. In some embodiments, decreased ocular function includes age-related retinal degeneration.

[0025] In some embodiments, age-related conditions may include muscular disorders, and the present invention includes methods for treating such muscular disorders in subjects requiring treatment. In various configurations, muscular disorders that can be treated according to this teaching include, but are not limited to, muscle weakness, muscle atrophy, muscle wasting, and muscle weakness. In various configurations, muscular disorders that can be treated according to this teaching include, but are not limited to, sarcopenia, dynapenia, cachexia, muscular dystrophy, myotonia, spinal muscular atrophy, and myopathy. Muscular dystrophy may be, for example, Duchenne muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Emery-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, or oculopharyngeal muscular dystrophy. In some configurations, myotonia may be myotonic dystrophy, congenital myotonia, or congenital paramyotonia. In some configurations, myopathy may be Vethlem myopathy, congenital muscle fiber type inequality, progressive ossifying fibrodysplasia, hyperthyroidism, hypothyroidism, minicore myopathy, polynuclear myopathy, myotubular myopathy, nemaline myopathy, periodic paralysis, hypokalemic myopathy, or hyperkalemic myopathy. In some configurations, muscle disease may be acid maltase deficiency, carnitine deficiency, carnitine palmityltransferase deficiency, deblancher enzyme deficiency, lactate dehydrogenase deficiency, mitochondrial myopathy, myoadenylate deaminase deficiency, phosphorylase deficiency, phosphofructokinase deficiency, or phosphoglycerate kinase deficiency. In some configurations, muscle disease may be sarcopenia, dynapenia, or cachexia. In some configurations, muscle disease may be sarcopenia.

[0026] Embodiments for preventing and treating age-related conditions may include preventing age-related functional decline in subjects requiring such treatment. In various configurations, age-related functional decline may result from or be related to, in non-limiting examples, loss of appetite, low glucose levels, muscle weakness, malnutrition, or age-related loss of appetite. Other non-limiting age-related conditions that may be treated by the compositions described herein may include diabetes (e.g., type 2 diabetes) and obesity.

[0027] In various embodiments, the present invention includes administering the compositions described herein to promote NMN / NAD+ production in a subject.

[0028] The therapeutically effective dose refers to the amount of the active ingredient that provides the desired result. The exact dosage is determined by the practitioner in light of factors relevant to the subject requiring treatment. The dosage and administration are adjusted to provide a sufficient level of the active ingredient or to maintain the desired effect. Factors that may be considered include the severity of the disease state, the subject's overall health, the subject's age, weight, and sex, diet, timing and frequency of administration, drug combinations, sensitivity to response, and tolerance / response to therapy. In some embodiments, the composition is administered to the subject in doses that provide NAMPT and / or variants of about 10 mg to about 500 mg per day, or about 50 mg to about 500 mg. In some embodiments, the subject is human. In various embodiments, the subject is human.

[0029] The present invention also relates to processes for preparing various compositions described herein. In various embodiments, the method includes separating vesicles from a culture medium containing components selected from the group consisting of adipocytes, blood, and plasma.

[0030] In various embodiments, the culture medium is a culture containing adipocytes. The adipocytes may overexpress genes encoding NAMPT (e.g., Nampt) or its biosynthetic precursor.

[0031] In various embodiments, the culture medium is a culture medium containing blood or plasma.

[0032] In various embodiments, the separation process includes centrifugation (e.g., ultracentrifugation). In some embodiments, the separation process includes exosome isolation techniques.

[0033] In various embodiments, the vesicles of the compositions described herein are derived synthetically or semi-synthetically. For example, NAMPT and / or its variants may be produced by recombinant technology. Subsequently, NAMPT and / or its variants and lipids may be combined, for example, in an aqueous solvent.

[0034] NAMPT and NAMPT variants In various embodiments, the composition includes NAMPT. In some embodiments, NAMPT includes wild-type NAMPT of SEQ ID NO: 1. In some embodiments, NAMPT includes wild-type NAMPT of SEQ ID NO: 2. [Table 1] TIFF0007850416000002.tif72170

[0035] In various embodiments, the composition includes variants of NAMPT. For example, two single-amino acid variants of the NAMPT protein, K53R and K53Q, have been reported. K53 is acetylated on iNAMPT, and SIRT1 deacetylates this lysine, making NAMPT more readily secreted. The K53R variant is secreted approximately three times more than the wild-type NAMPT protein, while the K53Q variant shows a significant decrease in secretion. Since K53R does not alter the enzymatic activity of NAMPT, the K53R variant may exhibit better efficiency in being packaged into exosomes and delivered to target tissues.

[0036] Therefore, a variant of NAMPT may include an amino acid sequence having an arginine or glutamine residue (particularly an arginine residue) at the position corresponding to position 53 of wild-type NAMPT in SEQ ID NO: 1, and the remaining amino acid sequence of the variant may have at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or at least 99.99% sequence identity with SEQ ID NO: 1. In some embodiments, a variant of NAMPT may include an amino acid sequence having an arginine or glutamine residue (particularly an arginine residue) at the position corresponding to position 53 of wild-type NAMPT in SEQ ID NO: 2, and the remaining amino acid sequence of the variant may have at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or at least 99.99% sequence identity with SEQ ID NO: 2.

[0037] In various embodiments, the variant of NAMPT contains at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or at least 99.99% sequence identity with wild-type NAMPT of SEQ ID NO: 1 or SEQ ID NO: 2, and further contains at least one amino acid substitution that removes an acetylation site compared to wild-type NAMPT. In some embodiments, the variant of NAMPT is secreted from cells more efficiently than wild-type NAMPT, or is packaged into exosomes more efficiently than wild-type NAMPT.

[0038] NAMPT variants may also include those with the following sequences: [Table 2] TIFF0007850416000004.tif232170TIFF0007850416000005.tif41168

[0039] After describing the present invention in detail, it will be apparent that modifications and variations are possible without departing from the scope of the invention as defined in the appended claims. [Examples]

[0040] The following non-limiting examples are provided to further illustrate the present invention.

[0041] material and method The following materials (Table 1) and methods were used to carry out the experiments in the following examples. [Table 3] TIFF0007850416000007.tif232169TIFF0007850416000008.tif25170

[0042] Animal models C57BL / 6J mice were bred in our laboratory using mice purchased from Jackson Laboratories or obtained from NIH aged rodent colonies. The juvenile (4–6 months old) and aged (18–26 months old) mice used in each experiment were matched in age and source. Cre-inducible STOP-NAMPT mice and adiponectin-Cre mice were provided by Joseph Baur of the University of Pennsylvania and Evan Rosen of Beth Israel Deaconess Medical Center, respectively. All strains were backcrossed to a C57BL / 6J background. Throughout the study, heterozygous ANKI mice were produced by crossing heterozygous adiponectin-Cre mice with homozygous STOP-Nampt mice. Both male and female ANKI mice were tested for eNAMPT and tissue NAD. +Quantification, circular analysis, sleep fragmentation count, ERG analysis, and their characterization, including lifespan, were used. Only male ANKI mice were used for glucomycotabolism and islet morphometric analysis due to their more robust phenotype. Unless otherwise specified, all mice were ad libitum-fed a standard diet (LabDiet5053; LabDiet, St. Louis, MO) and housed in 4-5 groups at 22°C with a 12 / 12-hour light-dark cycle. Cages and bedding were changed weekly. Mice were regularly monitored for health and there were no viral or parasitic infections during this study.

[0043] Human subjects The human plasma samples used for eNAMPT quantification were obtained from male subjects aged 37 to 80 years.

[0044] cell culture HEK293 cells were obtained from ATCC (Manassas, VA) and maintained in DMEM (Sigma Aldrich, St. Louis, MO) supplemented with 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. Pre-OP9 adipocytes were maintained in α-MEM (Sigma Aldrich, St. Louis, MO) supplemented with 20% FBS and penicillin-streptomycin. All cells were maintained at 37°C and 5% CO2. Pre-OP9 adipocytes were differentiated into fully differentiated adipocytes by culturing them in α-MEM for 48 hours with 0.2% FBS, 175 nM insulin, and 900 μM oleic acid bound to albumin. Primary hypothalamic neurons were isolated from E14 embryos and cultured in basal neural medium (Sigma Aldrich, St. Louis, MO) supplemented with 10% FBS, 2 mM L-glutamate, and B27. HEK293 was derived from female fetuses. The sex of the mice from which OP9 preadipocytes were derived is unknown. Primary hypothalamic neurons were isolated from both sexes' embryos.

[0045] Lifespan and hazard ratio analysis All animals were kept in the animal facility of this study with unrestricted access to standard laboratory food and water. Mice prepared for survival studies were not used for any other biochemical, physiological, or metabolic analyses. All mice in the aging cohort were carefully examined daily. Lifespan endpoints were determined when each mouse was either dead or euthanized according to IACUC guidelines. Autopsies were performed immediately after death or euthanasia by the University of Washington Mouse Pathology Core. Age-related mortality (q x The hazard ratio (Hz) was calculated by dividing the number of animals surviving at the end of each interval by the number of animals surviving at the beginning of the interval. The hazard ratio (Hz) was given by Hz = 2q x / (2-q x The natural logarithm of Hz was calculated using ( ) and plotted against time.

[0046] physical activity Spontaneous motor activity was evaluated at the University of Washington Animal Behavior Core. Briefly, individual mice were placed in clear polystyrene containers surrounded by pairs of 4x8 matrix photocells that quantified the total number of steps taken. Another set of photocells was placed 7 cm above the floor to quantify vertical standing movements. Wheel running activity was evaluated by placing mice in individual cages with running wheels and housing them in a circadian cabinet under a 12:12 light-dark cycle. Mice were acclimatized for two weeks prior to the measurement of wheel running activity.

[0047] sleep analysis Mice were anesthetized with isofluorane, and screw electrodes were surgically implanted in the skull for electroencephalography (EEG), and stainless steel wire electrodes were surgically implanted in the nuchal muscle for electromyography (EMG). Mice were allowed to recover from surgery for 3 days, followed by acclimatization in recording cages for 2 weeks. EEG / EMG recordings were performed consecutively for 2 days. Epochs of 10-second EEG / EMG signals were visually scored as wakefulness [low amplitude delta (1–4 Hz) and theta (4–8 Hz) frequencies with high EMG activity], NREM sleep [high amplitude delta with no EMG activity], and REM sleep [low amplitude rhythmic theta activity with no EMG activity]. Scorers were blinded for genotype during quantification.

[0048] Metabolic evaluation In the glucose tolerance test, mice were given a single dose of dextrose (1 g / kg body weight) intraperitoneally after an overnight fast in an aspen bed. Blood was collected from the tail vein at each time point to measure glucose and insulin levels. In the insulin tolerance test, mice were given insulin (0.70 units / kg body weight) intraperitoneally, and blood was collected from the tail vein to measure blood glucose levels. Plasma insulin levels were quantified using the Singulex assay at the University of Washington's Core Laboratory for Clinical Research. EchoMRI was performed at the Diabetes Model Phenotyping Core of the University of Washington's Diabetes Research Center.

[0049] Electroretinography Mice anesthetized with a mixture of ketamine and xylazine were subjected to ERG using the UTAS-E3000 Visual Electrodiagnostic System. ERG waveform quantification was performed using an existing Microsoft Excel macro, where wave a amplitude was defined as the difference between the mean baseline and the most negative point of the mean trace, and wave b amplitude was defined as the difference between the most negative point and the most positive point of the wave peak.

[0050] Small-scale cohort prospective lifespan analysis Female C57BL / 6J mice aged 26–28 months were obtained from NIA aging colonies. Plasma was collected from tail vein blood, and eNAMPT levels were quantified. Subsequently, the mice were housed in groups of four per cage and were not handled except for daily examinations. The number of days from blood collection to death was calculated as the remaining lifespan.

[0051] eNAMPT Western blot analysis Under ketamine-xylazine anesthesia, plasma was collected from the tail vein by capillary or cardiac puncture using a syringe pre-treated with heparin sulfate. The blood was spun down at 3000 × g. 2 μl of freshly collected plasma was incubated with 200 μl of 1x sample buffer at 95°C for 10 minutes, and then stored at -30°C until use. Immediately before analysis, 5 μl of each sample was added to 45 μl of 1x sample buffer and incubated further at 95°C for 30 minutes. This 30-minute boil was necessary to make the eNAMPT band individually quantifiable. Plasma eNAMPT was detected as a double when the SDS-PAGE was run for a sufficiently long time. 10 μl of the final mixture was separated on 4–15% SDS-PAGE and analyzed by Western blotting using anti-NAMPT polyclonal antibody (Bethyl) in the case of mice and anti-NAMPT monoclonal antibody (Adipogen) in the case of humans. The NAMPT antibody was used at a 1:1000 dilution. All other antibodies were used at a 1:100 dilution.

[0052] Gene expression analysis RNA was extracted using the RNeasy mini-kit (QIAGEN) and converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo). Quantitative real-time RT-PCR was performed using the StepOnePlus system (Applied Biosystems), and relative expression levels for each gene were calculated by normalizing the Gapdh levels and then the mean of the control mice.

[0053] Purification and characterization of EVs The extracellular viable cells (EVs) used in this study were isolated according to the manufacturer's instructions, either by ultracentrifugation or using a whole exosome isolation kit from plasma (ThermoFisher Scientific). Mouse plasma was isolated by centrifuging blood at 1000×g for 10 minutes. EVs were also collected in vitro by preparing serum-free α-MEM in fully differentiated OP9 adipocytes for 48 hours. The isolated plasma and OP9 conditioned medium were centrifuged at 1000×g for 10 minutes. The supernatant was centrifuged again at 2000×g for 20 minutes. The resulting supernatant was further centrifuged at 10,000×g for 30 minutes before EV isolation.

[0054] For EV isolation from plasma by ultracentrifugation, plasma was diluted 1:1 in PBS and centrifuged at 100,000 × g for 2 hours. The supernatant was collected for Western blot analysis, and the remaining pellet was resuspended in a volume of PBS equal to the starting plasma volume and centrifuged again at 1,000,000 × g for 2 hours. For EV isolation from OP9 conditioned medium by ultracentrifugation, the medium was centrifuged at 100,000 × g for 2 hours. Again, the supernatant was collected for Western blot analysis, and the remaining pellet was resuspended in a volume of PBS equal to the starting volume of the medium. The resuspended EVs were centrifuged again at 100,000 × g for 2 hours. The final pellets of EVs from both plasma and OP9 were resuspended in 50 μl of PBS.

[0055] For EV isolation using a whole exosome isolation (TEI) kit, unless otherwise specified, EVs were resuspended in the same volume of PBS as the plasma used to isolate them. The supernatant obtained after EV isolation was collected as the soluble protein fraction. The quality of the isolated EVs was confirmed by measuring the levels of EV marker proteins [Alix (Santa Cruz Biotechnology), TSG101 (Santa Cruz Biotechnology), CD63 (Santa Cruz Biotechnology), CD81 (Santa Cruz Biotechnology), and CD9 (BD Bioscience)] and non-EV proteins [transferrin (abcam), albumin (abcam), adiponectin (abcam), and adipsin (R&D)]. 40 μg of protein from total plasma, the isolated EV fraction, and the supernatant / soluble protein fraction were loaded onto SDS-PAGE gels and evaluated by Western blotting.

[0056] Sucrose gradient fractionation analysis of EV Extracellular viable cells (EVs) were prepared by ultracentrifugation at 100,000 × g for 2 hours or by a TEI kit. The isolated EVs were diluted in 90% sucrose solution to a final concentration of 82%. The EVs were then layered at the bottom, followed by a layer of sucrose solution ranging from 82% to 10% on top. The samples were centrifuged at 100,000 × g for 20 hours to collect six fractions. Each fraction was diluted 1:100 in PBS and pelletized by centrifugation at 100,000 × g for 2 hours. The pellets from each fraction were then resuspended in equal volumes of PBS and subjected to analysis by Western blotting.

[0057] Proteinase K digestion assay Proteinase K was added to 50 μl of plasma at a final concentration of 1 μg / μl and incubated at 37°C for 10 minutes. Subsequently, 25 μl of PBS and 15 μl of exosome precipitation reagent (ThermoFisher Scientific) were added, and the mixture was incubated on ice for 30 minutes. The mixture was centrifuged at 1000 × g, and the precipitated EV was analyzed by Western blotting.

[0058] Proteomic analysis of plasma extravasation cells (EVs) Plasma was isolated from EDTA-supplemented blood isolated from 6- and 24-month-old wild-type B6 female mice and 24-month-old control and ANKI female mice. EVs were isolated from 400 μl of plasma by ultracentrifugation and reconstituted in water. Proteins were extracted and analyzed by progenesis LC-MS (NonLinear Dynamics). Protein identification was performed using Mascot Server v2.4 (Matric Science). A list of identified proteins was generated with a minimum 95% peptide threshold, a minimum 95% protein threshold, and a minimum of two peptides, with a false protein detection rate of 0.5%. Of the 248 proteins identified above the threshold, 181 proteins were identified in previous proteomics studies of EVs / exosomes based on EVpedi.org.

[0059] Isolation of primary hypothalamic neurons Hypothalamuses from E16-E18 embryos were dissected and placed on ice in Hibernate E medium. The hypothalamuses were digested in 0.25% trypsin-EDTA (Sigma) supplemented with DNase I (Sigma) at 37°C for 15 minutes. After adding an equal volume of DMEM supplemented with 10% FBS, the cells were gently dissociated by pipetting until no clumps remained. The cells were collected by centrifugation at 450 × g for 5 minutes at room temperature. The cells were washed and resuspended in basal neural medium containing 10% FBS, 2% B27, 2M L-glutamine, and antibiotics. The cells were placed directly on wells or on coverslips pre-coated with poly-l-lysine (Sigma). Two days after isolation, the cells were treated with 10 μM Ara-C (Sigma) for at least 4 days, or until non-neuronal cells were removed.

[0060] EV internalization assay The isolated extracellular viable cells (EVs) were resuspended in PBS. BODIPY TR ceramide in DMSO was added to the EVs or PBS at a final concentration of 100 μM and incubated at 37°C for 1 hour. Unintegrated dyes from the labeled EVs were removed by exosome spin column (ThermoFisher Scientific) according to the manufacturer's instructions. The purified EV or PBS solution was added directly to primary hypothalamic neurons growing on coverslips and incubated for 30 minutes. After incubation, the cells were washed in PBS and fixed in 4% paraformaldehyde.

[0061] Recombinant NAMPT-containing EV generation and their internalization assay The isolated extracellular viable cells (EVs) were resuspended in 1 μg / μl of FLAG-tagged recombinant NAMPT protein (recNAMPT) and incubated overnight at 37°C. recNAMPT-containing EVs were isolated from the mixture by adding 0.2 volumes of exosome precipitation reagent (ThermoFisher Scientific). recNAMPT-containing EVs were reconstituted in the same volume of PBS as the starting plasma.

[0062] EV injection post-circular assay In the EV injection experiment, 20-month-old male and female mice were acclimatized with a 6-day simulated injection. To measure circling activity before treatment, the mice were intraperitoneally injected with 100 μl of PBS for 4 days. Subsequently, the same mice were injected with 100 μl of resuspended EV purified from 200 μl of plasma collected from 4-6 month-old mice, and the EV was resuspended in PBS. All injections were performed around 5:30 p.m.

[0063] Lifespan study of mice injected with EV Female C57BL / 6J mice, 25 months old, were obtained from the National Institute on Aging (NIA). Mice were sorted by body weight, and pairs of mice with similar body weights were assigned to each group. Four mice were housed per cage. Extracellular viable (EVs) were isolated from the plasma of 4- to 12-month-old wild-type mice using a TEI kit. In this lifespan study, the use of a TEI kit was necessary to achieve the highest yield of EVs from the limited number of mice available. EVs isolated from 500 μl of plasma were resuspended in 100 μl of PBS and administered to mice weekly by intraperitoneal injection, starting at 26 months of age.

[0064] Data Analysis Results are expressed as mean ± SEM. All statistical tests were performed using GraphPad Prism5. Significance between two groups was assessed by Student's t-test. Normality of data was assessed graphically. Comparisons between multiple groups were performed using one-way ANOVA with Tukey's post-hoc test. 24-hour plasma eNAMPT levels in mice aged 6–18 months were analyzed using two-way repeated measures ANOVA. Linear regression analysis was used to analyze plasma eNAMPT levels in mice and humans across different age groups. Spontaneous motor activity and circle running activity were compared using Wilcoxon's signed-rank test. ERG signals were analyzed using two-way repeated measures ANOVA with Bonferroni post-hoc test. Gehan-Breslow-Wilcoxon test was used for statistical analysis of lifespan. Fisher's exact test was used to compare the proportion of causes of death. Statistical comparisons of circle running activity before and after EV injection treatment were performed using paired t-tests. Sample size and other statistical parameters are shown in the figures and text. * p<0.05, ** p<0.01, *** p<0.001. Significant significance was concluded at p<0.05.

[0065] Example 1: In both mice and humans, plasma eNAMPT levels decrease with age. Previous studies have shown that adipose NAMPT expression decreases with age (Yoshino et al., 2011). We consistently found that iNAMPT protein expression levels in isolated adipocytes decreased from 6 months to 18 months of age (Figure 8A). Considering that adipose tissue is a major source of circulating eNAMPT (Yoon et al., 2015), we investigated whether circulating eNAMPT levels changed during mouse aging. Plasma eNAMPT levels decreased significantly by 33% and 74%, respectively, in both female and male mice from 6 months to 18 months of age (Figure 1A). In young (6-month-old) mice, calculated plasma eNAMPT concentrations were higher in females (55–123 ng / μl) than in males (29–52 ng / μl). However, in aged (18-month-old) mice, both males and females showed a significant decrease in circulating eNAMPT levels throughout the day (Figures 1B and 8B).

[0066] The age-related decline in plasma eNAMPT levels raised the possibility that plasma eNAMPT levels could be a valuable surrogate biomarker for aging. Therefore, we measured plasma eNAMPT levels across several different age groups in mice and humans. In both mice and humans, we found that plasma eNAMPT levels declined linearly with age (Figure 1C), suggesting that the underlying processes of eNAMPT secretion and its potential significance during aging may be conserved in both species. To further evaluate the potential association between plasma eNAMPT levels and aging, we asked whether a decline in plasma eNAMPT levels could predict a higher mortality risk and remaining lifespan in mice in a small prospective study. Interestingly, the number of days each mouse survived after the eNAMPT measurement date was highly correlated with its plasma eNAMPT level (Figure 1D). Higher plasma eNAMPT levels were associated with longer remaining lifespan. These results led to an intriguing hypothesis that circulating eNAMPT may play a significant role in regulating not only the aging process but also mammalian lifespan.

[0067] Example 2: Adipose tissue-specific overexpression of Nampt resulted in increased plasma eNAMPT levels and NAD levels in multiple tissues during aging. + Maintains biosynthesis. To investigate the role of eNAMPT in aging and lifespan control, we examined an aging cohort of adipose tissue-specific Nampt knock-in (ANKI) mice (Yoon et al., 2015). At 4 months of age, plasma eNAMPT levels did not differ between ANKI mice and control mice under ad libitum feeding conditions (Figure 2A). Young ANKI mice showed significantly higher plasma eNAMPT levels only in response to fasting (Yoon et al., 2015). When they reached 24 months of age, plasma eNAMPT levels were maintained at 3.3-fold and 3.6-fold higher levels in ANKI female and male mice compared to age-matched control mice, respectively (Figure 2B). Plasma eNAMPT levels in 18-month-old ANKI mice were comparable to those in 6-month-old control mice (Figure 9A). Adipose tissue Nampt overexpression was maintained at approximately 1.5 times higher in aged ANKI mice compared to control mice, confirming that Nampt overexpression was within the physiological range and suggesting that the ANKI model is physiologically effective (data not shown).

[0068] In 20-month-old ANKI mice that were allowed to feed freely, NAD was found in the hypothalamus, hippocampus, pancreas, and retina of female mice. + An increase in NAD levels was observed, but in males, it was only in the pancreas and retina. + An increase in NAD levels was observed (Figures 2C and 9B). + It should be noted that the tissues showing increased levels were those with relatively very low levels of iNAMPT (Revollo et al., 2007, Stein et al., 2014, Yoon et al., 2015). These results suggest that adipose tissue-specific overexpression of Nampt is associated with increased circulating eNAMPT levels and tissue NAD levels in multiple tissues, including the hypothalamus, hippocampus, pancreas, and retina. + This suggests that it may mitigate the age-dependent decline in levels.

[0069] Example 3: Aged ANKI mice show significant improvements in physical activity and sleep quality. In aging ANKI female mice, hypothalamic NAD + Given the increasing levels and the importance of hypothalamic SIRT1 activity in regulating physical activity and sleep quality during aging (Satoh et al., 2013, Satoh et al., 2015), these age-related physiological characteristics were investigated in aging ANKI female mice. Consistent with the age-related decline in circulating eNAMPT levels, 18-month-old wild-type mice showed significantly reduced circle running activity during dark periods compared to 6-month-old wild-type mice (Figure 10A). 4-month-old ANKI female mice exhibited similar levels of circle running activity during dark periods as age-matched control mice, while 18-month-old ANKI female mice showed significantly enhanced circle running activity compared to age-matched control mice, similar to the activity levels of 6-month-old wild-type mice (Figures 3A and 10A). In addition, spontaneous motor activity in open fields was evaluated in these mice. Consistent with circling activity during dark periods, aged ANKI female mice showed significantly higher total walking and standing activity compared to age-matched control mice (Figure 3B). However, aged ANKI male mice showed higher NAD activity in the hypothalamus. + Consistent with the lack of increase, there were no significant differences in total walking activity or standing activity compared to age-matched control mice (Figure 10B) (Figure 2C).

[0070] In humans, an increase in the number of sleep-wake transitions with age is well documented, a phenomenon known as sleep fragmentation (Mander et al., 2017). Consistent with such changes in aged humans, 20-month-old wild-type mice also showed an increased number of transitions between non-REM (NREM) sleep cycles and wakefulness cycles compared to 4-month-old wild-type mice (Figure 3C, left panel), indicating a significant increase in sleep fragmentation with age in mice. There was no difference in the number of transitions between REM sleep cycles and wakefulness or NREM sleep cycles (data not shown). Interestingly, compared to age-matched control mice, aged ANKI mice showed a significant decrease in the number of transitions between NREM sleep and wakefulness cycles, which remained at a similar level to that seen in 4-month-old wild-type mice (Figure 3C, right panel), suggesting that aged ANKI mice maintain better sleep quality.

[0071] These age-related activity and sleep characteristics are regulated by hypothalamic SIRT1 through the regulation of its downstream target genes, the orexin 2 receptor (Ox2r) and PR domain 13 (Prdm13) (Satoh et al., 2013, Satoh et al., 2015). Ox2r expression is important for regulating running activity during dark periods (Satoh et al., 2013), while Prdm13 expression is important for maintaining sleep quality (Satoh et al., 2015). Therefore, we examined the mRNA expression levels of Ox2r and Prdm13 in the hypothalamus of age-matched control and ANKI female mice. Consistent with the observed improvements in physical activity and sleep quality, hypothalamic Ox2r and Prdm13 expression levels were significantly increased in aged ANKI female mice compared to age-matched control mice (Figure 3D). These results suggest that age-related declines in circulating eNAMPT levels are linked to hypothalamic NAD + We show that it contributes to a decline in levels and SIRT1 activity, leading to age-related declines in physical activity and sleep quality, and that these functional declines can be improved by increasing circulating eNAMPT.

[0072] Example 4: Aged ANKI mice show significant improvements in glucose-stimulated insulin secretion, photoreceptor function, and cognitive function. NADs in the pancreas, retina, and hippocampus + Since levels were increased in aged ANKI mice (Figure 2C), we suspected they might also exhibit improvements in glucose metabolism, retinal function, and cognitive function. First, we performed intraperitoneal glucose tolerance tests (IPGTTs) in aged ANKI mice and control mice. Moderate but significant improvement in glucose tolerance was observed in aged ANKI male mice (Figure 4A). During the IPGTT, we also detected a significant increase in glucose-stimulated insulin secretion at 30 minutes (Figure 4B). The results of the insulin tolerance test were not different between aged ANKI mice and control mice, both showing severe insulin resistance (Figure 11A), suggesting that the improvement in glucose tolerance in aged ANKI mice was mainly due to increased insulin secretion. Interestingly, aged ANKI mice possessed a higher total number of pancreatic islets compared to age-matched controls (Figures 4C and D). In addition, these pancreatic islets observed in aged ANKI males remained smaller than those observed in age-matched controls (Figure 4E). These results suggest that NAD is involved in promoting glucose-stimulated insulin secretion in pancreatic β-cells and protecting them from stress. + This is consistent with the role of biosynthesis and SIRT1 (Kitamura et al., 2005, Moynihan et al., 2005, Ramsey et al., 2008, Revollo et al., 2007). Aging ANKI female mice also maintained a higher total number of pancreatic islets, but pancreatic NAD +Consistent with much larger fluctuations in levels (Figure 2C), no improvement in glucose tolerance was observed (data not shown). Aged ANKI female mice showed a moderate increase in body weight and fat mass, while aged ANKI male mice showed no difference (Figures 11B and 11C). Furthermore, their food intake was not significantly different compared to their age-matched controls (Figure 11D). We also examined the circulating levels of pro-inflammatory cytokines in aged ANKI mice. Except for a slight increase in IL-2 and a moderate decrease in G-CSF in aged ANKI females, there were no significant changes in circulating pro-inflammatory cytokine levels in either aged ANKI males or females (Figure 11E). Therefore, the ANKI phenotype observed in glucose metabolism is independent of body weight, fat, or pro-inflammatory cytokine levels.

[0073] Next, electroretinography was performed to examine retinal function under test conditions primarily involving rods and cones. Compared to age-matched control mice, aged ANKI mice showed significantly higher dark-adapted wave a amplitude at 5 dB and a tendency toward higher dark-adapted wave a amplitude at -4 and 0 dB (Figure 4F, left panel), indicating improved rod photoreceptor function. Similarly, improved rod photoreceptor function also led to a tendency toward enhanced dark-adapted wave b (Figure 4F, center panel). Furthermore, a tendency toward enhanced light-adapted wave b amplitude was observed in aged ANKI mice, suggesting enhanced cone function (Figure 4F, right panel). Interestingly, these changes are remarkably similar to those observed with long-term NMN administration (Mills et al., 2016).

[0074] Furthermore, contextual fear conditioning tests were performed on aged ANKI mice and age-matched control mice to evaluate their non-spatial hippocampus-dependent learning and memory abilities. Both types of mice showed similar responses during baseline and day 1 training tests (Figure 11F, first graph). However, during the first minute of the day 2 contextual fear conditioning test, aged ANKI mice exhibited significantly higher levels of freezing behavior compared to age-matched controls (Figure 11F, second graph). Similar trends were observed at 2 and 3 minutes during this test. Throughout the baseline and day 3 auditory cue tests, aged ANKI mice and age-matched control mice showed no significant differences (Figure 11F, third graph). Their hippocampal NAD + Consistent with the increase, these results suggest that aged ANKI mice maintain better hippocampus-dependent cognitive function compared to their age-matched controls. Therefore, in summary, these findings suggest that systemic NAD + By enhancing biosynthesis, this provides further support for the physiological significance of eNAMPT in maintaining tissue function in aging tissues such as the hypothalamus, hippocampus, pancreas, and retina.

[0075] Example 5: ANKI female mice exhibit a significant extension of median lifespan and delayed aging. Maintaining higher eNAMPT levels significantly mitigates age-related functional decline in aging ANKI mice; therefore, a cohort of ANKI and control mice was established to examine their lifespan. When freely fed a standard diet, ANKI female mice showed a statistically significant extension of median lifespan (13.4%) (controls 693 days vs. ANKI 786 days, Gehan-Breslow-Wilcoxon test, χ²). 2 =6.043, df=1, p=0.014) (Figure 5A Table 2). [Table 4]

[0076] Their maximum lifespan was no different from that of control mice (Table 2). Interestingly, ANKI female mice showed a significant delay in age-related mortality up to approximately 2 years of age (Figure 5B). However, as they approached the end of their lifespan, the difference in age-related mortality no longer existed, which may explain the lack of extension of maximum lifespan. Neoplasms were the leading cause of death, but the incidence and types of neoplasms were no different between ANKI mice and control mice (Table 3). [Table 5]

[0077] In contrast to females, the oldest 10% ANKI males showed a significantly longer maximum lifespan compared to control mice, but ANKI male mice did not exhibit lifespan extension (Figure 5A and Table 2), and there was no difference in age-related mortality throughout most of their lifespan (Figure 5B). These results demonstrate that maintaining youthful levels of circulating eNAMPT is important for slowing aging and extending healthy lifespan in mice, despite significant sex differences.

[0078] Example 6: Plasma eNAMPT is localized only in extracellular vesicles. Circulating eNAMPT in tissue NAD + How it enhances biosynthesis remains unclear at present. eNAMPT enhances NAD in a tissue-specific manner. + The finding that it enhances biosynthesis suggests that circulating eNAMPT enhances NAD in its target tissues. +This suggests that eNAMPT may directly contribute to biosynthesis. In recent years, the transport mechanism of microRNAs by extracellular vivipars (EVs) from one tissue to another has attracted much attention as an important mechanism of inter-tissue interaction (Whitham et al., 2018; Ying et al., 2017; Zhang et al., 2017). Therefore, we asked whether eNAMPT can also be transported by EVs in systemic circulation. EVs were purified from mouse plasma by conventional ultracentrifugation or using a polymer-based whole exosome isolation (TEI) kit. The yield of EVs from the TEI method was much higher than that from ultracentrifugation, but both methods clearly showed that eNAMPT was highly enriched in the EV fraction compared to whole plasma or the remaining non-EV fraction (Figure 6A). Localization of eNAMPT in EVs was also confirmed by enrichment of several EV markers, including TG101, CD63, CD81, and CD9, as well as depletion of transferrin and albumin. Furthermore, the EV fractions from TEI or ultracentrifugation were further purified by suspension in a sucrose density gradient. In both EV fractions, eNAMPT was clearly detected in a third fraction from the sucrose density gradient with several other EV markers, including Alix, TSG101, CD63, CD81, and CD9 (Figures 6B and 12A). In addition, the density of the fraction carrying eNAMPT-containing EVs isolated by TEI was measured. eNAMPT was co-purified with Alix, one of the EV markers, in density fractions previously reported for EVs in the range of 1.10–1.15 (Figure 12B) (Ying et al., 2017). We also investigated whether eNAMPT in human plasma was present in the EVs. Consistent with mouse eNAMPT, human plasma eNAMPT was mainly present in the EV fraction (Figure 6C). The proper enrichment of EVs from mouse and human plasma was further confirmed by electron microscopy (Figures 12C and 12D), indicating that the types of EVs purified from mouse and human plasma match those characterized as small EVs (Durcin et al., 2017). Unfortunately, attempts to immunogold label eNAMPT contained in the EVs failed due to the lack of suitable antibodies available for this purpose.Therefore, we investigated whether eNAMPT in plasma is protected from protease treatment by its localization within extracellular proteins (EVs). While plasma treatment with proteinase K almost completely digested circulating plasma proteins such as transferrin and immunoglobulin light chains, eNAMPT and the EV marker TSG101 exhibited resistance to proteinase K digestion (Figure 6D). Furthermore, when the lipid bilayer of the EVs was dissolved by the addition of detergent (Triton-X), proteinase K treatment was able to eliminate eNAMPT and TSG101. These findings further confirmed that eNAMPT secreted into the bloodstream was encapsulated in EVs. Using cultured OP9 adipocytes, we also confirmed that fully differentiated adipocytes secrete EVs containing eNAMPT and other EV marker proteins, but without other secretory proteins such as adiponectin and adipsin (Figure 12E).

[0079] We found that the eNAMPT content in extracellular proteins (EVs) dramatically decreased in mice aged 6–22 months (Figure 6E). Furthermore, we found that the eNAMPT content in EVs from 24-month-old ANKI mice was significantly higher than that of age-matched control mice (Figure 6F). These observed changes in eNAMPT levels in EVs were compared between young (6-month-old) and aged (24-month-old) mice, and between aged ANKI mice and control mice, with proteomic comparisons of EV-containing proteins showing significant changes in only a small fraction (2–3%) of EV-containing proteins (181 identified proteins) under these conditions, and none of these proteins showed significant changes in NAD. + The results were physiologically significant because they demonstrated that the changes were not related to metabolism (Figure 12F). In summary, these results indicate that eNAMPT is carried by EVs in the circulation of mice and humans, and that changes in plasma eNAMPT levels observed in aging or ANKI mice are due to changes specific to eNAMPT contained in EVs.

[0080] Example 7: eNAMPT contained in EV is internalized into cells, and NAD +It directly enhances biosynthesis. After demonstrating eNAMPT localization within EVs, it was confirmed that eNAMPT contained in EVs is internalized into the cell, and NAD is produced within the cell. + We investigated whether biosynthesis could be enhanced. First, isolated EVs were labeled with BODIPY TR ceramide, a red fluorescent dye capable of labeling the lipid bilayer of EVs. Then, primary hypothalamic neurons were incubated with these BODIPY-labeled EVs. Primary hypothalamic neurons were labeled only when BODIPY-labeled EVs were added, but not when the control BODIPY-treated medium was added, suggesting that EVs were incorporated into primary hypothalamic neurons (Figure 7A). Figure 7. eNAMPT contained in EVs directly enhances NAD+ biosynthesis in primary hypothalamic neurons, improving age-related decline in physical activity and extending lifespan in mice.

[0081] Next, bacterial-produced FLAG-tagged recombinant NAMPT alone or FLAG-tagged recombinant NAMPT encapsulated in EVs was added to primary hypothalamic neurons. Interestingly, only FLAG-tagged NAMPT contained in EVs was internalized into the cytoplasmic fraction of primary hypothalamic neurons (Figure 7B). When FLAG-tagged recombinant NAMPT is incorporated into purified EVs, these EVs with additional amounts of NAMPT are found to enhance intracellular NAD in primary hypothalamic neurons. + We were able to increase the level (Figure 7B, right panel). Cellular NAD + To further investigate the effect of eNAMPT contained in EV on biosynthesis, isotope-labeled nicotinamide (D-4-NAM) was used in primary hypothalamic neurons to study NAD + Biosynthesis rates were measured. Both EVs (protein concentration of 1 μg / μl), purified from mouse plasma by ultracentrifugation and TEI, were compared to the control in terms of NAD + A similar increase was observed in biosynthesis (Figure 7C). NAD +To investigate whether the observed effect on biosynthesis was due to the enzymatic activity of eNAMPT contained in EV, we used EV purified from OP9 adipocyte culture medium by ultracentrifugation. We confirmed that EV purified by ultracentrifugation from OP9 adipocytes was properly internalized into primary hypothalamic neurons (Figure 13A). Using this OP9 adipocyte line, we demonstrated that concentrated EV from OP9 culture medium, although not EV-depleted supernatant, showed a significant enhancement of NMN biosynthesis in primary hypothalamic neurons (Figure 13B). Next, we compared the effects of EV purified from control and Nampt knockdown (Nampt-KD) OP9 adipocyte culture medium on NMN biosynthesis in primary hypothalamic neurons. NAMPT expression was reduced by 80% with EV secreted from Nampt-KD OP9 adipocytes (Figure 13C). While EV purified from the culture medium of control OP9 adipocytes clearly showed enhancement of NMN biosynthesis, EV purified from the culture medium of Nampt-KD OP9 adipocytes did not show enhancement of NMN biosynthesis in primary hypothalamic neurons (Figure 7D), and cellular NMN / NAD + We demonstrated that these effects of EVs, which stimulate biosynthesis, are primarily due to eNAMPT contained in EVs.

[0082] Internalization of eNAMPT contained in EVs into the cytoplasm of primary hypothalamic neurons was also investigated using mouse plasma and purified EVs from 6 and 18-month-old mice (Figures 13D and 7E). The amount of internalized NAMPT in the cytoplasm reflected the amount of eNAMPT contained in the original plasma or purified EVs, indicating that EVs from young mice can deliver more eNAMPT to cells compared to those from aged mice. Next, NAD in primary hypothalamic neurons + We compared the ability of purified EVs from young and aged mouse plasma to stimulate biosynthesis. +The increase in levels was significantly higher in EVs from 6-month-old mice compared to those from 20-22-month-old mice (Figure 7F). Furthermore, EVs from ANKI male and female mice showed increased intracellular NAD levels in primary hypothalamic neurons compared to those from age-matched control mice. + We were able to increase the level (Figure 7G). These results suggest that eNAMPT contained in EVs that are internalized into target cells increases the NMN / NAD level within those target cells. + This provides compelling evidence that it may contribute to enhancing biosynthesis.

[0083] Example 8: Supplementation with eNAMPT contained in EV enhances running activity and extends lifespan in aging mice. eNAMPT contained in EV is used in intracellular NAD in primary hypothalamic neurons. +Considering that levels could be enhanced, we hypothesized that supplementation with eNAMPT-containing EV could transmit similar anti-aging effects to aging wild-type mice, as observed in aging ANKI mice. To test this possibility, EV purified from the plasma of 4-6 month old mice was intraperitoneally injected into 20-month old wild-type female mice for four consecutive days. Notably, supplementation with EV purified from the plasma of younger mice significantly enhanced circling activity in aging mice during dark periods compared to age-matched control mice injected with PBS (Figure 13E and 7H). Interestingly, while circling activity during dark periods was clearly increased, activity during light periods was significantly decreased, suggesting that these EV-injected aging mice may sleep better. To further confirm whether this effect is attributable to eNAMPT contained in EV, we compared circling activity in aging wild-type female mice by injecting control and EV purified from Nampt-KD OP9 adipocyte culture medium. While EV purified from control OP9 medium again showed a significant increase and decrease in circle running activity during darkness and light periods, EV purified from Nampt-KD OP9 medium did not exhibit these effects (Figures 7I and 13F), demonstrating that this effect is mediated by eNAMPT contained in EV. A mild enhancement of circle running activity was also observed in aged male mice (Figure 13G), suggesting that male mice can also respond to eNAMPT supplementation in high levels of EV.

[0084] Next, we tested whether eNAMPT-containing extracellular viable (EV) purified from the plasma of young mice could extend the lifespan of aging mice. EV purified from young to middle-aged (4-12 months old) mice was injected once a week into 26-month-old female mice. Notably, supplementation with EV purified from young to middle-aged mice significantly extended the lifespan of aging mice (Figure 7J). The median lifespan was extended by 10.2% (840 days for vehicle-injected mice, 926 days for EV-injected mice, Gehan-Breslow-Wilcoxon test, χ²). 2(=11.10, df=1, p=0.0009), two mice were still alive when the results were recorded. The mean maximum lifespan of the four longest-living mice in each group was 881±63.6 and 999±47 days for vehicle-injected mice and EV-injected mice, respectively (Student's t-test, p=0.0016, 13.3% extension). Aging mice injected with EV generally maintained a much healthier appearance and higher activity levels compared to age-matched control mice injected with vehicle (Figure 7K). In summary, these findings suggest that supplementation with eNAMPT contained in EV is an effective anti-aging intervention to mitigate age-related functional decline and extend lifespan in mice.

[0085] The results of Examples 1-8 indicate that NAD is important in controlling the aging process in mice and in determining healthy lifespan and lifespan. + This highlights the importance of a novel EV-mediated inter-tissue interaction mechanism for delivering the biosynthetic enzyme eNAMPT to specific tissues. Age-related declines in eNAMPT levels in circulating EVs are linked to NAD levels in these target tissues, including the hypothalamus, hippocampus, pancreas, and retina. + It limits availability and tissue function. Furthermore, supplementing aging mice with eNAMPT contained in EVs has been shown to mitigate age-related physiological declines during aging, either genetically or pharmacologically, and to extend the lifespan of the mice.

[0086] Since the hypothalamus is suggested to function as a higher-order aging control center in mammals (Satoh et al., 2013, Zhang et al., 2013, Zhang et al., 2017), it was hypothesized that eNAMPT secreted from adipose tissue plays a crucial role in influencing the aging process and ultimate lifespan. To address this hypothesis, we generated adipose tissue-specific Nampt knock-in (ANKI) mice (Yoon et al., 2015) and characterized their aging phenotype. Interestingly, aging ANKI mice exhibited youth-level circulating eNAMPT and increased NAD levels in multiple tissues, including the hypothalamus, hippocampus, pancreas, and retina. + The levels were maintained, and significant improvements were observed in physical activity, sleep quality, cognitive function, glucose metabolism, and photoreceptor function. Due to these beneficial effects on aging, ANKI mice, particularly females, showed a significant extension of healthy lifespan. Surprisingly, we found that eNAMPT was carried in extracellular vesicles (EVs) via the blood circulation in both mice and humans. eNAMPT contained in EVs is internalized in primary hypothalamic neurons and then absorbed intracellularly by NAD + Biosynthesis was enhanced. Injection of eNAMPT-containing EVs purified from young mice or cultured adipocytes (not from Nampt knockdown adipocytes) enhanced circulating activity and extended lifespan in aging mice. These findings indicate a novel inter-tissue interaction mechanism driven by EV-mediated delivery of eNAMPT. This new physiological system, mediated by eNAMPT contained in EVs, is related to systemic NAD + It plays a crucial role in maintaining biosynthesis and counteracting age-related physiological decline, suggesting that eNAMPT, contained in EV, may be a potential anti-aging biological agent in humans.

[0087] Examples 1–8 of this specification demonstrate that EV-mediated systemic delivery of eNAMPT mitigates age-related functional decline, delays age-related mortality, and extends healthy lifespan and life expectancy in specific target tissues, including the hypothalamus, hippocampus, pancreas, and retina, in mice. A remarkable finding in this study is that eNAMPT contained in EVs is internalized in target cells and enters NMN / NAD cells. + While it enhances biosynthesis, the NAMPT protein alone cannot be internalized by itself. This provides a crucial solution to the long-standing debate regarding the physiological importance and function of eNAMPT in mammals. eNAMPT is involved in systemic NAD + It functions as a biosynthetic enzyme and in the hypothalamus, NAD + While eNAMPT can enhance SIRT1 activity and neuronal activity (Revollo et al., 2007, Yoon et al., 2015), it has also been reported to function as a pro-inflammatory cytokine (Dahl et al., 2012). Considering that circulating eNAMPT is almost entirely contained within extracellular viable cells (EVs) under physiological conditions, and that only eNAMPT contained within EVs is properly internalized into the cytoplasmic fraction of cells, the physiological relevance and function of eNAMPT are systemic, particularly in tissues with relatively low levels of iNAMPT, such as the hypothalamus, hippocampus, pancreas, and retina, where it interacts with NMN / NAD + This suggests that it is to maintain biosynthesis. The exact mechanism by which eNAMPT-containing EVs specifically target these tissues needs to be elucidated, but this EV-mediated systemic delivery of eNAMPT is related to systemic NAD in mammals. + This is a novel inter-tissue interaction mechanism that maintains homeostasis and regulates the aging process and lifespan.

[0088] NAMPT-mediated NAD + A systemic decline in biosynthesis limits tissue function during aging. In mammals, NAMPT is the major NAD, starting with nicotinamide, a form of vitamin B3. + It is a rate-limiting enzyme in the biosynthetic pathway. Currently, systemic NAD is used.+ Availability declines dramatically with age, and age-related declines in iNAMPT levels are observed in many organizations with NAD. + It is well established that this contributes to the limitation of availability (Yoshino et al., 2018). Here, circulating eNAMPT levels decrease with age in mice and humans, and eNAMPT-mediated NAD + NAD in specific tissues that depend on biosynthesis + This indicates limitations in availability. Adipose tissue-specific overexpression of Nampt maintains circulating eNAMPT levels and results in significant enhancements of physical activity, sleep quality, glucose-stimulated insulin secretion, retinal photoreceptor function, and cognitive function in aging mice. These remarkable anti-aging effects of eNAMPT also contribute to an extension of median lifespan in mice. No significant side effects of eNAMPT, including inflammation and cancer risk, were observed in aging ANKI mice, contradicting the proposed primary function of eNAMPT as a pro-inflammatory cytokine.

[0089] Since circulating eNAMPT levels decline with age, an individual's ability to maintain high levels of circulating eNAMPT must be crucial for maintaining functional homeostasis of tissues over time, and this is likely to determine each individual's healthy lifespan. Results from a small prospective study provide compelling support for this concept, showing a significant correlation between circulating eNAMPT levels and remaining lifespan. eNAMPT secretion from adipose tissue is linked to NAD + Considering that it is regulated in a SIRT1-dependent manner (Yoon et al., 2015), fatty NAD +The reservoir and / or turnover of adipose tissue circulate eNAMPT levels and may thus be a key determinant for lifespan. Interestingly, the life-extending effect of dietary restriction has been reported to be inversely correlated with fat reduction measured at middle and late age, suggesting that certain fat-related factors are important for survival and life extension under dietary restriction (Liao et al., 2011). Based on these results, it would be very interesting to investigate whether eNAMPT secreted from adipose tissue is a significant factor in the delayed aging and life-extending effects of dietary restriction.

[0090] Gene replacement for eNAMPT delays aging and extends healthy lifespan in mice. Aging ANKI mice have increased NAD levels in the hypothalamus, hippocampus, pancreas, and retina. + It shows a significant enhancement of levels and organizational function. Previously, NAMPT-mediated NAD + Biosynthesis and NAD + We demonstrated that dependent sirtuins play a crucial role in regulating these tissue functions. In the hypothalamus, NAD + / SIRT1 signaling is crucial for controlling the aging process and determining lifespan (Satoh et al., 2013). In the hippocampus, NAMPT plays a vital role in the function of excitatory neurons (Stein et al., 2014), particularly neurons in the CA1 region (Johnson et al., 2018). In pancreatic β-cells, NAMPT and SIRT1 are important for regulating glucose-stimulated insulin secretion (Moynihan et al., 2005, Revollo et al., 2007). In the retina, NAMPT and mitochondrial sirtuins SIRT3 / 5 are essential for the function of rod and cone photoreceptor neurons (Lin et al., 2016, Mills et al., 2016). These tissues are NAD + It most likely represents the group of organizations most vulnerable to degradation. eNAMPT also has appropriate NAD +Other tissues may be targeted to maintain biosynthesis. Given that adipose tissue is a major source of circulating eNAMPT (Yoon et al., 2015), it will be important to further elucidate the inter-tissue interactions between adipose tissue and other tissues through EV-mediated eNAMPT delivery.

[0091] Interestingly, the phenotype of aged ANKI mice overlaps with that of aged BRASTO mice (Satoh et al., 2013). In particular, enhanced circling activity and sleep quality are observed in both aged ANKI and BRASTO mice. Consistent with these phenotypes, hypothalamic expression levels of Ox2r and Prdm13, two SIRT1 target genes responsible for these phenotypes (Satoh et al., 2013, Satoh et al., 2015), are significantly increased in both mouse models. Nevertheless, BRASTO mice exhibit both extended median and maximal lifespan, while ANKI mice show only an extension of median lifespan. This discrepancy between BRASTO and ANKI mice suggests an intriguing possibility: that SIRT1 levels in hypothalamic neurons primarily determine their maximal function, thereby limiting maximal lifespan, while circulating eNAMPT levels modulate the degree of hypothalamic neuron function, thereby altering median lifespan. Given that continuous supplementation with eNAMPT-containing EV extends the median and maximum lifespan of aging mice, the effect of eNAMPT in ANKI mice may be inhibited at a very late stage of aging by reducing adipose tissue mass.

[0092] Example 9 Primary neurons were isolated from mouse embryos at E16 and treated in vitro after 7 days in basal neuronal medium (NB) containing the following combinations of additives: Untreated - Neurons were left in standard basal neuronal culture medium containing B27, N2, and glutamine supplementation; NB - Basal neuronal medium without additives; 200 μl EV - EV extracted from 200 μl mouse plasma, FK866 (10 nM)-NAMPT inhibitor; NMN - 250 μM nicotinamide mononucleotide (NAD+ precursor); SN - Supernatant serum recovered from plasma after EV extraction, combined with NB in ​​a 1 / 2 ratio. NAD+ levels were measured after 30 minutes of applicable treatment using an NAD / NADH-Glo fluorescence kit. Figure 14 shows the induction of NAD+ biosynthesis in cultured primary mouse hippocampal neurons by treatment with eNAMPT contained in extracellular vesicles (EVs). Neurons cultured in nutrient-depleted NB-only medium showed decreased NAD+ levels, which were partially rescued by EV supplementation. These data suggest that EV is sufficient to increase neuronal NAD+ content. This increase was blocked by FK866 treatment, suggesting that the EV-induced increase in NAD+ is NAMPT-dependent.

[0093] Primary hippocampal glial cultures were isolated from p2 offspring, and poorly adhering microglia were removed by shaking. Extracellular viable cells (EVs) isolated from mouse plasma were labeled with the sphingolipid dye Bodipy-TR-ceramide. Astrocyte enriched cultures were treated with these labeled EVs for 30 minutes, followed by fixation and immunofluorescence staining for the astrocyte marker GFAP. Figure 15 shows low levels of EV uptake in primary mouse GFAP+ astrocytes and more substantial EV uptake in GFAP cells. Low levels of Bodipy dye can be observed in GFAP+ astrocytes (white arrows), indicating some EV uptake by astrocytes. However, more substantial staining is clearly seen in GFAP cells, some of which exhibit clear microglial morphology (white arrows). These data suggest that astrocytes can take up EVs, but their affinity for EVs may be lower than that of other cell types.

[0094] Figure 16 shows significant EV uptake by microglia. Primary mouse hippocampal microglia cells (see Figure 15) were reseeded from astrocyte-enriched cultures and similarly treated with Bodipy-labeled EVs. Consistent co-localization of Bodipy signaling with immunofluorescence labeling of the microglia marker IBA1 indicates considerable EV uptake by primary microglia. These data suggest that microglia have a high affinity for EV uptake, at least in culture.

[0095] When describing elements of the present invention or preferred embodiments thereof, the articles “a,” “an,” “the,” and “said” are intended to indicate the presence of one or more of the elements. The terms “comprising,” “including,” and “having” are intended to be comprehensive and mean that additional elements other than those listed may exist.

[0096] From the above, it will be seen that some of the objectives of the present invention are achieved, and other advantageous results are attained. Since various modifications can be made to the above methods, processes, and compositions without departing from the scope of the present invention, all subject matter included in the above description and shown in the accompanying drawings is intended to be construed as illustrative and not as limiting.

Claims

1. A composition for increasing NMN or NAD+ biosynthesis in a subject, comprising extracellular nicotinamide phosphoribosyltransferase (eNAMPT) and / or its variants and extracellular vesicles, wherein the extracellular vesicles encapsulate the eNAMPT and / or its variants, and the variant of eNAMPT comprises at least 99% sequence identity with wild-type NAMPT of SEQ ID NO: 1 or SEQ ID NO: 2, and further comprises an amino acid sequence having an arginine residue at a position corresponding to position 53 of wild-type NAMPT of SEQ ID NO: 1, or an amino acid sequence having an arginine residue at a position corresponding to position 53 of wild-type NAMPT of SEQ ID NO:

2.

2. The composition according to claim 1, characterized in that the composition comprises a plurality of extracellular vesicles containing eNAMPT, wherein the vesicles have an average particle size of about 10 nm to about 200 nm, about 10 nm to about 100 nm, or about 20 nm to about 100 nm.

3. The composition according to claim 1 or 2, wherein the concentration of eNAMPT and / or its variant in the composition is about 1% by weight to about 20% by weight.

4. The composition according to any one of claims 1 to 3, wherein the composition comprises eNAMPT.

5. The composition according to any one of claims 1 to 3, wherein the variant of eNAMPT comprises an amino acid sequence having an arginine residue at a position corresponding to position 53 of wild-type NAMPT of SEQ ID NO: 1, and the remaining amino acid sequence of the variant comprises at least 99% sequence identity with SEQ ID NO:

1.

6. The composition according to any one of claims 1 to 3, wherein the variant of eNAMPT comprises an amino acid sequence having an arginine residue at a position corresponding to position 53 of wild-type NAMPT in SEQ ID NO: 2, and the remaining amino acid sequence of the variant comprises at least 99% sequence identity with SEQ ID NO:

2.

7. The composition according to any one of claims 1 to 3, wherein the variant of eNAMPT has at least 99.9% sequence identity with the wild-type NAMPT of SEQ ID NO: 1 or SEQ ID NO:

2.

8. The composition according to any one of claims 1 to 7, wherein the composition does not contain or is essentially free from adipocytes, blood, and / or plasma.

9. A pharmaceutical composition comprising the composition according to any one of claims 1 to 8 as an active ingredient for increasing NMN and / or NAD+ biosynthesis in a target or cell.

10. A pharmaceutical composition for preventing or treating age-related conditions in a subject, The pharmaceutical composition according to claim 9, wherein the age-related condition includes a physiological condition selected from the group consisting of decreased physical activity, decreased sleep quality, decreased cognitive function, decreased glucose metabolism, decreased visual acuity, and combinations thereof.

11. A process for preparing the composition according to any one of claims 1 to 8 or the pharmaceutical composition according to any one of claims 9 or 10, A process comprising subjecting a culture medium containing the lipid and eNAMPT and / or its variants to a separation process to obtain a concentrated vesicle fraction, wherein the concentration of the vesicles in the concentrated vesicle fraction is higher than the concentration of the vesicles in the culture medium.

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