Microrna engineered vesicles to promote cell reprogramming

Abstract

In multicellular organisms, complex secondary epigenetic regulatory systems are crucial for determining cell function and gene expression patterns across different organs and tissues, involving noncoding microRNAs (miRNAs), methylated DNA marks, and both methylated and acetylated histones. An intricate system of epigenetic gene regulation is not only fundamental to maintaining balanced and healthy cell function but also seems intrinsically linked to the functional decline observed during aging, environmental damage, and disease. The disclosed system uses prescribed sets of miRNA mimics and miRNA inhibitors that can stimulate the transient protein output of the OCT4, SOX2, and KLF4 protein transcription products. Each set of miRNAs is loaded into extracellular vesicles or nanocarrier vesicles to form nano bio-therapeutics that are designed to reprogram and rejuvenate aging tissues and organs. The therapeutical aim is to rejuvenate by reprogramming tissues and organs while minimizing the risks of cancer.

Description

[0001] MicroRNA Engineered Vesicles to Promote Cell Reprogramming

[0002] Cross-Reference To Related Applications

[0003] This application claims benefit from US provisional application No. 63 / 551,513, filed February 8, 2024 and US provisional application No. 63 / 647,622, filed May 15, 2024, each of which is incorporated by reference herein for all purposes.

[0004] Field Of Art

[0005] The disclosed system relates generally to epigenetic gene regulation to restore tissue and organ vitality while minimizing the risks of oncogenesis, and more specifically to a system using miRNAs that induce the transient cellular production of Oct-4, Sox-2, and KLF-4 without reverting to the induced pluripotent state of embryonic stem cells, and being loadable into artificial or natural vesicles to form miRNA engineered vesicle therapeutics that are designed to reprogram and rejuvenate aging tissues and organs.

[0006] Background

[0007] All life relies on a genetic blueprint for replication, development, and adaptive responses to environmental changes. The genetic code is stored in DNA and remains largely uniform across the various cells within an organism's body. However, in multicellular organisms, a complex secondary epigenetic regulatory system also plays a pivotal role. The epigenetic regulatory system, which is crucial for determining cell function and gene expression patterns across different organs and tissues, involves noncoding microRNAs (miRNAs), methylated DNA marks, and both methylated and acetylated histones. These elements work together in a sophisticated feedback mechanism, controlling the specialized patterns of DNA folding and gene expression in each tissue and organ. This intricate system of epigenetic gene regulation is not only fundamental to maintaining balanced and healthy cell function but also is intrinsically linked to the functional decline observed during aging, environmental damage, and disease. The current system uses a prescribed set of miRNA mimics and miRNA inhibitors that can induce the transient expression of Oct-4, Sox-2, and KLF-4. Each set of miRNAs is loaded into artificial or natural vesicles (artificial nanocarriers or natural extracellular vesicles) to form miRNA engineered vesicle therapeutics that are designed to reprogram and rejuvenate aging tissues and organs. The therapeutical aim is to restore tissue and organ vitality while minimizing the risks of oncogenesis. The reprogramming therapeutic system disclosed herein prioritizes maximal effectiveness and safety, representing a significant advancement in the field of regenerative therapeutics.

[0008] To understand epigenetic reprogramming technology, it is helpful to give some background on aging and development. Multipotent adult stem cells typically undergo epigenetic changes to differentiate into progenitor cells that form all the hundreds of differentiated tissue types (such as heart, liver, lung, muscle, kidney, neurons, skin cells, etc.) in a mature adult animal. Apart from these developmentally programmed epigenic events, aging itself involves epigenetic changes to DNA (methylation marks) and histones via enzymes that affect their state of methylation or acetylation [1 -31. These DNA and histone epigenetic changes affect the open or closed folding of the chromatin DNA that determines gene expression levels. Typically, genes are generally active in open chromatin regions and inactive in closed regions.

[0009] This epigenetic regulation of genes occurs in both somatic cells [3, 4] and adult stem cells [5], Programmed cell changes, lifestyle, stress, and dietary intake can all affect the epigenetic chromatin state [6-81, leading to progressive alterations in gene expression as the body develops or ages. Moreover, the activity of the Long Interspersed Element-1 (L1), as part of the large transposable element group, is modulated by miRNAs to increase genomic instability and the risk of age-related diseases [9, 101. As a result of these age-related epigenetic changes, gene expression typically moves away from optimal youthful fitness largely due to the epigenetic drift in the chromatin state forged by epigenetic regulation by miRNAs in response to the micro-environmental influences with the passage of time. Thus, the specific configuration (e.g. DNA marks and histone epigenetic changes of chromatin) of the youthful chromatin gene structures are slowly disturbed with time, leading to more dysfunctional gene expression patterns. This is consistent with the miRNA theory of aging, which proposes that epigenetic “drift” with aging is caused by changes in the youthful miRNA expression patterns brought on by miRNA stress signaling responses of cells to multiple micro-environmental stress events that sporadically occur with the passage of time [111. Of course, there are many existing theories of aging, including the recent information loss theory of aging

[0012] ), which is currently gaining popularity. Apart from any particular theory of aging, the key translational question is how does one safely and effectively reprogram the epigenetic state to stop or reverse aging in humans?

[0010] To get around the epigenetic drift in older cells, in the 1990s, Applicant studied the use of embryonic stem cells from in vitro fertilized human eggs that are pluripotent and can form all tissue types. These embryonic stem cells (ES cells) have a functional age of zero and thus were of great interest in culturing human ES cells ex vivo. However, it soon became apparent that the injection of ES cells in humans could be problematic as there are ethical concerns related to human embryo destruction and ES cells are not genetically the same as the cells of a patient and thus are typically rejected by the immune system. Moreover, ES cells are known to spontaneously form teratoma cancer cells.

[0011] A breakthrough discovery in 2007 reversed the epigenetic changes in older cells by transfecting an individual’s fibroblasts with only four genes OSKM (OCT4, SOX2, KLF4, and c-MYC) to prepare induced Pluripotent Stem (iPS) cells [131. These iPS cells have reversed epigenetic changes in an individual’s adult somatic cells back into their beginning embryonic state and have been used like ES cells to clone mice, dogs, and horses with normal lifespans. Unfortunately, reprogramming efficiency is typically very low and iPS cells are even more likely than ES cells to generate tumors [14-161. These factors, plus the need for extensive reprogramming to form progenitor cells that can replace various adult tissues [17- 191, has long thwarted iPS use in the clinic.

[0012] One solution to the problems with ES and iPS cells is to use the patient’s own autologous Adipose Mesenchymal stem cells (AD-MSCs), which are presently the most common stem cell product in commercial use. For example, autologous AD-MSCs can be injected into aged or injured joints because of their safety and minimal FDA regulation. However, most adults have declining numbers of functional adult stem cells that can be used for regenerative purposes [20-241 and the MSCs extracted from a patient cannot be expanded efficiently as they typically senesce in culture [2], Therefore, current regenerative therapies using MSCs often disappoint due to the low dosage of functional MSCs, especially if those MSCs are extracted from older or unhealthy individuals.

[0013] To get around the low-functioning autologous MSCs from older adults, heterologous umbilical cord mesenchymal stem cells (UC-MSCs) have been used with generally good outcomes. The UC-MSCs are neonatal cells that have youthful stem cell vigor, which is more efficacious than the autologous AD-MSCs from older adults. Nevertheless, heterologous UC- MSCs can still promote unwanted immune reactions in some patients and UC-MSCs contain fewer cells than typically delivered with AD-MSCs from abdominal fat. Both these factors have limited the use of UC-MSCs. In summary, existing cell technology is limited in providing the full benefits of youthful stem cell treatments for treating disease.

[0014] To address these long-standing impediments to stem cell regenerative therapeutics, Applicant attempted to identify ways to "reverse age" each person’s own adult stem cells to a younger state via epigenetic drug reprogramming in vitro with the eventual goal of their use in clinical trials. At that time in 2021-2022 there was no effective way to reliably reprogram adult stem cells back to a younger state without generating safety risks employing viral expression vectors using at least 3 of the OSKM genes, as some cells may reach the iPS stage while others remain in a relatively unchanged epigenetically old state. Nevertheless, some scientists had transfected inducible OSKM gene viral vectors into mice and had significant success in safely reversing epigenetic age if the OSKM genes are only expressed intermittently [25, 261 ■ Even better results were obtained with inducible viral expression vectors using OSK genes that drop c-Myc from OSKM to minimize cancer risks [271. Given the difficulties of gene therapy using the OSK or OSKM viral vectors and the safety issues with the potential generation of iPS cells [281, Applicant focused on epigenetic drugs to see if epigenetic drugs might be safer and more effective than expressing the OSKM or OSK genes.

[0015] After screening a limited set of epigenetic drugs to determine a combo that might reduce epigenetic age in older human AD-MSC cells, Applicant discovered one strong hit from its screens, which comprised four epigenetic kinase drug inhibitors: Go6983, SB525334, PD0325901, and Chroman 1. The experiment was repeated with several controls to verify success in age reversal. First, AD-MSCs from a 49-year-old female were cultured ex vivo in DMEM / F12 media with 5% fetal calf serum in a 3% oxygen cell incubator. Four epigenetic drugs were added to the standard cell media at sufficient doses that effectively inhibit 4 cellular enzymes for a period of 6 to 24 days with fresh media and drugs added every 4 days. The drugs used and their doses comprised: 1 uM Go6983, 1.4 uM SB525334, 0.04 uM PD0325901, and 10 nM Chroman 1. After drug incubation, the epigenetic drugs were washed out and the cells were cultured in standard media for 4 days without drug treatments. Treated cells were checked for epigenetic age using the SkinBlood, GrimFit, and Horvath DNAmAge epigenetic clocks at the Clock Foundation (Irvine, CA), which can be viewed in Figures 1 and 2 below. The epigenetic clock data clearly shows a significant reduction of cellular age of about 4 to 6 years.

[0016] While the age reversal on direct exposure of cultured stem cells to epigenetic drugs is an important discovery, it is not clear if this result of 3 to 6 years of age reductions using cocktails of 4 or more epigenetic drugs can lead to chemical drug treatments in humans, as these potent epigenetic drugs could have toxic side effects on multiple tissues and organs.

[0017] In other data not shown, telomere length was also tested on treated samples and found that exposure to the epigenetic drugs shortened telomere length, which argues that the drug cocktail was not a fully successful epigenetic age reduction treatment. The Yang et al. paper did not report on telomere effects using their six drug cocktails

[0029] , so it is unknown how telomeres changed in their case.

[0018] Another promising epigenetic reprogramming therapeutic is Extracellular Vesicles (EVs) derived from neonatal or young stem cells. EVs are widely known to play important roles in cell-to-cell communication, cancer biology, immune responses, growth signaling, and cellular reprogramming. EVs exhibit different activities based on their origin. While EVs released from cancerous or senescent cells promote dysfunctional cellular changes, those from neonatal or young stem cells are believed to facilitate cellular rejuvenation [27-291. Spanning diameters of 40 to 1000 nm, EVs encapsulate a diverse array of cellular factors, including enzymes, growth factors, proteins, mRNAs, and miRNAs. Their membrane-bound structure enables them to traverse cell membranes seamlessly, including penetrating the blood-brain barrier. Notably, EVs excel as nanocarriers for pharmaceuticals and therapeutic agents, attributed to their inherent cell-targeting capabilities, minimal immunogenicity, and superior biodegradability

[0030] . EVs sourced from young stem cells are being increasingly recognized for their potential in treating a multitude of diseases and age-related ailments.

[0019] However, there are notable challenges in harnessing EVs from young stem cells for therapeutic applications. The inherent heterogeneity in EV populations, with varying cargo compositions, directly influences therapeutic outcomes. The methods employed in EV isolation are crucial, as they significantly affect both the quality and quantity of EVs, factors that are vital for clinical usage. The production of EVs at a scale suitable for clinical applications poses a significant financial and technical challenge, while variability in efficacy from batch to batch - owing to cellular degradation over passages or microbial contamination - remains concerning. Moreover, the potential for immune responses triggered by some EVs necessitates rigorous testing. Regulatory complexities also present significant obstacles in the development and approval of EV-based therapies. Additionally, the presence of toxic EVs released from senescent cells in older individuals may negate the mild beneficial effects of administered youthful stem cell-derived EVs [32, 33]. One recent study shows that treatment with young pork EVs halved the epigenetic ages of blood, heart, and liver tissue

[0031] , Since pigs can carry harmful viral threats such as swine influenza, hepatitis and pathogenic E, there are concerns about their use as EV treatments. However, all mammalian cells can carry viruses and pork EVs have many strong benefits that make them a strong choice for mammalian derived EVs.

[0020] This disclosure describes Applicant’s approach to reprogramming epigenetic age through the use of microRNAs (miRNAs or miRs). MiRNAs constitute a large group, with over 2600 human miRNAs cataloged. These small non-coding RNAs, typically comprising 18-25 base pairs, play a significant role in post-transcriptional gene regulation by either repressing translation or degrading target mRNAs. While most miRNA studies focus on screening miRNAs as markers for disease states, there is a substantial body of research dedicated to investigating miRNAs as potential therapeutic agents. These proposed miRNA therapeutics encompass synthetic double-stranded miRNAs, often referred to as miRNA mimics, which are utilized to restore endogenous miRNA levels. Additionally, there are antisense miRNA inhibitors, consisting of single-stranded RNA molecules, designed to reduce the expression of specific target miRNAs. The field is witnessing a growing number of miRNA-based therapies undergoing preclinical testing, targeting a wide range of age- related diseases and disorders, including Alzheimer's disease, inflammatory diseases, stroke recovery, and skin aging [34-381.

[0021] The quest for the most effective and safest therapeutic to reverse epigenetic aging in vivo and enhance cellular and organ rejuvenation in humans remains ongoing. Current insights suggest a lack of established therapies capable of safely reversing epigenetic age. The complexities and challenges associated with neonatal stem cell-based epigenetic therapeutics, epigenetic drugs, or inducible OSKM and OSK viral gene vector systems make reliance on these strategies precarious for developing safe and effective in vivo age-reversal treatments and cell rejuvenation. Although Extracellular Vesicles (EVs) from young stem cells show promise, significant hurdles remain. Notably, producing these stem cell derived EVs at scale is both financially and technically demanding, and consistency across different batches is a major concern.

[0022] The system disclosed herein comprises designing engineered extracellular vesicles or nanocarrier vesicles loaded with 2 or more miRNA therapeutics that stimulate transient endogenous OSK genes in cells while suppressing the induction of iPS cells, and expands on US provisional application No. 63 / 551,513, filed February 8, 2024 and US provisional application No. 63 / 647,622, filed May 15, 2024, each of which are incorporated by reference herein for all purposes. The system disclosed herein is built on the miRNA Theory of Aging, wherein changes in miRNA patterns are the main drivers of the aging process

[0039] . The miRNAs are major determinants of tissue and organ types [40-421 that are nearly fully developed in the newborn. While miRNAs are required for cell differentiation during pre- neonatal development [43, 44], miRNA induced changes continue at slower rates throughout childhood and adult life. Moreover, miRNA are critical factors in how cells respond to changes in the environment and to all types of stress [45, 46], Applicant theorizes aging is caused by the plethora of adaptive changes in the cell miRNA patterns due to the miRNA responses to all types of stress that occur throughout an individual’s lifespan. More evidence for the miRNA theory of aging is found in a newly published paper

[0047] showing that miR-302 has the potential to reverse cellular senescence. Note that the miR-302 was loaded into human embryonic stem cells (hESCs), which likely added critical miRNAs to get their rejuvenation and age reversal. The system disclosed herein provides a blueprint to identify the full complement of miRNAs needed for age reversal (those that stimulate OCT4, SOX2, and KLF4 or load miRNAs into young animal derived EVs (like pork EVs) to supply the missing miRNAs.

[0023] Summary of the Disclosure

[0024] An aspect of the disclosed system is to provide for rejuvenating therapeutics comprising extracellular vesicles (EVs) or nanocarrier vesicles (nanocarriers) engineered by loading a set of two or more types of microRNAs (miRNAs) that induce transient cellular protein production of OCT4, SOX2, and KLF4 but which do not reprogram cells back to the induced pluripotent state of embryonic stem cells.

[0025] Another aspect of the disclosed system is to provide for rejuvenating therapeutics comprising EVs or nanocarriers that have been loaded with mature miRNAs encoding at least two precursor miRNA genes that induce transient cellular protein production of OCT4, S0X2, and KLF4 but which do not reprogram cells back to the induced pluripotent state of embryonic stem cells.

[0026] Yet another aspect of the disclosed system is to provide rejuvenating therapeutics comprising EVs or nanocarriers that have been loaded with inducible DNA expression plasmid vectors encoding at least two precursor miRNA genes that induce transient cellular protein production of OCT4, S0X2, and KLF4 but which do not reprogram cells back to the induced pluripotent state of embryonic stem cells.

[0027] Another aspect of the disclosed system is to provide formulations to epigenetically reprogram cells which utilize nanocarriers or EVs to efficiently and safely transport the mature miRNA or miRNA genes through the circulatory system.

[0028] Another aspect of the present system is the use of pork or grape EVs loaded with a known set of miRNAs.

[0029] Another aspect of the disclosed system is to provide miRNA engineered EVs that can safely produce potent therapeutics for rejuvenation in vivo. Yet another aspect of the disclosed system is to utilize a miRNA system to adjust epigenetic cell responses for all types of cell stress and environmental changes.

[0030] Another aspect of the disclosed system is to utilize miRNAs that can transiently activate 0CT4, S0X2, and KLF4 to make epigenetic changes in the cell programming to adjust to changes in the environment.

[0031] Another aspect of the disclosed system is to utilize sets of two or more miRNAs to reprogram old cells by transient activation of the three Yamanaka genes 0CT4, S0X2, and KLF4.

[0032] Yet another aspect of the disclosed system is to utilize mature miRNAs rather than the miRNA DNA genes to minimize the cancer risks incurred using plasmid vectors.

[0033] Another aspect of the present system is to enable the generation of plasmid miRNA vectors to maximize the potential tools of the miRNA therapeutics in treating multiple diseases and extending lifespan.

[0034] Another aspect of the system disclosed herein is to use EVs as natural carriers for therapeutic sets of miRNA types to safeguard miRNA and plasmid DNA during passage in the blood stream and facilitate the integration of all the needed miRNA types in each EV that fuses into each cell.

[0035] Another aspect of the present system is to use differing miRNA types in a set to reprogram cells in a consistent manner.

[0036] Another aspect of the system disclosed herein is the development of miRNAs engineered EVs as cell reprogramming therapeutics that do not require changes in the DNA genetic code or risky transfections of exogenous DNA or gene editing. An aspect of the present therapeutic system is to provide for miRNA engineered nanocarriers or EV vesicles that are expected to have low side effects, low immunogenicity, low cancer risks, good membrane permeability, and tissue targeting potential.

[0037] Another aspect of the present system is to couple the miRNAs, known for epigenetic regulation, with the nano scale cell to cell communication role of vesicles to generate a therapeutic potential for in vivo epigenetic reprogramming.

[0038] Another aspect of the disclosed system is to guide some miRNA sets to targeted adult stem cells in tissues or organs.

[0039] Description of the Figures

[0040] Fig 1 compares duplicate samples of adipose stem cells treated with Control (0 drug) and four NT9 treatments with increasing drug exposure (samples 1 to 4) data for each of two methylated CpGs epigenetic Clocks analyzed by the Clock Foundation in Irvine, CA. The GrimFitAge and Skin&Blood clocks gave similar results with declines of ~4 years and unpaired t-tests of t = 0.0125 and t = 0.011 respectively.

[0041] Fig 2 compares duplicate samples of Adipose Control (0 drug) and four NT9 treatments with increasing drug exposure (samples 1 to 4) data for the Clock. The data show ~6 years reduction of Horvath DNAmAge epigenetic age for the longest drug exposure. An unpaired t-test analysis gives significance of t = 0.001.

[0042] Fig 3 shows MA Plot of common genes (black) vs differentially expressed genes (non-black - 1.5%) for Control vs T reated. Only the epi genes (about 1.5% of the genes) changed expression levels with NT9 treatment.

[0043] Fig 4 depicts the quality of the grape EVs which was checked by Nanoparticle Tracking Analysis (NTA). The NTA analysis shows a variety of EV sizes with an average EV diameter of 149 nm for the grape EVs. The EV yield was some 10 ml of 10A12 EVs / ml.

[0044] Fig 5 depicts the quality of the EV morphology as checked by Transmission Electron Microscopy (TEM).

[0045] Fig 6 depicts Multidimensional Scaling (MDS) analysis of samples. MDS was conducted to visualize the distance / similarity between T1 , T2, C4, C6. Top 500 genes with highest variance among samples were used to make this plot.

[0046] Fig 7 illustrates the incidence of the most prevalent chronic diseases, risk of death (the age specific mortality rate) and healthspan for over 300,000 UK participants. The disease incidence increases exponentially with age at approximately the same rates for all the age-related disease states. The diseases and health indications are Healthspan, Cancer, Death, Myocardial Infarction (Ml), Diabetes, Chronic Obstructive Pulmonary Disease (COPD), Stroke, Congestive Heart Failure (CHF), and Dementia

[0053] . Description of the Disclosed Embodiments

[0047] The miRNA system appears to be the universal epigenetic regulating system that nature has selected to adjust epigenetic cell responses for all types of cell stress and environmental changes. The miRNAs play a key role in reprogramming gene expression and thus can make epigenetic changes in the cell programming to adjust to changes in the environment. For example, the miRNA expression pattern in the brain shows a high specificity for developmental stage and cell type

[0048] . It is Applicant’s theory that miRNAs play a crucial role in regulating the state of cellular aging. Therefore, changes in miRNAs are critical to reprogramming aging. There are over 2600 miRNAs that are estimated to control some 1 / 3 of the human genes, but only a relatively few miRNAs can transiently activate OCT4, SOX2, and KLF4. Since a single miRNA type can directly or indirectly target hundreds of genes, two or more miRNA types can have huge effects on reprogramming gene expression.

[0048] The system disclosed herein utilizes sets of two or more miRNAs to reprogram old cells by transient activation of the three Yamanaka genes OCT4, SOX2, and KLF4. For example, the highly conserved polycistronic miR-302 / 367 family cluster of 5 miRNAs (miR-302a, miR-302b, miR-302c, miR-302d, and miR-367) has hundreds of direct and indirect targets and can reprogram mouse and human cells to an embryonic iPS cells state by targeting the endogenous OCT4 and SOX2 mRNAs and is 100 times more efficient than standard Yamanaka OCT4, SOX2, KLF4, and cMYC DNA transfection methods in helping generate iPS cells under the right conditions. [491. All miR-302 family members have the same seed sequence of 5-AAGTGC-3' except for miR-367

[0050] . This miR-302 cluster has also been put into a Dox inducible plasmid expression vector and transfected into adult human hair follicle stem cells to generate embryonic induced Pluripotent stem (iPS) cells wherein the miR-302 family reprograms the hair follicle cells to express OCT4, SOX2, KLF4, and NANOG [511. The miR-302 cluster of miRNAs has also been combined with miR-200c and miR-369 to reprogram mouse and human somatic cells into iPS cells using the mature miRNAs instead of viral or plasmid constructs [521. Since miRNAs have the potential to reprogram adult hair follicle cells into embryonic iPS cells, there does not appear to be any barrier to reprogramming adult cells back to youthful functional states like fetal or newborn cells.

[0049] The system disclosed herein prefers mature miRNAs rather than the miRNA DNA genes to minimize the cancer risks incurred using plasmid vectors [521. However, plasmid miRNA vectors are generated to maximize the potential tools of the miRNA therapeutics in treating multiple diseases and extending lifespan. While mature miRNAs and miRNA DNA are often injected directly or with nanocarriers into the blood stream, the system disclosed herein prefers the use of EVs as natural carriers for therapeutic sets of miRNA types to safeguard miRNA and plasmid DNA during passage in the blood stream and facilitate the integration of all the needed miRNA types in each EV that fuses into each cell. The copy number balance of differing miRNA types in a set is critical to reprogramming cells in a consistent manner and EVs may organically carry some of the critical miRNAs.

[0050] The system disclosed herein provides for the development of miRNAs engineered EVs as cell reprogramming therapeutics that do not require changes in the DNA genetic code or risky transfections of exogenous DNA or gene editing. Thus, the proposed miRNA engineered nanocarriers or EV vesicles are expected to have low side effects, low immunogenicity, low cancer risks, good membrane permeability, and tissue targeting potential. Coupling the miRNAs, known for epigenetic regulation, with the nano scale cell to cell communication role of vesicles generates a powerful therapeutic potential for in vivo epigenetic reprogramming. Moreover, vesicles have some innate targeting capacity, which can help guide some miRNA sets to targeted adult stem cells in tissues or organs.

[0051] The system disclosed herein utilizes pork or grape EVs loaded with a known set of miRNAs that may effectively reprogram many of the more than 30 trillion cells in the human body. Moreover, miRNA engineered EVs can safely produce potent therapeutics for rejuvenation in vivo. The system disclosed herein comprises designing engineered EVs or nanocarriers loaded with 2 or more miRNA therapeutics (either as matured miRNAs or as DNAs that code for the miRNAs) that stimulate transient OSK gene expression. Since the induced OSK gene expression is transient, this method naturally suppresses the induction of iPS cells. The system disclosed herein is partially built on the miRNA Theory of Aging, wherein changes in miRNA patterns are the main drivers of the aging process

[0039] . The miRNAs are major determinants of tissue and organ types [40-421 that are nearly fully developed in the newborn. While miRNAs are required for cell differentiation during pre- neonatal development [43, 44], miRNA induced changes continue at slower rates throughout childhood and adult life. Moreover, miRNA are critical factors in how cells respond to changes in the environment and to all types of stress [45, 46], As stated above, Applicant believes that aging is caused by the plethora of adaptive changes in the cell miRNA patterns in responses to all types of stress that occur throughout an individual’s lifespan.

[0052] In one embodiment, therapeutics for epigenetic reprogramming comprise at least one subset of cells, tissues, or organs from a mammal. The therapeutics comprise vesicles engineered by loading a set of two or more types of microRNAs (sense or antisense miRNAs) that induce transient cellular production of OCT4, SOX2, and KLF4 but which do not reprogram cells back to the induced pluripotent state of embryonic stem cells. Either sense / antisense miRNAs or DNA coding sense / antisense miRNAs are loaded into EVs or nanocarriers. Since a single miRNA type can directly or indirectly target hundreds of genes, two or more sense / antisense miRNA types can have huge effects on reprogramming gene expression. For example, the highly conserved polycistronic miR-302 / 367 family cluster of 5 miRNAs (miR-302a, miR-302b, miR-302c, miR-302d, and miR-367) has hundreds of direct and indirect targets and can reprogram mouse and human cells to an embryonic iPS cells state by targeting the endogenous OCT4 and SOX2 mRNAs and is 100 times more efficient than standard OCT4, SOX2, KLF4, and cMYC DNA transfection methods in helping generate iPS cells under the right conditions [491. All miR-302 family members have the same seed sequence of 5-AAGTGC-3' except for miR-367

[0050] . However, the miR-302 family induces OCT4 and SOX2 but not KLF4 [511, so at least one other miRNA type such as anti-miR-128-3p

[0054] , anti-miR-7-5p [55, 56], anti-miR346 [571, or anti-miR-145

[0058] to induce KLF4 is required. Since the reprogramming does not include any viral vectors, these miRNAs engineered EVs are expected to have low side effects, low immunogenicity, and low cancer risks. However, use of DNA coding for the miRNAs does have a somewhat increased risk of promoting cancer.

[0053] In another embodiment, the vesicles for the epigenetic reprogramming therapeutics comprise extracellular vesicles or nanocarrier vesicles loaded with at least two microRNAs (sense or antisense miRNAs) that induce transient cellular production of OCT4, SOX2, and KLF4 but which do not reprogram cells or tissues back to the induced pluripotent state of embryonic stem cells. Mature miRNAs and / or antisense miRNAs that code the various mature miRNAs or antisense miRNAs are used. Taking the miR-302 family described above, EVs are loaded with at least one miRNAs comprising miR-302a, miR-302b, miR-302c, or miR-302d along with a KLF4 producing miRNA such as anti-miR-128-3p, anti-miR-7-5p, anti- miR346, or anti-miR-145.

[0054] The vesicles for the epigenetic reprogramming therapeutics could also comprise extracellular vesicles or nanocarrier vesicles loaded with at least two microRNA genes (coding for sense or antisense miRNAs) that induce transient cellular production of OCT4, SOX2, and KLF4 but which do not reprogram cells or tissues back to the induced pluripotent state of embryonic stem cells. Here, the system uses DNA plasmids that code the various miRNA genes or anti-genes along with an inducible promoter to turn on the miRNA genes or anti-genes. Taking the miR-302 family described above, EVs are loaded with the whole miR-303 set in a DNA plasmid comprising miR-302a, miR-302b, miR-302c, and miR-302d along with an inducible promoter and KLF4 producing miRNA genes such as anti-miR-128- 3p, anti-miR-7-5p, anti-miR346, or anti-miR-145. While an embodiment comprising miRNA DNA to engineer vesicles may have higher cancer risks due to the potential for integration of the plasmid DNA into the patient’s cells, the benefits may be worth the slightly increased risks with life threatening diseases like cancer and cardiovascular disease due to the potential for longer lasting miRNAs and the ability to better control the miRNA expression vectors with inducible promoters.

[0055] In another embodiment, the reprogramming comprises epigenetic reprogramming of at least one subset of cells, tissues, or organs that reduces epigenetic cellular age, as verified by reduced epigenetic clock age using DNA methylation at specific CpG sites in the genome or by changes in gene expression patterns. The miRNA loaded EV therapeutics are expected to change cell DNA methylation at specific CpG sites that will affect epigenetic aging. Currently, the preferred epigenetic clocks for human cells comprise DNAm Age, GrimFit Age, and Skin&Blood (Fig 1 and 2), but different clocks are typically used for cells from mice and other mammals. Gene expression patterns change with age, so transcriptome analysis can be used to see if the biological age of a subgroup of cells has been significantly reduced and if the treatment makes the transcription pattern more like young adult cells.

[0056] As disclosed, treatment of a subset of cells, tissues, or organs can promote more functional multipotent adult stem cells and renewal of progenitor cells but does not reprogram cells or tissues back to an induced pluripotent state. The multipotent adult stem cells (e.g. all types of mesenchymal stem cells) and the progenitor cells are primary targets of the present system of therapeutics. There are 4 reasons for this. Adult stem cells and most progenitor cells are easier to reprogram as most already have primed some of the needed genes (e.g. OCT4 and KLF4 are sometimes expressed at zero to low levels in mesenchymal stem cells) and the cells can undergo rapid division. Second, adult stem and progenitor cells are needed to replace senescing or damaged differentiated cells. Unfortunately, the functions of adult stem and progenitor cells decline with age. Third, differentiated cells are performing many essential organ functions (e. g. heart, liver, and lung tissues) and there could be harmful side effects if many fibroblasts, chondrocytes, keratinocytes, pancreatic islet cells, hepatocytes, muscle cells, and immune cells are diverted from producing their needed cell products by reprogramming to a more stem like state. Fourth, most fully differentiated cells have a low or nonexistence level of proliferation which makes reprogramming more difficult. The capacity of stem and progenitor cells to replicate and divide strongly suggests that they can be reprogrammed by a therapeutic as disclosed. Moreover, progenitor cells may be reprogrammed to adult stem cells by said therapeutic.

[0057] While the reprogramming of adult stem cells and progenitor cells are primary targets for the disclosed therapeutics, the generation of pluripotent embryonic stem cells by said therapeutic poses a significant risk of cancer and thus would be discarded if cancer cells or iPS cells do arise from any of the preclinical testing of the therapeutics. A quick test of cancer tendency would be the ability to form rapidly growing colonies in petri dishes. However, the best test of potential cancer cells comprises injecting cells treated with miRNA engineered EVs into nude mice and check for the growth of cancer cells over the following 3- 8 weeks.

[0058] Mature miRNAs or DNA coding miRNAs are directly transfected into isolated vesicles using electroporation, passive encapsulation, or transfection reagents such as Lipofectamine, FuGENE, ExoFect, Polyethylenimine, and calcium chloride. Both EVs and nanocarriers are vesicles that can encapsulate a diverse array of cellular factors, including enzymes, growth factors, proteins, mRNAs, and miRNAs. Their membrane-bound structure enables them to traverse cell membranes seamlessly, including penetrating the blood-brain barrier. Notably, both EVs and nanocarriers excel as carriers for pharmaceuticals and therapeutic agents, attributed to their minimal immunogenicity. However, loading charged miRNAs and DNAs across the bilayer membrane of the vesicle can be more complicated. During the formation of nanocarriers, RNA or DNA can be efficiently encapsulated passively or electroporation can be used to create transient pores in the lipid bilayer, allowing RNA or DNA to enter the nanocarriers. In the case of EVs, electroporation works efficiently as does the use of transfection reagents like Lipofectamine, FuGENE, ExoFect, Polyethylenimine (PEI), and calcium chloride.

[0059] ExoFect with grape-derived EVs was used for Applicant’s first experimental work. A set of 2 or more miRNA types are loaded together into each batch of EVs in high copy number (> 500 copies) that may exceed the mixed cargo of native miRNAs in the EVs. This use of individual sets of 2 or more types of miRNAs in each EV helps to ensure that each of the genes OCT4, SOX2, and KLF4 are in the therapeutic window of intermittent expression between too little and too much. It should be noted that grape EVs, like other plant EVs likely require a full set of added miRNAs, since grape EVs are unlikely to organically have needed human factors for age regulation. Applicant continues to develop young pork EVs which should supply some organic age lowering miRNAs.

[0060] Some embodiments of loaded human miRNA sets comprise Anti-miR-128-3p plus at least one miRNA mimic from the set of the miR-302 / 367 family of miR-302a-3p, miR-302b-3p, miR-302c-3p, miR-302d-3p, and miR-367-3p. Table 1 below shows 14 miRNA sets that can induce OCT4, SOX2, and KLF4, along with the last 3 miRNAs that have epigenetic affects that may balance the OSK genes. The highly conserved polycistronic miR-302 / 367 family cluster of 5 miRNAs (miR-302a, miR-302b, miR-302c, miR-302d, and miR-367) has hundreds of direct and indirect targets and can reprogram mouse and human cells. All miR-302 family members have the same seed sequence of 5-AAGTGC-3' except for miR-367

[0050] . The miR-302 family induces OCT4 and SOX2 but not KLF4

[0051] , so anti- miR-128-3p [541 is added to generate KLF4. Anti-miR-128-3p is a favored KLF4 activator because it also activates hnRNPAI (heterogeneous nuclear ribonucleoprotein A1), which is required for cell proliferation, telomerase activity, and L1 transposable element activation HP].

[0061] Some embodiments of loaded human miRNA sets may also comprise at least one miRNA type from set A comprising Anti-miR-346, Anti-miR-7, Anti-miR-145, Anti-miR-128, miR-200a, miR-200b, miR-200c; plus at least one miRNA type from set B of the miR-302 / 367 family (miR-302a-3p, miR-302b-3p, miR-302c-3p, miR-302d-3p, miR-367-3p). Table 1 below shows a prospective list of 17 miRNAs that are linked to the regulation of one or more OCT4, SOX2, and KLF4 (OSK) genes and other cell epigenetic factors. The main OSK genes are typically activated by two large families: miR-200s DNA (rows 1-3) and miR-302 / 367 (rows 4-8) that support stem cells function [591. Other miRNAs (rows 9-14) suppress one or three of the OSK genes, but their anti-miRNAs can activate one or three of the OSK genes. The last 3 rows (15-17) show miRNAs that modify epigenetic functions that may help balance or modify the miRNAs that directly target the OSK genes. For example, methyltransferase 3a and 3b (Dnmt 3a and Dnmt 3b) are part of a family of highly conserved DNA methyltransferases that catalyze 5-methylcytosine methylation. Regulatory domains of Dnmt 3a, b allow interactions with histone methyltransferase to influence gene expression. Epigenetic Factors include the DNA repair p53 gene, and the p21 gene that inhibits the activity of the cyclin-dependent kinases (CDKs) which are involved in epigenetic changes.

[0062] Table 1. List of 17 human miRNAs that are linked to the regulation of one or more of OCT4, SOX2, and KLF4 genes and 3 other epigenetic factors. As discussed earlier, several groups have transfected OSKM or OSK gene viral vectors into mice and had some success in safely reversing epigenetic age and rejuvenating some organs if the OSKM or OSK genes are expressed intermittently [25-271. However, this approach uses exogenous viral or plasmid DNA, which presents genetic risks due to potential DNA insertion into the genomic DNA. The system disclosed herein may also use plasmid DNA but also includes loading sets of mimic miRNA and anti-miRNA types (Table 1) into EVs that transiently boost OCT4, SOX2, and KLF4 expression in at least one subset of cells, tissues, or organs. Not only does this miRNA / EV system remove the genomic risks of foreign DNA insertion, but it employs the natural regulatory system that cells use to program their epigenetic state. For example, the miR-302 / 367 family cluster (miR-302a, miR-302b, miR-302c, miR-302d, miR-367) plays a central role in maintaining embryonic stem cell pluripotency and can help generate higher OCT4 and SAX2 levels. The reprogramming capacity of the miR-302 family is demonstrated by its capacity to generate iPS cells under specific conditions that are a 100 times more potent than OSKM [49, 601. Recently, miR-302b was shown to reverse cellular senescence in a newly published paper

[0047] . The miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 , and miR-429) is also an important family cluster for promoting sternness in adult stem cells [611. The miR-200 family also promotes OCT4 and SOX2 and can generate iPS cells if used along with the miR-302 family and miR-369 [611. The three members of the miR-200a,b,c family and the four members of the miR-302a,b,c,d family are strong promoters of sternness and proliferation, so any one of these miRNAs could be a core component for the cellular reprogramming therapeutics.

[0063] As disclosed, loaded gene precursor miRNAs comprise an inducible DNA expression plasmid vector encoding the gene precursor of at least one miRNA type from set A comprising Anti-miR-346, Anti-miR-7, Anti-miR-145, Anti-miR-128, miR-200a, miR-200b, miR-200c; plus the gene precursor for the miR-302s family (comprising miR-302a-3p, miR-302b-3p, miR-302c-3p, miR-302d-3p. The system can use DNA plasmids that code the various miRNA genes or anti-genes along with an inducible promoter to turn on the miRNA genes or anti-genes. Taking the miR-302 family described above, EVs are loaded with the whole miRNAs in a DNA plasmid comprising miR-302a, miR-302b, miR-302c, miR-302d along with an inducible promoter and the KLF4 producing Anti-miR-346, Anti-miR-7, Anti- miR-145, and Anti-miR-128. As mentioned above, while a system using miRNA DNA to engineer EVs may have slightly higher cancer risks due to the potential for integration of the plasmid DNA into patient’s cells, the benefits may be worth the slightly increased risks in lethal diseases due to the potential for longer lasting miRNAs, more different miRNA types, and the ability to better control the miRNA expression vectors with inducible promoters. There are four basic tissue types (connective, epithelial, muscle, and neural) and more than 20 organs (e.g. the heart, brain, lungs, liver, kidneys, stomach, intestines, pancreas, skin, and thyroid) in the human body. Since different tissues or organs express their own set of miRNAs and have their own epigenetic expression pattern, it may not be the case that any one set of miRNAs can rejuvenate all tissues and organs in the body. For example, inducible vectors expressing the OSK or OSKM genes in all cells of mice can rejuvenate many tissues and organs in the older mice but also induces death due to cell dysplasia in the intestinal epithelium

[0062] . Thus, the system disclosed herein delineates multiple sets of miRNA genes that individually may rejuvenate cells in some subsets of cells, tissues, or organs but cause dysfunction in others. Having multiple sets of miRNAs raises the probability that all subsets of cells, tissues, and organs can be rejuvenated if the subsets of miRNAs can be targeted to specific tissues or organs using differing miRNAs and / or different vesicles.

[0064] As disclosed, the regulation of OCT4, SOX2, and KLF4 proteins comprises mimics or inhibitors of mouse, rat, dog, or cat miRNA analogs of the human miRNAs with linkage to each mouse, rat, dog, or cat version of OCT4, SOX2, and KLF4. Many of the human miRNAs are unchanged in the mouse, rat, dog, or cat. For example, the miR-200 and the miR-302 family clusters are highly conserved, so human-like miR-200 and the miR-302 family clusters are also found in many other mammals.

[0065] The disclosed system contemplates novel unknown therapeutic miRNA sets that are linked to the regulation of the OCT4, SOX2, and KLF4 proteins comprising inhibitor or mimics of human miRNAs in the database mirbase.org that have yet to be analyzed for their capacity to regulate the OCT4, SOX2, and KLF4 protein products. There are over 2600 human miRNAs, and many have unknown functions. As more research is done on these mRNAs, it is contemplated that the present system would be able to expand the miRNA sets discussed herein to produce more potent and / or varied therapies to treat many more disease states as well as overall aging.

[0066] Extracellular Vesicles can be derived from many scalable animal sources such as pork blood, bovine milk, or chicken eggs. As discussed in the background section, EVs are membrane bound particles of 40 to 1000 nm in diameter that excel as nanocarriers for pharmaceuticals and therapeutic agents, attributed to their inherent cell-targeting capabilities, minimal immunogenicity, and superior biodegradability

[0030] . Since the production of large numbers of EVs from cultured cells grown on plates or in suspension culture poses financial and technical challenges, the present system proposes multiple EV systems to improve the production capacity of the commonly used EV choices. Choosing a high-capacity scalable EV system is helped by the fact that the miRNA / EV therapeutic does not require any part of the native load of proteins, DNAs, mRNAs, or miRNAs that exist in each EVs produced from an animal or plant source. Thus, EVs from animals or plant sources may be explored. When considering sources that are scalable and immunologically inert, it will be recognized by one having skill in the art to choose those that can be harvested in large quantities and have a low risk of carrying components that might trigger adverse immune reactions. For example, EVs derived from Pork [311, bovine milk, or colostrum (milk from the first milking after giving birth) provide high numbers of EVs and is safe, low cost, and scalable. EVs purified from bovine milk have been injected into mice with no adverse events [63, 641. Another potential choice with high scalability is chicken eggs (both egg yolk and egg white have EVs). Pork EVs are considered favorable as they organically have factors for mammalian reprogramming

[0031] ,

[0067] The Extracellular Vesicles can also be derived from scalable plant products such as the juice of grape, grapefruit, lemon, carrots, ginger, broccoli, or apple. One problem with animal derived EVs is that they can carry viruses and other pathogens that can infect humans. Plant-derived EVs (or exosome-like nanocarriers) are attractive due to their inherent lack of mammalian pathogens, making them potentially safer and less immunogenic for human use. Grapes, lemons, carrots, ginger, and broccoli have been shown to be scalable and immunologically safe. The current favored plant EV sources are grape and grapefruit juice, as these have apparent higher EV levels (1760 mg / kg in grape, 2210 mg / kg in grapefruit [651. Like animal EVs, grape EVs and grapefruit EVs also have some cross species biocompatibility without causing local or systemic side effects. For example, treating healthy mice with typical fruit EVs does not alter their liver enzymes, kidney function, or intestinal immune function [65, 661 ■ Moreover, pathological changes in the brain, heart, liver, spleen, lung, and kidney tissues are not observed. Work with red grape EVs as carriers of two miRNAs of interest is discussed below.

[0068] The Extracellular Vesicles can be derived from any mammalian cell line or tissue. Other cells could be used to help rejuvenate tissues or organs that do not reprogram well using milk or plant derived EVs. Mammalian cells include induced pluripotent stem cells (iPS cells). For many uses in mammals, umbilical cord mesenchymal stem cells (UC-MSCs) or placental tissue will be the preferred neonatal cells. Neonatal foreskin stroma is another favored stem cell source, as both UC-MSCs and neonatal foreskin stroma show similar patterns of morphology, surface protein expression, multipotent sternness, non- immunogenetic, and genetic stability. However, cultured human cells would be much more expensive to prepare.

[0069] The Nanocarrier Vesicles can be produced synthetically to be carriers of DNAs or miRNAs. Nanocarriers are synthetic vesicles that are made of lipid-based carriers like liposomes, micelles, niosomes, transfersomes, ethosomes, polymerosomes, and many other materials. They can be engineered to various sizes and specifications and have been used for drug delivery. Choosing between nanocarriers (e.g. liposomes) and EVs as carriers of RNA / DNA in the bloodstream depends largely on the specific application. Nanocarriers offer some advantages over EVs as carriers of RNA or DNA, including greater customizability, scalability, encapsulation efficiency, and ease of surface modification. However, the choice between nanocarriers and EVs depends on the application, as EVs are preferred if from cells that organically have reprogramming factors (e. g. h-ESCs, umbilical cord cells, and pork blood). There are several commercial companies that make custom nanocarriers, but the costs are typically considerably more than pork, milk, and plant derived EVs. To keep production in house and perhaps save time and money, one can buy liposome producing machines to do liposome production.

[0070] Testing miRNA Engineered EVs Effectiveness in Programming Cells

[0071] High quality synthetic miRNA mimics and miRNA inhibitors can be purchased commercially online, so manufactures of quality miRNAs are easily obtained. Applicant currently utilizes AcceGen miRNA. Depending on which vesicles are used (plant or mammalian cells), the production of EVs can vary somewhat but multiple centrifugations and size exclusion chromatography (SEC) typically yield higher EV purity. Applicant prepared EVs from freshly squeezed grape juice to get high EV count and lower costs.

[0072] Red Grape EV extraction methods were developed as follows. Red grapes are juiced using a cold pressed masticating juicer and then mixed with an equal volume of 1 X PBS. The mix is then subjected to a series of centrifuge runs: low-speed centrifugation (3000 x g for 10 min at 4o C) to remove pelleted cells, fibers, and large particles; intermediate speed centrifugation (20,000 x g for 30 min at 4°C) to remove large debris and subcellular organelles; high speed centrifugation (100,000x g for 2.0 hours) to pellet EVs. The centrifuge pellet (CP) pellets are each dissolved in 2 ml of PBS and then pooled with other CP pellets and spun at 3,000 x g for 10 min at 4o C to spin out the undissolved debris. The CP supernatant is filtered through a Sterile Syringe PES 0.22 urn Filter. The sterile CPS samples are frozen slowly in 1 ml samples and stored at -80° C. One ml of the frozen CP prep (CPS.2_0.22) is sent on dry ice to a commercial company for NTA testing. Applicant is in the process of preparing EVs from pork blood to directly compare pork EVs to grape EVs.

[0073] EV quality control. Preps of EVs are pooled together and given a batch number. A sample of each batch is sent off to Alpha Nano Tech for size and yield of EVs / ml by diluting ~5 ul of rEV / s into 5 ml of PBS-SLI for analysis of EVs by Nanoparticle Tracking Analysis (NTA). EV yield was about 10A12 EVs / ml. Morphology is analyzed by Transmission electron microscopy (TEM). A recent example of NTA for the grape EVs is shown in Fig. 4 and grape EV TEM is shown in Fig. 5.

[0074] Loading miRNA into grape EVs. Human miR-302a-3p miRNA Agomir (miR-302a) and miR-128-3p miRNA Antagomir (anti-miR-128) were ordered from Accegen. Both miRNA types were dissolved into a 20 uM solution. To minimize EV damage to the EM membrane, the preferred method of miRNA loading is to incubate the miRNA with the grape EVs using Exo-Fect Exosome Transfection Kit from System Biosciences (SBI), as this transfection kit loaded tested well in getting the miRNA using 10A8 EVs.

[0075] Stem Cell Transfection with miRNA engineered EVs. T25 cell samples with Adipose Mesenchymal Stem Cells (AD-MSCs) were used for a test of cell reprogramming effectiveness using miRNA loaded EVs of the final four transfected AD-MSC groups: (T1) 0.4 uM of miR-303a in 10A8 EVs; (T2) 0.4 uM of miR-303a plus 0.2 uM of anti-miR-128 in 10A8 EVs); (C4 Control) 10A8 EVs but no miRNA; and (C6 Control) no 10A8 EVs and no miRNA in PBS. The T25 flasks were seeded with 200,000 AD-MSC cells in 4 ml of DMEM / F12 supplemented with 5% bovine fetal cafe serum. After 24 hours, labeled T25 flasks were treated with 300 ul of one of the 4 treatments (T 1 , T2, C4, or C6) and then cultured for 5 days of treatment. The cells were then counted for cell density and viable cells before being passed for expansion in T75 flasks. Culture continues for another 4 weeks to grow enough cells for Total RNA-seq and age methylated CpG studies. Duplicate lines of the same 4 treatments were sent to Zymo Research for Total RNA-Seq to determine the gene expression pattern for each treatment.

[0076] Figure 6 depicts Multidimensional Scaling (MDS) analysis of samples. MDS was conducted to visualize the distance / similarity between T1 , T2, C4, C6. Top 500 genes with highest variance among samples were used to make this plot.

[0077] Table 2 suggests that the addition of grape EVs in C4, miR-302a in T1, and miR-302a plus miR-180 in T2 have resulted in unique patterns of gene expression for C4, T 1 , and T2 from the starting control C6. This indicates that miRNAs are capable of reprogramming gene expressions of human stem cells (C6). It is also interesting that T2 vs C4 has over 7 times as many genetic differences as does T1 vs C4. Adding multiple miRNAs apparently can have bigger effects. Table 3 - 5 further explore the reprogrammed gene expression.

[0078] Table 2 below shows genes that have significant differences in the 4 groups.

[0079] Group 1 versus Higher Higher Not differentially

[0080] Group 2 Group 1 Group 2 expressed

[0081] C6_vs_C4 0 32 30289

[0082] T1_vs_C4 3 0 30318

[0083] T1_vs_C6 79 1 15545

[0084] T2_vs_C4 12 10 23244

[0085] T2_vs_C6 54 2 30265

[0086] T2 vs T1 2 6 30313

[0087] Table 3 below lists the significant gene expression changes for the 3 genes in T1 vs C4.

[0088] Mean Log2 Fol Significance

[0089] Gene counts change Rate Gene Functions

[0090] RASD1 178 1.87 4.2e-05 Modulates stem cell differentiation pathways

[0091] Tumor suppressor and lineage of stem cell

[0092] RAB3A 72 1.87 4.6e-01 Has role in regulating vesicle trafficking potentially affects stem cell differentiation

[0093] FGFR2 217 1.82 8.6e-07 Help mesenchymal stem cells regulate their proliferation, differentiation, and self-renewal Table 4 below lists significant gene expression changes for 12 genes in T2 vs C4.

[0094] Mean Log2 Fold False Gene Functions

[0095] Gene name counts change discovery

[0096] D.USP1 13775 0.75 1.3e-03 A tumor suppressor, controlling cell growth and self-renewal - counteracts cancer stem cells

[0097] ACTA2 16665 0.84 1.6e-12 Codes for alpha-smooth muscle actin in stem cells

[0098] DEPPI 1672 0.90 2.8e-03 Stem cell survival and differentiation via its impact on mitochondrial function and autophagic activity

[0099] CCND2 3011 0.90 1 ,3e-06 Crucial role in regulating the cell cycle and enhancing the regenerative potential of stem cells

[0100] OAS3 358 1.36 1.1e-04 Maintains cellular homeostasis and protects against viral attacks by triggering a degradation of viral RNA

[0101] MX1 276 1.45 1.1e-04 Serves as a marker for identifying these cells with important roles in bone maintenance and repair

[0102] IGFBP2 403 1.53 1.2e-08 Promotes survival, proliferation, and maintenance of various stem cell populations

[0103] FGFR2 217 1.56 3.0e-04 Help mesenchymal stem cells regulate their proliferation, differentiation, and self-renewal

[0104] TCIM 206 1.66 1.8e-05 Key regulator in the lineage commitment to bone or fat cells by mesenchymal stem cells

[0105] NPPB 132 1.81 3.0e-04 Expressed in cardiac progenitor cells within stem cell populations - plays role in regulating cardiac function

[0106] OAS2 166 1.88 7.1e-06 Primarily known for its role in the innate immune response against viral infections

[0107] RSAD2 45 2.34 4.7e-02 Key antiviral defense mechanism in the innate immune response within stem cells Table 5 shows 10 genes that are differentially expressed higher in C4 than in T2.

[0108] Mean Log2 Fol False Gene Functions

[0109] Gene name counts change discovery

[0110] EGR2 292 -1.81 9.3e-10 Role in regulating stem cell differentiation and neural development by acting as a transcription factor

[0111] EGR3 319 -1.68 7.1e-06 Role in regulating the proliferation of hematopoietic stem cells (HSCs), acting as a limiting factor that inhibits growth

[0112] FOSB 8659 -1.40 2.3e-08 Role in regulating stem cell proliferation and differentiation by acting as a transcription factor in neural stem cells

[0113] CXCL2 231 -1.32 5.0e-02 Role in attracting and mobilizing stem cells by interacting with the CXCR2 receptor on their surfaces

[0114] SPQN2 694 -1.03 2.8e-02 Role in cell adhesion, migration, and differentiation, often showing increased expression in various cancers

[0115] PE K 851 -1.03 3.5e-03 Role in stem cell proliferation, differentiation, and migration through its production of enkephalin peptides as opioids

[0116] MAFB 653 -1.01 5.0e-02 Role in regulating stem cell differentiation, particularly in the development of myeloid lineages like macrophages

[0117] HAPL.N1 3575 -0.94 2.3e-08 Promotes extracellular matrix environment that supports stem cell proliferation, differentiation, and migration

[0118] FOS 14079 -0.80 4.9e-09 Plays crucial role in the early activation of stem cells, particularly in muscle regeneration

[0119] EGR1. 18990 -0.80 2.2e-13 Influencing stem cell differentiation, proliferation, and maintenance of a quiescent state Table 6 shows that Myc and Notch3 are expressed at higher levels in T4 than in C4 or C6, while the p21 senescent gene marker (CDKN1A) is higher in C4 or C6 than in T2.

[0120] Group C4 Value Standard

[0121] Gene T2 Value C6 Value Error Gene Functions

[0122] MYC 3024 2238 0.054 Regulates stem cell self-renewal,

[0123] 1779 1.2E-11 proliferation, reprogramming and maintenance of pluripotency

[0124] NOTCH3 13999 11161 0.034 Regulates the function and

[0125] 10568 0.035 maintenance of stem cells, particularly in relation to their differentiation

[0126] CDKN1A 8534 10969 0.032 p21 gene with crucial role in

[0127] 10007 0.032 regulating stem cell behavior by acting as a cell cycle inhibitor

[0128] The vast majority of the genes that are over- or under-expressed in Tables 3, 4, and 6 are involved in stem cell functions such as proliferation, differentiation, or migration. Since these are the basic functions of young adult stem cells, it appears that both miR-302a and miR-180 may promote stronger stem cell functional than do controls C4 and C6. Note that Table 5 shows genes that are higher in C4 than in T2 and these appear somewhat less focused on stem cells and more negative to cell growth. In Table 6, all gains in Myc expression in T2 are also observed in T1, suggesting that miR-302a is the miRNA that is upregulating Myc. In the case of Notch3, most of the gain in gene expression comes from T2, indicating that miR-180 is the major inducer of Notch3. In the case of CDKN1A, this is the known p21 senescence marker, which is higher in both C4 and C6, indicating that miR-180 is the main driver of the decreased expression of CDKN1A in T2.

[0129] Of note, Applicant observed about 60% faster rate of growth in the T2 cells that were treated with both miR-302a and miR-180 (T2). Young stem cells can grow faster than older ones. Assuming the reprogrammed T2 AD-MSCs have strengthened stem cell functions, this changed programming should lead to improved healthspan and / or younger biological age. Applicant plans to repeat the experiments above using pork derived EVs and then test both types of miRNA engineered EVs for their ability to reprogram epigenetic age. The in vitro platform of reprogramming vesicles with miRNAs has great advantages over current reprogramming therapeutics, as it is quicker, less expensive, more efficient, and safer than the previous genetic methods of intermittent reprogramming with transfections of plasmids or AAV virus expressing OCT4, SAG2, and KLF4. Uses of miRNA-Vesicle Therapeutics. The risks of common chronic disease, mortality risks, and healthspan as a function of age. Using clinical histories of 300,447 British (UK) adults of ages 37 to 73, a study

[0053] found that the risks of chronic age-related diseases double every 8 years (Fig 7). This doubling of morbidity risks is very close to the doubling of mortality risk every 8 years of adult life that was found in Gompertz law and with the doubling of mortality risks observed in the US social security annual death rates that double every 8 years of adult life. This amazing correspondence between risks of age- related disease and mortality risks suggests that aging is the underlying cause of many age- related diseases. If this correspondence is true and the miRNA-EV therapeutics of the present system are safe and even partly effective, the miRNA-EV therapeutics will be a game changer in reversing most chronic age-related diseases, while extending healthspan.

[0130] It is contemplated that humans and animals treated with the therapeutic disclosed herein could have improved function in an organ, faster healing from bodily injury or disease, lower chronic inflammation, lower risks of age-related diseases, moderation of disease symptoms, lower levels of glucose, reduced hypertension, or any combination thereof. Dietary restriction (dietary fasting) is currently the only clinically proven intervention that increases life span by several years

[0069] , In animal trials, dietary restriction has extended lifespan by 30% to 50% with an improvement in health span, lower inflammation, reduced risks of age-related diseases, and extended both mean and maximum lifespan

[0069] , The therapeutics of the present system should meet or exceed the success of dietary restriction by reprogramming and rejuvenating older adult stem cells.

[0131] It is contemplated that the present therapeutic can be topically applied to the skin to significantly improve or totally reverse burns, wounds, scars, rough brown patches (seborrheic keratosis), rough pinkish patches (actinic keratosis), dry, thinning, sagging, winkling, or discolored skin, or age spots, skin tags, warts, and hair loss or any combination thereof. The skin is the largest organ in the body and is easiest to reach using topical treatments. Studies have shown that EVs have efficacy in cases of skin aging, atopic dermatitis, and wounds

[0070] , Because the therapeutics of disclosed herein are nano scale EVs loaded with regenerative miRNAs, they should have good success as topical products. As skin is a specific organ, some skin-specific miRNAs should be combinable with the disclosed system. For example, hair loss may be reversed with miR-218-5p [711.

[0132] It is contemplated that the miRNA engineered extracellular vesicle therapeutic described herein can be applied to the brain to prevent, stop, or reverse human Alzheimer’s Disease, Vascular dementia, Lewi Body dementia, or Frontotemporal dementia or any combination thereof. Age is the main risk factor for Alzheimer’s disease (AD)

[0072] , While early onset AD is typically due to mutations in genes like A and tau, the vast majority of AD cases are late onset disease, which is likely due to the accumulation of age-related dysfunction. Effective treatments for Alzheimer’s disease require a powerful multipath approach that remediates many of the major hallmarks of the aging process itself. For example, miR-128 is strongly correlated to cognitive function

[0073] while the miR-302 family along with miR-200b play important roles in animal models of AD

[0038] , These data suggest that the therapeutics disclosed herein are interesting therapeutics to test as an AD therapeutic.

[0133] It is contemplated that the miRNA engineered extracellular vesicle therapeutic described herein can be used to prevent, stop, or reverse diseases comprising arthritis, atherosclerosis, autoimmune diseases, cancer, cardiac arrhythmia, chronic heart failure, Chronic Obstructive Pulmonary Disease (COPD), diabetes, kidney disease, immune deficiency diseases, liver disease, muscle atrophy, osteoporosis, prostate enlargement, stroke, or other age-related diseases. These common diseases typically have exponentially rising risks as a function of age. Most current treatments for these age-related diseases only treat symptoms of the disease and do not provide cures. The miRNA engineered extracellular vesicle therapeutics of claim 1 should help prevent or treat these diseases but may be specialized to the disease as to miRNA composition and dosing. Application of therapeutic will depend on disease and vary as to intravenous injection, specific organ injection, nasal spray to brain, inhalation to lungs, or oral capsules.

[0134] It is contemplated that humans or mammals treated with the therapeutic disclosed herein would have significantly longer mean healthspans and lifespans. While it is premature to speculate that a single miRNA engineered vesicle therapeutic disclosed herein can extend mean healthspan or lifespan, a combination of different therapeutics to differing subsets of cells, tissues, or organs should have a significant chance to extend mean healthspans and lifespans.

[0135] It is contemplated that the miRNA engineered extracellular vesicle therapeutics described herein may be applied by intravenous injection, specific organ injection, nasal spray to brain, inhalation to lungs, oral capsules, lyophilized or freeze-dried powder for oral intake, or topical application to skin. There are over 30 trillion cells in the average human body and scores of cells and organs with differing epigenetic states, so reaching all cell niches may require differing therapeutic applications and multiple treatments.

[0136] Although the present therapeutic specifies 2 or more types of miRNAs in each vesicle, there may be cases where a treatment of the 2 types of miRNAs can be provided at separate times of the day or in using different organ injections, it may be best to only do one type of miRNA in each vesicle as long as the daily therapeutic load uses a minimum of 2 types of miRNAs per 24 hour period.

[0137] In summary, the present system provides for engineered vesicle therapeutics comprising a prescribed set of miRNA mimics and miRNA inhibitors (microRNAs) that induces a transient expression of OCT4, S0X2, and KLF4, and which do not reprogram cells back to an induced pluripotent state of embryonic stem cells, wherein each set of miRNAs is loaded into natural or artificial vesicles to form miRNA engineered vesicle therapeutics, said therapeutics epigenetically reprogramming at least one subset of cells, tissues, or organs from a mammal to reverse epigenetic drift which can lead to cellular dysfunction, aging, or disease.

[0138] The vesicles comprise extracellular vesicles or nanocarrier vesicles. The extracellular vesicles can be derived from scalable plant products such as the juice of grape, grapefruit, lemon, carrots, ginger, broccoli, or apple. The extracellular vesicles can also be derived from scalable animal sources such as porcine blood, bovine milk, or chicken eggs. Further, the extracellular vesicles can be derived from any mammalian cell line or tissue. The nanocarrier vesicles may comprise liposomes. The nanocarrier vesicles can be produced synthetically to be carriers of DNAs or miRNAs.

[0139] The prescribed set of microRNAs comprises a set of two or more types of microRNAs (sense or antisense miRNAs or both). In addition, the prescribed set of microRNAs may comprise a set with at least two microRNAs (sense or antisense miRNAs or both). Further, the extracellular vesicles or nanocarrier vesicles can be loaded with at least two microRNA genes (coding for sense or antisense miRNAs or both).

[0140] In some embodiments, the prescribed set of microRNAs comprises mature miRNAs or DNA coding miRNAs directly transfected into isolated vesicles using electroporation, passive encapsulation, or transfection reagents such as Lipofectamine, FuGENE, ExoFect, Polyethylenimine, and calcium chloride. In some embodiments, the prescribed set of microRNAs comprises loaded human miRNA sets comprising Anti-miR-128-3p plus at least one miRNA mimic from the set of the miR-302 / 367 family of miR-302a-3p, miR-302b-3p, miR-302c-3p, miR-302d-3p, and miR-367-3p. In some embodiments, the prescribed set of microRNAs comprises loaded human miRNA sets comprising at least one miRNA type from set A comprising Anti-miR-346, Anti-miR-7, Anti-miR-145, Anti-miR-128, miR-200a, miR-200b, miR-200c; plus at least one miRNA type from set B of the miR-302 / 367 family (miR-302a-3p, miR-302b-3p, miR-302c-3p, miR-302d-3p, miR-367-3p). In some embodiments, the prescribed set of microRNAs comprises loaded precursor miRNAs comprising an inducible DNA expression plasmid vector encoding the gene precursor of at least one miRNA type from set A comprising Anti-miR-346, Anti-miR-7, Anti-miR-145, Anti- miR-128, miR-200a, miR-200b, miR-200c; plus the gene precursor for the miR-302s family (comprising miR-302a-3p, miR-302b-3p, miR-302c-3p, miR-302d-3p). In some embodiments ,one or more types of microRNAs delivered in a second treatment within 12 hours using one or more different types of miRNA could be loaded into the vesicles. The therapeutics of the present system provides epigenetic reprogramming of at least one subset of cells, tissues, or organs that reduces epigenetic cellular age, as verified by reduced epigenetic clock age using DNA methylation at specific CpG sites in the genome or by changes in gene expression patterns. In addition, the therapeutics can provide reprogramming of a subset of cells, tissues, or organs promotes more functional multipotent adult stem cells and renewal of progenitor cells but it does not reprogram cells or tissues back to an induced pluripotent state.

[0141] Disclosed is a system of providing therapeutics for epigenetic reprogramming of at least one subset of cells, tissues, or organs from a mammal comprising vesicles engineered by loading a prescribed set of microRNAs that induces a transient expression of OCT4, S0X2, and KLF4, and which does not reprogram cells back to an induced pluripotent state of embryonic stem cells, whereby said therapeutics restore the youthful or healthy state of said cells, tissues or organs, potentially reversing damage, aging, or disease.

[0142] Although the present system has been described with reference to the disclosed embodiments, modifications and variations can be made and still the result will come within the scope of the disclosure. No limitation with respect to the specific embodiments disclosed herein is intended or should be inferred.

[0143] References Villeponteau B: The heterochromatin loss model of aging. Exp Gerontol 1997, 32:383- 394. Bork S, Pfister S, Witt H, Horn P, Korn B, Ho AD, Wagner W: DNA methylation pattern changes upon long-term culture and aging of human mesenchymal stromal cells. Aging Ce / / 2010, 9:54-63. Ravaioli F, Bacalini MG, Franceschi C, Garagnani P: Age-Related Epigenetic Derangement upon Reprogramming and Differentiation of Cells from the Elderly. Genes (Basel) 2018, 9. Walker MP, LaFerla FM, Oddo SS, Brewer GJ: Reversible epigenetic histone modifications and Bdnf expression in neurons with aging and from a mouse model of Alzheimer's disease. Age (Dordr) 2013, 35:519-531. Przybilla J, Rohlf T, Loeffler M, Galle J: Understanding epigenetic changes in aging stem cells-a computational model approach. Aging Ce / / 2014, 13:320-328. Spolnicka M, Pospiech E, Adamczyk JG, Freire-Aradas A, Peplonska B, Zbiec-Piekarska R, Makowska Z, Pieta A, Lareu MV, Phillips C, et al: Modified aging of elite athletes revealed by analysis of epigenetic age markers. Aging (Albany NY) 2018, 10:241-252. Lawn RB, Anderson EL, Suderman M, Simpkin AJ, Gaunt TR, Teschendorff AE, Widschwendter M, Hardy R, Kuh D, Relton CL, Howe LD: Psychosocial adversity and socioeconomic position during childhood and epigenetic age: analysis of two prospective cohort studies. Hum Mol Genet 2018. Najar RA, Wani NA, Bhat JA, Dar NJ, Rahat B, Gupta AP, Kaur J, Kaur J, Hamid A: Modulation of dietary folate with age confers selective hepatocellular epigenetic imprints through DNA methylation. J Nutr Biochem 2018, 53:121-132. Pedersena IM, Zisoulisa DG: Transposable elements and miRNA: Regulation of genomic stability and plasticity. Mob Genet Elements 2016,

[0144] 6:10.1080 / 2159256X.2152016.1175537. Fung L, Guzman H, Sevrioukov E, Idica A, Park E, Bochnakian A, Daugaard I, Jury D, Mortazavi A, Zisoulis DG, Pedersen IM: miR-128 Restriction of LINE-1 (L1) Retrotransposition Is Dependent on Targeting hnRNPAI mRNA. Int J Mol Sci 2019, 20. Mendell JT, Olson EN: MicroRNAs in stress signaling and human disease. Ce / / 2012, 148:1172-1187. Lu YR, Tian X, Sinclair DA: The Information Theory of Aging. Nat Aging 2023, 3:1486- 1499. Omole AE, Fakoya AOJ: Ten years of progress and promise of induced pluripotent stem cells: historical origins, characteristics, mechanisms, limitations, and potential applications. PeerJ 2018, 6:e4370. Lin SL, Ying SY: Mechanism and Method for Generating Tumor-Free iPS Cells Using Intronic MicroRNA miR-302 Induction. Methods Mol Biol 2018, 1733:265-282. Menendez S, Camus S, Herreria A, Paramonov I, Morera LB, Collado M, Pekarik V, Maceda I, Edel M, Consiglio A, et al: Increased dosage of tumor suppressors limits the tumorigenicity of iPS cells without affecting their pluripotency. Aging Ce / / 2012, 11 :41- 50. Trosko JE: Induction of iPS cells and of cancer stem cells: the stem cell or reprogramming hypothesis of cancer? Anat Rec (Hoboken) 2014, 297:161-173. Xu M, Shaw G, Murphy M, Barry F: Induced Pluripotent Stem Cell-Derived Mesenchymal Stromal Cells Are Functionally and Genetically Different From Bone Marrow Derived- Mesenchymal Stromal Cells. Stem Cells 2019. Cypris O, Frobel J, Rai S, Franzen J, Sontag S, Goetzke R, Szymanski de Toledo MA, Zenke M, Wagner W: Tracking of epigenetic changes during hematopoietic differentiation of induced pluripotent stem cells. Clin Epigenetics 2019, 11 :19. Nishihara K, Shiga T, Nakamura E, Akiyama T, Sasaki T, Suzuki S, Ko MSH, Tada N, Okano H, Akamatsu W: Induced Pluripotent Stem Cells Reprogrammed with Three Inhibitors Show Accelerated Differentiation Potentials with High Levels of 2-Cell Stage Marker Expression. Stem Cell Reports 2019, 12:305-318. Capogrossi MC: Cardiac stem cells fail with aging: a new mechanism for the agedependent decline in cardiac function. Circ Res 2004, 94:411-413. Brusnahan SK, McGuire TR, Jackson JD, Lane JT, Garvin KL, O'Kane BJ, Berger AM, Tuljapurkar SR, Kessinger MA, Sharp JG: Human blood and marrow side population stem cell and Stro-1 positive bone marrow stromal cell numbers decline with age, with an increase in quality of surviving stem cells: correlation with cytokines. Meeh Ageing Dev2010, 131 :718-722. Artegiani B, Calegari F: Age-related cognitive decline: can neural stem cells help us? Aging (Albany NY) 2012, 4:176-186. Hell RC, Ocarino NM, Boeloni JN, Silva JF, Goes AM, Santos RL, Serakides R: Physical activity improves age-related decline in the osteogenic potential of rats' bone marrow- derived mesenchymal stem cells. Acta Physiol (Oxf) 2012, 205:292-301. Eliazer S, Brack AS: Stem cells: Cause and consequence in aged-muscle decline. Nature 2016, 540:349-350. Browder KC, Reddy P, Yamamoto M, al. e: In vivo partial reprogramming alters age- associated molecular changes during physiological aging in mice. . Nat Aging Chondronasiou D, Gill D, Mosteiro L, al. E: Multi-omic rejuvenation of naturally aged tissues by a single cycle of transient reprogramming. Aging Cell 2022,

[0145] 00 : https: / / doi.Org / 10.1111 / acel.13578 Lu Y, Brommer B, Tian X, Krishnan A, Meer M, Wang C, Vera DL, Zeng Q, Yu D, Bonkowski MS, et al: Reprogramming to recover youthful epigenetic information and restore vision. Nature 2020, 588:124-129. Kim Y, Jeong J, Choi D: Small-molecule-mediated reprogramming: a silver lining for regenerative medicine. Exp Mol Med 2020, 52:213-226. Yang JH, Petty CA, Dixon-McDougall T, Lopez MV, Tyshkovskiy A, Maybury-Lewis S, Tian X, Ibrahim N, Chen Z, Griffin PT, et al: Chemically induced reprogramming to reverse cellular aging. Aging (Albany NY) 2023, 15:5966-5989. Sun D, Zhuang X, Xiang X, Liu Y, Zhang S, Liu C, Barnes S, Grizzle W, Miller D, Zhang HG: A novel nanoparticle drug delivery system: the anti-inflammatory activity of curcumin is enhanced when encapsulated in exosomes. Mol Ther 2010, 18:1606-1614. Horvath S, Singh K, Raj K, Khairnar SI, Sanghavi A, Shrivastava A, Zoller JA, Li CZ, Herenu CB, Canatelli-Mallat M, et al: Reversal of biological age in multiple rat organs by young porcine plasma fraction. Geroscience 2024, 46:367-394. Lananna BV, Imai SI: Friends and foes: Extracellular vesicles in aging and rejuvenation. FASEB Bioadv 2021, 3:787-801. Robbins PD: Extracellular vesicles and aging. Stem Cell Investig 2017, 4:98. Bansal A, Prathap R, Gupta S, Chaurasia A, Chaudhary P: Role of microRNAs in stroke recovery. J Family Med Prim Care 2019, 8:1850-1854. Gerasymchuk M, Cherkasova V, Kovalchuk O, Kovalchuk I: The Role of microRNAs in Organismal and Skin Aging. Int J Mol Sci 2020, 21. Scalavino V, Liso M, Cavalcanti E, Gigante I, Lippolis A, Mastronardi M, Chieppa M, Serino G: miR-369-3p modulates inducible nitric oxide synthase and is involved in regulation of chronic inflammatory response. Sci Rep 2020, 10:15942. Walgrave H, Zhou L, De Strooper B, Salta E: The promise of microRNA-based therapies in Alzheimer's disease: challenges and perspectives. Mol Neurodegener 2021, 16:76. Wang M, Qin L, Tang B: MicroRNAs in Alzheimer's Disease. Front Genet 2019, 10:153. Kinser HE, Pincus Z: MicroRNAs as modulators of longevity and the aging process. Human Genetics 2020, 139:291-308. Ludwig N, Leidinger P, Becker K, Backes C, Fehlmann T, Pallasch C, Rheinheimer S, Meder B, Stabler C, Meese E, Keller A: Distribution of miRNA expression across human tissues. Nucleic Acids Res 2016, 44:3865-3877. Liang Y, Ridzon D, Wong L, Chen C: Characterization of microRNA expression profiles in normal human tissues. BMC Genomics 2007, 8:166. Kriegel AJ, Liu Y, Liu P, Baker MA, Hodges MR, Hua X, Liang M: Characteristics of microRNAs enriched in specific cell types and primary tissue types in solid organs. Physiol Genomics 2013, 45:1144-1156. Galagali H, Kim JK: The multifaceted roles of microRNAs in differentiation. Curr Opin Cell B / o / 2020, 67:118-140. Ivey KN, Srivastava D: microRNAs as Developmental Regulators. Cold Spring Harb Perspect Biol 2015 Jul; 7(7): 2015, 7: a008144. Emde A, E H: miRNAs at the interface of cellular stress and disease. EMBO J 2014:1428-1437. Leung AK, Sharp PA: MicroRNA functions in stress responses. Mol Cell 2010, 40:205- 215. Bi Y, Qiao X, Cai Z, Zhao H, Ye R, Liu Q, Gao L, Liu Y, Liang B, Liu Y, et al: Exosomal miR-302b rejuvenates aging mice by reversing the proliferative arrest of senescent cells. Cell Metab 2025. Zolboot N, Du JX, Zampa F, Lippi G: MicroRNAs Instruct and Maintain Cell Type Diversity in the Nervous System. Front Mol Neurosci 2021 , 14:646072. Anokye-Danso F, Trivedi CM, Juhr D, Gupta M, Cui Z, Tian Y, Zhang Y, Yang W, Gruber PJ, Epstein JA, Morrisey EE: Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell 2011 , 8:376-388. Maraghechi P, Aponte MTS, Ecker A, Lazar B, Toth R, Szabadi NT, Gocza E: Pluripotency- Associated microRNAs in Early Vertebrate Embryos and Stem Cells. Genes (Basel) 2023, 14. Lin SL, Chang DC, Lin CH, Ying SY, Leu D, Wu DT: Regulation of somatic cell reprogramming through inducible mir-302 expression. Nucleic Acids Res 2011 , 39:1054-1065. Miyoshi N, Ishii H, Nagano H, Haraguchi N, Dewi DL, Kano Y, Nishikawa S, Tanemura M, Mimori K, Tanaka F, et al: Reprogramming of mouse and human cells to pluripotency using mature microRNAs. Cell Stem Cell 2011 , 8:633-638. Zenin A, Tsepilov Y, Sharapov S, Getmantsev E, Menshikov LI, Fedichev PO, Aulchenko Y: Identification of 12 genetic loci associated with human healthspan. Commun Biol 2019, 2:41. Farina FM, Hall IF, Serio S, Zani S, Climent M, Salvarani N, Carullo P, Civilini E, Condorelli G, Elia L, Quintavalle M: miR-128-3p Is a Novel Regulator of Vascular Smooth Muscle Cell Phenotypic Switch and Vascular Diseases. Circ Res 2020, 126:e120-e135. Shang Y, Zhu Z, Zhang Y, Ji F, Zhu L, Liu M, Deng Y, Lv G, Li D, Zhou Z, et al: MiR-7-5p / KLF4 signaling inhibits sternness and radioresistance in colorectal cancer. Cell Death Discov 2023, 9:42. Zhang G, Chen F, Wu P, Li T, He M, Yin X, Shi H, Duan Y, Zhang T, Wang J, et al: MicroRNA-7 Targets the KLF4 Gene to Regulate the Proliferation and Differentiation of Chicken Primary Myoblasts. Front Genet 2020, 11 :842. Miao X, Wu X, Shi W: MicroRNA-346 regulates neural stem cell proliferation and differentiation by targeting KLF4. Am J Transl Res 2017, 9:5400-5410. Xu N, Papagiannakopoulos T, Pan G, Thomson JA, Kosik KS: MicroRNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells. Cell 2009, 137:647-658. Balzano F, Cruciani S, Basoli V, Santaniello S, Facchin F, Ventura C, Maioli M: MiR200 and miR302: Two Big Families Influencing Stem Cell Behavior. Molecules 2018, 23. Lee YJ, Ramakrishna S, Chauhan H, Park WS, Hong SH, Kim KS: Dissecting microRNA- mediated regulation of sternness, reprogramming, and pluripotency. Cell Regen 2016, 5:2. Anastasiadou E, Ceccarelli S, Messina E, Gerini G, Megiorni F, Pontecorvi P, Camero S, Onesti MG, Trivedi P, Faenza M, et al: MiR-200c-3p maintains sternness and proliferative potential in adipose-derived stem cells by counteracting senescence mechanisms. PLoS One 2021, 16:e0257070. Sinclair: Cellular Repromming to reverse aging and promote tissue regeneration. US Patent Application 2023, US 2023 0338468:2- paragraph (0021). Herrmann IK, Wood MJA, Fuhrmann G: Extracellular vesicles as a next-generation drug delivery platform. Nat Nanotechno, 16:748-759. Munagala R, Aqil F, Jeyabalan J, Gupta RC: Bovine milk-derived exosomes for drug delivery. Cancer Lett 2016, 371 :48-61. Yi Q, Xu Z, Thakur A, Zhang K, Liang Q, Liu Y, Yan Y: Current understanding of plant- derived exosome-like nanoparticles in regulating the inflammatory response and immune system microenvironment. Pharmacol Res 2023, 190:106733. Hao S, Yang H, Hu J, Luo L, Yuan Y, Liu L: Bioactive compounds and biological functions of medicinal plant-derived extracellular vesicles. Pharmacol Res 2024:107062. Sangha GS, Weber CM, Sapp RM, Setua S, Thangaraju K, Pettebone M, Rogers SC, Doctor A, Buehler PW, Clyne AM: Mechanical stimuli such as shear stress and piezol stimulation generate red blood cell extracellular vesicles. Front Physiol 2023, 14:1246910. Thangaraju K, Neerukonda SN, Katneni II, Buehler PW: Extracellular Vesicles from Red Blood Cells and Their Evolving Roles in Health, Coagulopathy and Therapy. Int J Mol Sci 2020, 22. Flanagan EW, Most J, Mey JT, Redman LM: Calorie Restriction and Aging in Humans. Annu Rev Nutr 2020, 40:105-133. Yang GH, Lee YB, Kang D, Choi E, Nam Y, Lee KH, You HJ, Kang HJ, An SH, Jeon H: Overcome the barriers of the skin: exosome therapy. Biomater Res 2021, 25:22. Hu S, Li Z, Lutz H, Huang K, Su T, Cores J, Dinh PC, Cheng K: Dermal exosomes containing miR-218-5p promote hair regeneration by regulating beta-catenin signaling. Sci Adv 2020, 6:eaba1685. Liu RM: Aging, Cellular Senescence, and Alzheimer's Disease. Int J Mol Sci 2022, 23. Zhang M, Han W, Xu Y, Li D, XueQ.: Serum miR-128 Serves as a Potential Diagnostic Biomarker for Alzheimer’s Disease. Neuropsychiatr Dis Treat 2021, 17:269-275.

Claims

AMENDED CLAIMS received by the International Bureau on 06 August 2025 (06.08.2025)

1. Engineered vesicle therapeutics comprising: a prescribed set of miRNA mimics and miRNA inhibitors (microRNAs) that induces a transient expression of OCT4, SOX2, and KLF4, and which do not reprogram cells back to an induced pluripotent state of embryonic stem cells, wherein the prescribed set of microRNAs comprises loaded human miRNA sets comprising at least one miRNA type from set A comprising Anti-miR-346, Anti-miR-7, Anti-miR-145, Anti- miR-128, Anti-miR-126, Anti-miR-375; plus at least one miRNA type from set B of the miR-302 / 367 family (miR-302a-3p, miR-302b-3p, miR-302c-3p, miR-302d-3p, miR-367-3p), wherein each set of miRNAs is loaded into natural or artificial vesicles to form miRNA engineered vesicle therapeutics, said therapeutics epigenetically reprogramming at least one subset of cells, tissues, or organs from a mammal to reverse epigenetic drift which can lead to cellular dysfunction, aging, or disease.

2. The therapeutics of claim 1 , wherein the vesicles comprise extracellular vesicles or nanocarrier vesicles.

3. The therapeutics of claim 1 , wherein the prescribed set of microRNAs comprises a set of two or more types of microRNAs (sense or antisense miRNAs or both).

4. The therapeutics of claim 1 , wherein the prescribed set of microRNAs comprises a set with at least two microRNAs (sense or antisense miRNAs or both).

5. The therapeutics of claim 2, wherein the prescribed set of microRNAs comprises a set of two sense or antisense miRNAs or both.

6. The therapeutics of claim 2, wherein the extracellular vesicles or nanocarrier vesicles are loaded with at least two microRNA genes (coding for sense or antisense miRNAs or both).

7. The therapeutics of claim 1 , wherein reprogramming comprises epigenetic reprogramming of at least one subset of cells, tissues, or organs that reduces epigenetic cellular age, as verified by reduced epigenetic clock age using DNAmethylation at specific CpG sites in the genome or by changes in gene expression patterns.

8. The therapeutics of claim 1 , wherein reprogramming of a subset of cells, tissues, or organs promotes more functional multipotent adult stem cells and renewal of progenitor cells but does not reprogram cells or tissues back to an induced pluripotent state.

9. The therapeutics of claim 1 , wherein the prescribed set of microRNAs comprises mature miRNAs or DNA coding miRNAs directly transfected into isolated vesicles using electroporation, passive encapsulation, or transfection reagents such as Lipofectamine, FuGENE, ExoFect, Polyethylenimine, and calcium chloride.

10. The therapeutics of claim 1 , wherein the prescribed set of microRNAs comprises loaded human miRNA sets comprising Anti-miR-128-3p plus at least one miRNA mimic from the set of the miR-302 / 367 family of miR-302a-3p, miR-302b-3p, miR-302c-3p, miR-302d-3p, and miR-367-3p.

11. The therapeutics of claim 1 , wherein the at least one miRNA type from set A further comrpises miR-200a, miR-200b, miR-200c.

12. The therapeutics of claim 2, wherein the extracellular vesicles can be derived from scalable animal sources such as porcine blood, bovine milk, or chicken eggs.

13. The therapeutics of claim 2, wherein the extracellular vesicles can be derived from scalable plant products such as the juice of grape, grapefruit, lemon, carrots, ginger, broccoli, or apple.

14. The therapeutics of claim 2, wherein the extracellular vesicles can be derived from any mammalian cell line or tissue.

15. The therapeutics of claim 2, wherein the nanocarrier vesicles can be produced synthetically to be carriers of DNAs or miRNAs.

16. The therapeutics of claim 2, wherein the nanocarrier vesicles comprise liposomes.

17. The therapeutics of claim 1 , wherein the vesicles comprise grape extracellular vesicles or porcine extracellular vesicles.

18. The therapeutics of claim 6, wherein the prescribed set of microRNAs comprises loaded precursor miRNAs comprisingan inducible DNA expression plasmid vector encoding the gene precursor of at least one miRNA type from set A comprising Anti-miR-346, Anti-miR-7, Anti-miR-145, Anti- miR-128, miR-200a, miR-200b, miR-200c; plus the gene precursor for the miR-302s family (comprising miR-302a-3p, miR-302b-3p, miR-302c-3p, miR-302d-3p).

19. The therapeutics of claim 2, wherein one or more types of microRNAs delivered in a second treatment within 12 hours using one or more different types of miRNA could be loaded into said vesicles.

20. Therapeutics for epigenetic reprogramming of at least one subset of cells, tissues, or organs from a mammal comprising vesicles engineered by loading a prescribed set of microRNAs that induces a transient expression of OCT4, SOX2, and KLF4, and which does not reprogram cells back to an induced pluripotent state of embryonic stem cells, whereby said therapeutics restore the youthful or healthy state of said cells, tissues or organs, potentially reversing damage, aging, or disease.