Methods and composition for modulating a bioelectric pattern

Abstract

Disclosed herein are methods and compositions for modulating a bioelectric pattern in a subject, organ, cell, cell collective, or tissue in need thereof. In some embodiments, the modulation results in increasing the health and / or lifespan of a subject and / or has a therapeutic effect on a disease or condition that the subject is suffering from. In some embodiments, the bioelectric pattern is modulated by modulating the expression and / or activity of the hyperpolarization activate cyclic nucleotide gated potassium and sodium channel 2 (HCN2).

Description

Atty. Dkt. No.166118.01474 METHODS AND COMPOSITIONS FOR MODULATING A BIOELECTRIC PATTERN CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 619,147, filed January 9, 2024. The content of which is incorporated herein by reference in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (16611801474.xml; Size: 2,664 bytes; and Date of Creation: January 8, 2025) is herein incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0003] N / A. FIELD OF THE INVENTION

[0004] The disclosed technology is directed to methods and compositions for modulating endogenous bioelectric signaling and bioelectric patterns. Such modulation can be employed for a variety of healthcare related applications, including but not limited to increasing the health and / or lifespan of a subject. BACKGROUND

[0005] Aging is a natural process that involves morphological and functional changes in cellular and extracellular components leading to a progressive decline in most biological functions. The process of aging is influenced by a variety of factors, including genetics, lifestyle, and aspects of the environment. The decline of cellular function due to aging causes a gradual loss of physiological functions and increased susceptibility to a host of diseases, including cardiovascular diseases, cancer, neurodegenerative disorders, Type II diabetes, and infectious and non-infectious diseases, which negatively affect the quality of human life. Accordingly, there is a need in the art to arrest or slow aspects of the aging process to reduce these negative impacts and to improve the quality of life for the aging population. BRIEF SUMMARY OF THE INVENTION

[0006] Disclosed herein are methods and compositions for modulating Vmem of cells, thereby modulating bioelectric signaling and bioelectric patterning in a subject, organ, cell collective, or tissueAtty. Dkt. No.166118.01474 in need thereof. In some embodiments, the modulation results in increasing the health and / or lifespan of a subject and / or has a therapeutic effect on a disease or condition that the subject is suffering from. In some embodiments, the bioelectric signaling (bioelectric pattern) is modulated by increasing the expression and / or activity of hyperpolarization-activated cyclic nucleotide-gated ion channel (HCN channel), by administering to the subject (providing to the subject) a therapeutic agent that modulates ion channel activity, such as an HCN channel. In some embodiments, the HCN channel is one or more of HCN1, HCN2, HCN3 or HCN4. In some embodiments, the HCN channel is HCN2.

[0007] In some embodiments, a method for increasing the health and / or lifespan of a subject is provided. In some embodiments, the method comprise administering a therapeutic agent for modulating a bioelectric pattern in the subject by increasing the expression and / or activity of an HCN channel in the subject. In some embodiments, the HCN channel comprises an HCN2 channel. In some embodiments, an increased health and / or lifespan comprises one or more of: greater resistance to carcinogens, greater resistance to teratogens, greater resistance to disease and / or infection, greater resistance to the normal wear-and-tear of life including but not limited to cardiovascular disease, vascular dysfunction, muscle loss, bone loss, anemia, immune dysfunction, cognitive decline, non- cancerous regeneration of cells instead of cell senescence, reducing one or more of: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, disabled macroautophagy, deregulated nutrient-sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis.

[0008] In some embodiments, increasing the expression and / or activity of HCN2 comprises providing one or more therapeutic agents to the subject, comprising but not limited to one or more of: HCN2 nucleic acid (DNA and / or RNA, such as mRNA), HCN2 protein, HCN2 agonist / activator, and / or a compound that mimics the effect of HCN2 overexpression. In some embodiments, the HCN2 nucleic acid comprises a sequence encoding SEQ ID NO: 1, or a sequence encoding an amino acid with at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the HCN2 protein comprises SEQ ID NO: 1, or an amino acid having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 1. Thus, in some embodiments, the method comprises one or more of providing HCN2 nucleic acid to the subject, providing HCN2 protein to the subject, providing an HCN2 agonist / activator to the subject, providing a compound that mimics the effect of HCN2 overexpression to the subject. In some embodiments, providing HCN2 nucleic acid to the subject comprises administering a composition comprising HCN2 nucleic acid formulated with lipid nanoparticles, lipid / polymer nanoparticles, or as a vector, such as but not limited to a viral vector. In some embodiments, providing HCN2 protein to the subjectAtty. Dkt. No.166118.01474 comprises administering genetically modified cells expressing or overexpressing HCN2 protein. In some embodiments, an agonist of HCN2 is provided to the subject. In some embodiments, the HCN2 agonist is a direct agonist. In some embodiments, the HCN2 agonist is an indirect agonist (e.g., the indirect agonist effects a pathway member upstream of HCN2 expression or activity, e.g., an agonist of activator, or an antagonist of repressor). In some embodiments, one or more of lamotrigine, gabapentin, nicotinamide nononucleotide, resveratrol, and dexamethasone are provided to the subject. In some embodiments, one or more additional active agents, e.g., anti-aging agents, electroceutical agents, and / or additional ion channel modulators, are also provided, e.g., as combination therapy. In embodiments, microinjection is used to administer the HCN2 modulator and / or the one or more additional active agents.

[0009] In some embodiments, the HCN2 nucleic acid, HCN2 protein, the HCN2 agonist and / or the compound that mimics the effect of HCN2 overexpression is provided to the subject systemically. In some embodiments, the HCN2 mRNA, HCN2 protein, the HCN2 agonist and / or the compound that mimics the effect of HCN2 overexpression is provided to an organ, cell collective, or tissue of the subject.

[0010] In some embodiments, the subject is a non-diseased animal subject.

[0011] In some embodiments, the subject is in utero.

[0012] In some embodiments, the subject is diagnosed with, suspected to have, or is at higher risk of onset of a disease or disorder, and the bioelectric signaling (bioelectric pattern) is modulated to treat, prevent, delay onset, alleviate or ameliorate symptoms of the disease or disorder. In some embodiments, the subject is treated systemically with a therapeutic agent, i.e., the therapeutic agent for modulation of the bioelectric pattern is administered systemically. In some embodiments, the affected tissue, cell collective, or organ (or the tissue, cell collective, or organ likely to be affected) is treated with the therapeutic agent, and / or a tissue, cell collective, or organ adjacent or nearby to the affected tissue, cell collective, or organ is treated with the therapeutic agent, thereby increasing the expression and / or activity of HCN2 in the affected tissue, cell collective, or organ, and / or in the adjacent or nearby tissue, cell collective, or organ. In some embodiments, the treatment of a specific tissue, cell collective, or organ with the therapeutic agent results in modulation of the bioelectric pattern in one or more additional tissues, cell collectives, or organs. In some embodiments, the treatment of a specific tissue, cell collective, or organ with the therapeutic agent results in results in systemic modulation of the bioelectric pattern in the subject. By way of example but not by way of limitation, diseases or disorders include one or more of a defect caused by teratogen, carcinogen, or toxin exposure, cancer, a wound, cardiovascular disease or disorder, a neurological disease orAtty. Dkt. No.166118.01474 disorder, an inflammatory disease or disorder, an autoimmune disease or disorder, a metabolic disease or disorder.

[0013] In some embodiments, the neurological disease or disorder is one or more of dyskinesia, seizure disorder, epilepsy, ataxia, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, multiple sclerosis. In some embodiments, the inflammatory disease or condition comprises chronic inflammation. In some embodiments the metabolic disease or condition comprises Type 2 diabetes, obesity, metabolic syndrome.

[0014] In some embodiments, the therapeutic agent is administered multiple times per day, once per day, every other day, weekly, monthly, as needed as determined by a subject’s physician. In some embodiments, one or more additional active agents are administered to the subject, e.g., as in combination therapy.

[0015] In some embodiments, the subject is a human.

[0016] Disclosed herein are compositions for modulating bioelectric signaling (modulating a bioelectric pattern) and / or for increasing health and / or lifespan of a subject, comprising one or more of: HCN2 mRNA, HCN2 protein, HCN2 agonist / activator, a compound that mimics the effect of HCN2 overexpression. In some embodiments, the composition comprises a pharmaceutical carrier, and optionally one or more additional active agents. In some embodiments, the composition is formulated as a therapeutic composition. In some embodiments, the composition is formulated for systemic administration, or for local administration. In some embodiment, the composition is formulated for administration orally, by inhalation, injection, by microinjection, or transdermally. In some embodiments, one or more additional active agents are administered, e.g., as combination therapy.

[0017] Disclosed herein are genetically engineered cells comprising increased expression and / or activity of HCN2. In some embodiments, the cell comprises a mammalian cell. In some embodiments, the cell comprises an exogenous copy of an HCN2 gene operably linked to a promoter. In some embodiments, the engineered cell is in an organ, a cell collective, or a tissue. In some embodiments, the organ, cell collective, or tissue is engineered ex vivo, and is intended for transplant to a subject. In some embodiments, the cell is harvested from the patient, modified to include and express an exogenous copy of an HCN2 gene, and re-introduced into the patient. In some embodiments, the cell is harvested from a donor (not the patient), modified to include and express an exogenous copy of an HCN2 gene, and re-introduced into the patient.

[0018] Disclosed herein are methods to restore and / or establish and / or modulate bioelectric signaling (bioelectric patterns) in a subject in need thereof, the methods comprising modulating the expressionAtty. Dkt. No.166118.01474 and / or activity of HCN2 in the subject. In some embodiments, modulating the expression and / or activity of HCN2 comprises providing one or more therapeutic agents to the subject, comprising HCN2 nucleic, HCN2 protein, an HCN2 agonist / activator, a compound that mimics the effect of HCN2 overexpression. In some embodiments, the bioelectric patterns to be restored comprise a specific tissue, cell collective, or organ of the subject. In some embodiments, the tissue, cell collective, or organ comprises neural / nervous tissue (including but not limited to the brain, spinal cord, eyes), epidermis (skin), heart, liver, lungs, connective tissue, cartilage, muscle tissue, ovarian tissue, gastrointestinal tissue, pancreatic tissue, hematologic tissue, renal tissue, arterial tissue, venous tissue, immunological tissue, ocular tissue, stomach, esophagus, adrenal gland, colon, pituitary gland, testis. In some embodiments, the therapeutic agent is administered as a pharmaceutical composition, and is administered systemically, or is administered locally (e.g., to a specific region of the body, organ, cell collective, or tissue). In some embodiments, the subject is human. In some embodiments, one or more additional active agents are administered, e.g., as combination therapy.

[0019] In some embodiments, the methods and compositions disclosed herein further comprise an additional active agent, such as but not limited to one or more anti-aging agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.

[0021] FIG.1A and 1B. A) is a bar graph showing the effect of various drug treatment (T1-T11) on aging human organoids. Normal, human bronchial epithelial cells were used to generate the organoids for each of FIGS 1-4. The first bar in each sample represents the total number of organoids. The second bar represents the number of regenerated organoids produced after treatment. B) presents a table of the drug treatment compositions used in the experiment

[0022] FIG. 2. Shows images of organoids, and the chemical induction of regeneration in the aging organoid tissue. Fifteen new organoids were regenerated in the drug treatment group, while none were regenerated in the control group. Organoids typically exhibit senescence around day 40. The organoids in the images are at day 74 after initial induction of the organoids.Atty. Dkt. No.166118.01474

[0023] FIG. 3. Shows a magnified image of a human organoid after treatment. Regeneration (replication of the organoid) can be seen.

[0024] FIG. 4 . Shows images of organoids at various stages of regeneration with drug treatment. The first row shows untreated organoids undergoing senescence and decay. Rows 2-4 show regeneration of new organoids for senescent organoids. Organoids were characterized using RNAseq and immunohistochemistry. Cells display classical aging related changes in gene expression, cell senescence and death. Treatment resulted in regeneration of healthy organoids. Treatment was with gabapentin or lamotrigine, combined with nicotinamide mono nucleotide (NMN), and resveratrol.

[0025] FIG. 5. Is a graph illustrating the effects of HCN2 overexpression on lifespan in female Drosophila.

[0026] FIG. 6. Is graph illustrating the effects of HCN2 overexpression on lifespan in male Drosophila.

[0027] FIG. 7. Provides an image showing polarization state of human neonatal epithelial keratinocytes. Without treatment, the cell population typically experiences 15 population doublings before senescence, and exhibits a doubling time of 24-48 hours. Senescence is typically reached in about 20-30 days. Vmem Sensitive FLIM Dye – BeRST: longer lifetimes = depolarization; shorter lifetimes = hyperpolarization.

[0028] FIG. 8. Aging keratinocytes increasingly depolarize. Variability is the highest (day 17 and 20) prior to most depolarized state (day 27). Without wishing to be bound by theory, this may be a senescence transition period. Variability is lower on day 40 than on day 11.

[0029] FIG.9. Provides graphs showing that ouabain treatment causes depolarization except on day 27, where the cells are already at their most depolarized state. Variability is consistent with the date shown in FIG 8. Graphs represent day 11, 15, 17, 20, 27 and 40. The top graphs indicate Vmem fold change. The bottom graphs show variance.

[0030] FIG. 10. Provides results of K-mean cluster analysis which shows moderate clustering, and that neighboring cells appear to have more similar Vmem than distant cells. Images and data represent results from day 11, day 17, day 34 and day 40. On each image, the x-axis is labeled “Centroid_X”, each y-axis is labeled “Centroid_Y”, and each axis includes values of 0, 100, 200, 300, 400, and 500.

[0031] FIG.11. Bioelectricity in cells and non-neural cell groups. (A) Highly adaptable cells such as embryonic, stem, and cancer cells tend to have a relatively depolarized state, whereas mature and fully differentiated cells are hyperpolarized (data from

[0020] ). (B) The process of developmental bioelectricity involves ion channel proteins on cell surfaces, which contol the movement of ions and establish a resting potential (Vmem) across the membrane. This cell Vmem can also be communicatedAtty. Dkt. No.166118.01474 to other cells through electric synapses, or gap junctions. The resulting interactions between cell voltages lead to stable distributions of Vmem across tissues, as seen in the frog embryo face, where they determine the placement and number of craniofacial organs like eyes and mouth. These gradients can be manipulated using interventions that target ion channels, gap junctions, and small molecular signals like neurotransmitters.

[0032] FIG. 12A and 12B. A) Morphoceuticals discovery for aging. One possible approach to altering the bioelectrical patterns associated with diseases involves utilizing transcriptomic databases to identify potential ion channel targets within a particular tissue. This information can then be utilized as an input into a bioelectrical model to simulate the resulting bioelectrical pattern after intervention, which can be further analyzed to determine candidate drugs capable of moving the current bioelectrical state towards the desired pattern. Part of the image shown was reprinted with permission from the following reference: Cassandra DM Churchill, Philip Winter, Jack A Tuszynski, and Michael Levin. Eden–electroceutical design environment: ion channel tissue expression database with small molecule modulators. Iscience, 11:42–56, 2019. B) Illustrative corruption of the bioelectrical pattern and morphospace. The left panel provides an exemplary correlation between cancer resistance, regeneration and longevity quotient. The right panel provides an illustration of the transition, during aging, of the bioelectric pattern, the condition of the subject, and the morphospace traversed over time.

[0033] FIG.13A and 13B. A) Heatmap of HCN2 expression during aging in different tissue (GTex data sorted by end value); the heatmap of genetic expression is sorted by age (X axis, 20-70) and by tissue (Y-axis). B) HCN2 expression during aging in different tissue (GTex data sorted by descending ‘beginning minus’ end absolute value); the heatmap of genetic expression is sorted by age (X axis, 20-70 years) and by tissue (Y-axis).

[0034] FIG. 14A and 14B. A) Illustrative image showing voltage imaging of intestinal tissue harvested from Drosophila melanogaster. Intestinal tissue was harvested from control flies (young and old) and flies (young and old) engineered to overexpress HCN2 in intestinal tissue. The bioelectric pattern in the harvested tissue was analyzed by voltage imaging. B) Bioelectric sharpness (Moran’s I) in young control flies, old control flies, young HCN2-overexpressing flies, and old HCN2- overexpressing flies. In control flies, sharpness was reduced in older flies, as compared to younger flies (P-value: 0.01). Sharpness in old HCN2-overexpressing flies was increased, as compared to both old control flies (P-value: 0.0008) and young HCN2-overexpressing flies (P-value: 0.01).Atty. Dkt. No.166118.01474 DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention is described herein using several definitions, as set forth below and throughout the application.

[0036] Definitions

[0037] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”

[0038] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus ≤10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0039] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0040] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0041] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers toAtty. Dkt. No.166118.01474 groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

[0042] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0043] The term “compound”, as used herein, unless otherwise indicated, refers to any specific chemical compound disclosed herein. The compounds of the present disclosure include all stereoisomers (i.e., cis and trans isomers), tautomers, and all optical isomers of the present compound and related analogs in context (e.g., R and S enantiomers), as well as racemic, diastereomeric and other mixtures of such isomers, as well as all solvates and polymorphs of the compounds.

[0044] The term “patient” or “subject” refers to a mammal, preferably a human to which one or more of the present methods is applied.

[0045] As used herein, the terms “treat,” “treating,” “treatment” and the like refer to administration of a therapy which results in the slowing, alleviation, arrest, or reversal of one or more symptoms of a disease or condition of a subject in need of such treatment. A treated subject need not be fully “cured,” the disease or condition or symptom need not be fully arrested or alleviated to be considered “treated”. Any reduction, or any decrease in advance, of a sign or symptom exhibited by the subject is considered “treated”.

[0046] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, or topically. Administration includes self-administration and the administration by another. It is also to be appreciated that the various modes of treatment or prevention of medical conditions as described are intended to mean “substantial’, which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved. The language “provided to” or “administered to” are used interchangeably herein.

[0047] As used herein, the term “effective amount” or “pharmaceutically effective amount’ or “therapeutically effective amount of a composition, is a quantity sufficient to achieve a desiredAtty. Dkt. No.166118.01474 therapeutic and / or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in, the symptoms associated with a disease that is being treated. The amount of a composition of the invention administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. In some embodiments, an effective amount is an amount sufficient to modulate bioelectric signaling and / or bioelectric pattern. In some embodiments, an effective amount is an amount sufficient to increase the subject’s lifespan, tissue / organ structure, quality of life, and / or health. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions of the present invention can also be administered in combination with one or more additional therapeutic compounds.

[0048] As used herein, “lifespan” refers to the period of time during which a subject is alive. In some embodiments, the term “increased lifespan” with regard to subjects administered one or more treatments as disclosed herein (such as a therapeutic composition disclosed herein), means that the lifespan of a subject is increased relative to the lifespan of a control cohort. As used herein, “life expectancy” refers to an estimate of the number of remaining years of life of a subject at a given age. In some embodiments, the lifespan and / or life expectancy of the subject is increased at least 6 months, at least 1 year, at least two years, at least 3 years, at least 4 years, at least 5 years, or at least 10 years compared to a subject not administered the compositions.

[0049] As used herein, “health” refers to the physical and mental condition of a subject. In some embodiments, the term “increased health” or “increased healthspan” with regard to subjects administered one or more treatments disclosed herein (such as a therapeutic composition) refers to non-cancerous regeneration of cells instead of cell senescence, greater resistance to carcinogens, greater resistance to teratogens, greater resistance to the normal wear-and-tear of life including but not limited to muscle loss, bone loss, anemia, immune dysfunction, disease, and cognitive decline. A subject having increased health or increased healthspan will have an improved quality of life.

[0050] The term “cognitive function” is used to describe an endeavor or process by a patient or subject that involves thought or knowing. The diverse functions of the association cortices of the parietal, temporal and frontal lobes, which account for approximately 75% of all human brain tissue, are responsible for much of the information processing that goes on between sensory input and motor output. The diverse functions of the association cortices are often referred to as cognition, which literally means the process by which we come to know the world. Selectively attending to a particular stimulus, recognizing and identifying these relevant stimulus features and planning and experiencing the response are some of the processes or abilities mediated by the human brain which are related toAtty. Dkt. No.166118.01474 cognition. Compounds, compositions, and methods of treatment the present disclosure may be used to enhance cognition or reduce impairment of cognitive function. Methods of testing and documenting cognitive function in a subject are well-known in the art and include, without limitation, assessments of memory, problem solving / cognitive flexibility, and / or reaction time, such as the Mini-Mental State Examination (MMSE), Montreal Cognitive Assessment (MoCA), Mini-Cog, Alzheimer's Disease Assessment Scale - Cognitive (ADAS-Cog), Trail Making Test (TMT), and the Rey Auditory Verbal Learning Test (RAVLT). By evaluating cognition in a subject over time, impairment or improvement of cognitive function can be determined.

[0051] Impairment of cognitive function (“cognitive decline”) includes memory disorders and learning disorders, which are treatable according to the present technology, including but not limited to those disorders that result from aging, trauma, stroke, neurodegenerative disorders, or anxiety disorders. Examples of neurodegenerative disorders include, but are not limited to, those cognitive impairments associated with drug-induced states, neurotoxic agents, Alzheimer's disease, and aging. These conditions are readily recognized and diagnosed by those of ordinary skill in the art. The disease, condition, or disorder can be treated according to the methods and compositions disclosed herein, for example, by administering to the patient an effective amount of one or more compounds according to the present disclosure.

[0052] As used herein “Vmem” refers to membrane potential and relates to the voltage of one cell.

[0053] As used herein, “bioelectric pattern” refers to the spatial distribution of Vmem across many cells and relates to a multicellular phenomenon.

[0054] As used herein, “modulating bioelectric signaling” or “modulating a bioelectric pattern” referrers to altering Vmem by altering the membrane potential of multiple cells. Membrane potential can be modulated toward depolarization, polarization, or hyperpolarization. Modulation of the bioelectric signals results in modulation of a bioelectric pattern of multiple cells, such as in culture, in a tissue, in a cell collective, in an organ, or in an organism.

[0055] As used herein, the term “cell collective” refers to a group of cells capable of interacting to exhibit coordinated behavior and / or action.

[0056] Bioelectric Patterns, Disease, and Aging

[0057] Reference is made to Pio-Lopez and Levin (Aging as a morphostasis defect: a developmental bioelectricity perspective. doi.org / 10.31219 / osf.io / wkhx4, June 9, 2023), which is incorporated herein by reference in its entirety.

[0058] Maintaining order at the tissue level is crucial throughout the lifespan, as failure can lead to diseases such as cancer and an accumulation of molecular and cellular disorders. The most consistentAtty. Dkt. No.166118.01474 and pervasive result of these failures is aging, which is characterized by the progressive loss of function and decline in the ability to maintain anatomical homeostasis and reproduce. This leads to organ malfunction, diseases, and ultimately death. The traditional understanding of aging is that it is caused by accumulation of molecular and cellular damage resulting from energy metabolism and mitochondrial function, and that cell growth and lifespan are limited by replicative senescence due to shortening of telomeres.

[0059] Strategies to delay and potentially reverse the aging process have recently been explored and include blood factors, metabolic manipulations, senolytics, and cellular reprogramming. Methods and compositions are disclosed herein, which underlie, are intertwined with, or dovetail with each of the above explored strategies. The disclosed methods and compositions may be useful not only to alleviate many of the impacts of aging on quality of life, but also to treat and alleviate numerous disease and disorders common in the aging human population, such as cancer and cognitive impairment.

[0060] Bioelectric controls, such as voltage-sensitive channels, implement feedback loops that can break spatial symmetry and negative feedback for robustness. In addition, gap junctions (electrochemical synapses) enable bioelectric signals to be sent between cells in response to voltage and other ionic parameters, allowing for tissue-level feedback loops that respond to changes in large- scale patterns. These characteristics facilitate the precise orientation, scaling, and shaping of organs during embryogenesis, regeneration, and remodeling, through dynamic long-range coordination and anatomical decision-making. Specifically, it has been argued that these methods work because evolution exploits bioelectrical interfaces endogenously for modular control of anatomical homeostasis and repair processes. Using molecular-genetic and pharmacological techniques, researchers have been able to manipulate the distribution of voltage gradients to effect alternative anatomical structure / location during development, increase regeneration after injury, and cancer suppression. They have been able to induce the growth of entire organs from different types of tissue, change the direction of primary body axes, initiate regeneration of tails and limbs in non-regenerative conditions, alter the shape of regenerating heads in planaria to resemble those of different species, and convert oncogene-induced tumors into normal tissue. A variety of tools from behavioral and cognitive neuroscience have been harnessed for the control of tissue-level and organ-level information, which enables pushing much of the complexity onto the system itself via simple triggers. This top-down methodology for controlling anatomy opens promising avenues for therapeutic approaches to rejuvenation and the diseases of aging if we take the view that aging is a defect of morphostasis - a gradual drift from effective maintenance of anatomical homeostasis.Atty. Dkt. No.166118.01474

[0061] Developmental bioelectricity refers to signaling among non-excitable cells mediated by endogenous electricfields and differences in resting potential. These electric states arise due to specific ion channels and pump proteins, which uphold voltage gradients across the cell membrane. Such gradients elicit various cellular responses, including transcriptional and epigenetic responses (see FIG. 11B). Ion channels are selectively permeable; they differentiate between the electrical charge and size of ions, allowing movement of specific ions, such as K+, Na+, Ca2+, or Cl−, in one direction through their pore. Theflow of ions across membranes and the resulting changes in the bioelectrical pattern play a crucial role in many cellular processes, including muscle contraction, nerve signaling, secretion, tissue growth, cell proliferation and apoptosis. Ion channels play a critical role in the control systems of embryonic development and have been linked to several channelopathies of embryonic embryogenesis, which are disorders caused by mutations in ion channel genes. These conditions have been well-studied in various animal models such as fruitflies, zebrafish, mice, frogs, and human patients, implicating several ion channels, such as the potassium channel Kir2.1, and gap junctions (connexins). This research has helped to better understand the underlying mechanisms of these disorders and the role of ion channels in normal embryonic development. In addition, dysregulation of ionic gradients can contribute to age-related declines in physiological function and dysfunction of ion channels is often linked to organ failure during aging. Because of their central role, bioelectric signals are a popular target for induction of organ formation and regeneration.

[0062] Together, the voltage states induced by the action of channels, pumps, and gap junctions create a network of bioelectrical activity throughout the tissue, which ultimately triggers changes in gene expression and cell behavior (migration, differentiation, shape change, proliferation, apoptosis, etc.) that lead to specific morphogenetic outcomes. This bioelectric code translates patterns of voltage into information about organ size, shape, and positioning through mechanisms such as neurotransmitter gating, calcium signaling, and voltage-sensitive phosphatases. Analogous to the way electrical neural networks of the brain control muscle movement for behavioral goals, the ancient bioelectric networks of the body control cell activities to achieve morphogenetic outcomes, by storing and implementing gradually changing anatomical patterns.

[0063] Bioelectric control of single-cell function and differentiation

[0064] The state of Vmem in a cell and its neighbors is crucial in regulating cell behavior, along with other modes of signaling (see FIG. 11A). Usually, quiescent and differentiated cells exhibit strong polarization, while embryonic, stem, and tumor cells tend to be depolarized. However, many cells, much like neurons, have several Vmem values, with a unique set of voltage domains over their surface. Although the functional significance of voltage microdomains is yet to be determined, VmemAtty. Dkt. No.166118.01474 regulation is being employed in bioengineering to control cell connectivity differentiation, and wound healing.

[0065] Changes in Vmem are involved in controlling differentiation and proliferation in various cell types, including human mesenchymal stem cells, cardiomyocytes, iPSCs, vascular muscle, embryonic stem cells, myoblasts, as well as in neurotransmitter specification in the developing nervous system and heart. This implication of Vmem in migration, differentiation and proliferation is well- established. Crucially, the key role of bioelectricity extends far beyond the single cell state and underlies its control of large-scale order in regeneration, developmental defects, and disorders such as cancer.

[0066] Bioelectric control of large-scale anatomy

[0067] The dynamics of bioelectric signaling within the body play a crucial role in regulating diverse processes including wound healing, the formation of neural circuits, eye development, facial patterning, brain and tail size, and the determination of left-right and anterior-posterior body axis.

[0068] Manipulation of bioelectrical patterns has emerged as a promising approach for controlling morphogenesis and advancing regenerative medicine, especially because it offers the potential of using a high-level interface where simple signals can serve as triggers of complex, downstream self- limiting cascades that do not have to be micromanaged by the bioengineer. These interventions can be implemented at various levels, such as using electrotaxis to control cell movement in wound healing or by manipulating ion channels using different types of drugs known as morphoceuticals to establish specific bioelectric prepatterns for organogenesis or appendage induction. (See FIG.12).

[0069] Studies have demonstrated that malformations caused by mutations and teratogens can be reversed by enforcing the correct voltage maps using drugs, optogenetics, or channel misexpression. Additionally, profound defects in brain structure and function caused by teratogens can be reversed by restoring normal endogenous voltage pre-patterns in Xenopus.

[0070] Bioelectric signaling has also been found to be a viable method for inducing the regrowth of entire body parts. Studies on rats have demonstrated that limb regeneration can be enhanced through the use of bioelectric signals. Additionally, in a frog model of tail regeneration, complete regrowth of muscle, spinal cord, blood vessels, and skin has been observed subsequent to one-hour treatment with an ionophore, which induces a pro-regenerative state in the wound and does not need to be maintained during the subsequent regeneration process.

[0071] These bioelectrical networks in the tissues store the target morphology and coordinate cellular activity across distances. Because a cell’s bioelectric state is influenced by prior experiences, extracellular signaling, and signals from neighboring cells, these post-translational events areAtty. Dkt. No.166118.01474 invisible to traditional omics approaches. While complicating modeling and experimentation, this historicity property offers a unique target for biomedicine: reprogrammability.

[0072] Characteristics of aging cells with respect to Vmem

[0073] The inventors have previously shown that reinforcing the sharp bioelectric prepatterns resolves a variety of birth defects in frog embryos. The inventors hypothesize that resistance of the body to aging (small-scale morphogenesis that needs to happen throughout lifespan to resist progressive degradation) also requires a sharp bioelectric prepattern. The inventors hypothesize that if the prepattern is sharpened in the same way as was done for frog embryos, other organisms could exhibit longer healthspan, as well as resistance to carcinogens, teratogens, and the normal wear-and- tear of life.

[0074] Thus, the inventors propose to use genetic (DNA, RNA) or chemical (small molecule drugs) openers of ion channels, and in particular, a channel called HCN2, which due to its voltage-sensitive nature is a context-sensitive enhancer of bioelectric differences - a sharpen filter for bioelectric prepatterns. The inventors propose to use HCN2 proteins from different species (or functional variants or fragments thereof), to enhance healthspan, lifespan, and overall resistance to anything that undermines the target morphology of the body.

[0075] As shown in FIGS 7-10, aging human cells experience a change of Vmem, exhibit greater range of Vmem variance, and lose responsivity to stimuli. Regarding spatial dynamics, neighboring cells exhibit similar Vmem as compared to distant cells, and thus exhibit a clustering effect. As shown in the figures, aging cells undergo depolarization, and likely exhibit the greatest variance in Vmem when undergoing senescence. While both the youngest and oldest cells are able to respond to ouabain treatment as exhibited by depolarization, cell that are at their most depolarized state show no response.

[0076] Hyperpolarization Activated Cyclic Nucleotide Gated Potassium and Sodium Channels

[0077] Research has shown that developmental defects in the brain caused by mutations in key neurogenesis genes, such as Notch, can be rescued by the overexpression or activation of native HCN2 (hyperpolarization-activated cyclic nucleotide-gated) channels. As voltage-regulated ion channels, HCN2 channels are sensitive to their context, increasing hyperpolarization in slightly polarized cells but having no effect on depolarized cells, thus functioning as ”contrast enhancers” that sharpen weakened differences in Vmem across compartment boundaries, similar to how a sharpenfilter emphasizes order in a fuzzy image. In these studies, application of the HCN2 opener drugs was systemic, without the need to precisely control the spatial properties of delivery.

[0078] Hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels generate electrical rhythmicity in both specialized neuron and cardiomyocytes by evoking the pacemakerAtty. Dkt. No.166118.01474 current Ih (If, Iq). The primary activating stimulus for HCN channels is hyperpolarization of the plasma membrane, as proceeding in the repolarization phase of the action potential. In addition to this primary stimulus, sympathetic activity enhances the activation of HCN channels by the second messenger cAMP, binding to the channels. The resulting increase of the pacemaker current by cAMP binding evokes an acceleration of electrical rhythmicity. Four types of HCN channels have been described: HCN1, HCN2, HCN3, and HCN4. Of these, HCN2 and HCN4 are the main types expressed in heart. The presence of both hHCN2 and hHCN4 mRNAs in cardiac tissues raises the possibility that the two channels may form heteromeric complexes in vivo. HCN2 is primarily expressed in platelets, the frontal cortex, the spinal cord, and the retina. Increased expression of HCN2 has been reported in NSC Lung Cancer NCI-H460.

[0079] Table 1. HCN Isozyme Expression and Knockout Phenotype Isoform Expression (major sites) Phenotype Brain Heart Brain Heart Impaired motor learning, Hippocampus, enhanced HCN1 Purkinje cells, Sinoatrial node KO hippocampal- Not reported neocortex dependent learning and memory HCN2 Ubiquitous Ubiquitous KO Ataxia, absence Sinus epilepsy KO arrhythmia Olfactory HCN3 bulb, -- KO Phenotype not repo KO Not reported hypothalamus rted Embryonic lethal, embryos are bradycardic and unable Sinoatrial KO to speed up 4 T node, hear rate, HCN halamus, olfactory bulb atrioventricular KO Not reported non- node, Purkinje functional fibers pacemaker cells Temporarily Repetitive induced KO sinus pausesAtty. Dkt. No.166118.01474 Human Bradycardia mutation and QT- prolongation

[0080] In animal models, HCN2-deficient animals can be easily distinguished from their wild-type littermates because they are hypoactive and show a wide-based, ataxic gait. Electroencephalogram recordings coupled with video analyses clearly demonstrated that mutants suffer from absence epilepsy and sinus node dysfunction. Deletion of HCN2 in mice results in a cardiac phenotype. Mutants exhibit sinus arrhythmia characterized by varying RR intervals but otherwise unchanged electrocardiogram (ECG) parameters (PQ-, QRS-, QT-intervals). The same arrhythmia occurs in mice with cardiomyocyte-specific deletion of HCN2, ruling out the possibility that the phenotype has its origin in neuronal defects. Surprisingly, the deletion of HCN2 has no effect on heart rate. Mutant mice are neither bradycardic at rest nor have any impairment in speeding up heart rate during exercise or after administration of β-adrenergic agonists. These results indicate that HCN2 is not required for modulating basal as well as stimulated heart rate. In contrast, deletion of HCN4 in mice results in embryonic death. A separate study reported that HCN2 channels are a key determinant of myelin sheath length in the CNS. Reduction of HCN2 function via pharmacological blockade or generation of transgenic mice with two independent oligodendrocyte-specific HCN2 knock-out strategies reduced myelin sheath length. Researchers also reported that the lack of HCN2 facilitates the spontaneous occurrence of oscillatory activity in thalamocortical synaptic networks. Brain (or neural) oscillations” refers to the rhythmic and / or repetitive electrical activity generated spontaneously and in response to stimuli by neural tissue in the central nervous system.

[0081] Altered expression and / or activity of HCN2 has been implicated in age-related hearing loss, hypertrophic cardiomyopathy, atrial fibrillation, neuropathic pain, and inflammatory pain. Additional non-limiting age-related disease or conditions that would benefit from the methods and compositions disclosed herein include: amyotrophic lateral sclerosis, spinal muscular atrophy, Parkinson’s disease, Alzheimer’s disease, neuroinflammation, dyskinesia, seizure disorder, epilepsy, ataxia, Huntington’s disease, multiple sclerosis, cancer, defects caused by teratogen exposure, wounds, cardiovascular disease, neurological disease, inflammatory and / or autoimmune disease, metabolic disease, Type 2 diabetes, obesity, metabolic syndrome, genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, disabled macroautophagy, deregulated nutrient-sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, dysbiosis, aging and aging-related disorders, muscle loss, bone loss, anemia, immune dysfunction, neuropsychiatric disorders, learning disorders, memory disorders, attention deficitAtty. Dkt. No.166118.01474 disorder (ADD), attention deficit disorder with hyperactivity (ADD-HD), autism, pervasive development disorder, bipolar disorder, unipolar disorder, dysthymic disorder, post-partum depression, seasonal affective disorder, depression, and cognitive decline.

[0082] The amino acid sequence of human HCN2 is provided below, and it is contemplated that the full-length sequence, functional fragments thereof, or variants thereof will be useful in the context of the present disclosure. Similarly, HCN2 protein from species other than human, functional fragments thereof, and variants thereof are also contemplated.

[0083] Protein Sequence HCN2 [Homo sapiens] NCBI Reference Sequence: NP_001185.3 (SEQ ID NO: 1) 1 mdarggggrp gespgatpap gpppppppap pqqqpppppp papppgpgpa ppqhppraea 61 lppeaadegg prgrlrsrds scgrpgtpga astakgspng ecgrgepqcs pagpegparg 121 pkvsfscrga asgpapgpgp aeeagseeag pageprgsqa sfmqrqfgal lqpgvnkfsl 181 rmfgsqkave reqervksag awiihpysdf rfywdftmll fmvgnliiip vgitffkdet 241 tapwivfnvv sdtfflmdlv lnfrtgivie dnteiildpe kikkkylrtw fvvdfvssip 301 vdyiflivek gidsevykta ralrivrftk ilsllrllrl srliryihqw eeifhmtydl 361 asavmricnl ismmlllchw dgclqflvpm lqdfprncwv singmvnhsw selysfalfk 421 amshmlcigy grqapesmtd iwltmlsmiv gatcyamfig hataliqsld ssrrqyqeky 481 kqveqymsfh klpadfrqki hdyyehryqg kmfdedsilg elngplreei vnfncrklva 541 smplfanadp nfvtamltkl kfevfqpgdy iiregtigkk myfiqhgvvs vltkgnkemk 601 lsdgsyfgei clltrgrrta svradtycrl yslsvdnfne vleeypmmrr afetvaidrl 661 drigkknsil lhkvqhdlns gvfnnqenai iqeivkydre mvqqaelgqr vglfpppppp 721 pqvtsaiatl qqaaamsfcp qvarplvgpl algsprlvrr pppgpapaaa spgppppasp 781 pgapasprap rtspygglpa aplagpalpa rrlsrasrpl sasqpslphg apgpaastrp 841 assstprlgp tpaaraaaps pdrrdsaspg aaggldpqds arsrlssnl

[0084] The Inventors evaluated HCN2 expression during aging, in different human tissue. Data is shown in the heatmaps provided in FIG. 13A-B. Tissues evaluated included brain (cerebellum, caudate (basal ganglia), hippocampus, putamen (basal ganglia), substantia nigra, cerebellar hemisphere, hypothalamus, nucleus accumbens (basal ganglia), cortex, anterior cingulate cortex (BA24), spinal cord (cervical c-1), amygdala, and frontal cortex (BA9)), heart (left ventricle and atrial appendage), skin (sun exposed (lower leg), and not sun exposed (suprapubic)), testis, esophagus (muscularis, and mucosa), pituitary, adrenal gland, colon (sigmoid), and transformed fibroblast cells. Age of the tissue evaluated ranged from 20-70 years.

[0085] The effect of HCN2 overexpression was tested in model organism Drosophila melanogaster (Example 1). In female individuals overexpressing the HCN2 protein, greater mean longevity was exhibited (FIG.5). Only 50% of the wild-type females were alive after 50 days, while more than 80% of the HCN2 overexpressing females were alive at the same time point. More than 75% of the HCN2 overexpressing females were alive at 57 days as compared to less than 50% of the wild-type females. However, overexpression in male fruit flies had no apparent effect (FIG 6). The effect of intestinal tissue-specific HCN2 overexpression was also assessed in Drosophila melanogaster (Example 2;Atty. Dkt. No.166118.01474 FIGS. 14A and 14B). In control flies, sharpness was reduced in older flies, as compared to younger flies (P-value: 0.01). Sharpness in old HCN2-overexpressing flies was increased, as compared to both old control flies (P-value: 0.0008) and young HCN2-overexpressing flies (P-value: 0.01).

[0086] The effect of ion channel openers was also tested on human organoid tissue with respect to anti-aging. As shown in FIGS. 1-4, gabapentin, lamotrigine, and compositions comprising these compounds elicited organoid regeneration as opposed to senescence. Treatment resulted in regeneration of young healthy organoids from old, decaying organoids.

[0087] Methods and Compositions to Modulate Vmem and the Bioelectric Pattern

[0088] In some embodiments, methods and compositions are provided to modulate Vmem, and thereby modulate the bioelectric patterns in a subject in need thereof. In some embodiments, a bioelectric pattern is modulated to increasing the health and / or lifespan of a subject, and / or to treat or ameliorate a disease or conditions of the subject.

[0089] In some embodiments, one or more bioelectric patterns are modulated to sharpen or enhance the pattern. In some embodiments, the bioelectric patterns are modulated to more closely resemble a wild-type or control state, and in some embodiments, bioelectric patterns are modulated to exhibit a variance from a wild-type or control state. Bioelectric sharpness may be measured and / or quantified using any suitable method known in the art. In some embodiments, the bioelectric sharpness is measured by voltage imaging and / or calculation of Moran’s I.

[0090] In some embodiments, modulating a bioelectric pattern comprises modulating the Vmem of cells, such as by enhancing the expression and / or activity of one or more ion channel. In some embodiments, modulating a bioelectric pattern comprises modulating the Vmem of cells, such as by modulating the expression and / or activity of one or more ion channels. In some embodiments, the ion channel comprises a hyperpolarization-activated cyclic nucleotide-gated cannel (HCN channel). In some embodiments, the HCN channel comprises one or more of HCN1, HCN2, HCN3, or HCN4. In some embodiments, the HCN channel comprises HCN2.

[0091] In some embodiments, the expression and / or activity of an ion channel, such as an HCN2 ion channel is modulated by providing one or more ion channel modulators to cells, tissues, cell collectives, or organs, in vivo, ex vivo, or in vitro. In some embodiments, an ion channel modulator is provided locally or systemically to a subject. In some embodiments, ion channel modulators comprise one or more of: HCN2 DNA or mRNA, HCN2 protein, an HCN2 agonist / activator, a compound that mimics the effect of HCN2 overexpression.

[0092] By way of example, but not by way of limitation, in some embodiments, providing HCN2 nucleic acid (DNA or mRNA) to the subject comprises administering a composition comprisingAtty. Dkt. No.166118.01474 HCN2 nucleic acid formulated as one or more of naked DNA or RNA, with lipid nanoparticles, lipid / polymer nanoparticles, as a vector, such as a viral vector. Nucleic acid (DNA and RNA, e.g., mRNA) delivery strategies are well-known in the art and include, without limitation delivery of nucleic acid, formulation with lipid nanoparticles, formulation in protein-nucleic acid complexes, e.g., with cleavable linkers and / or cationic proteins which form complex with the nucleic acid, polymer nanoparticles, formulating nucleic acid with lipid / polymer nanoparticle hybrids, ex-vivo loading, cationic nano-emulsion, and providing the nucleic acid in vectors, such as via viral vectors. Microinjection of any of these forms of nucleic acid is contemplated herein.

[0093] Protein delivery strategies are also well known in the art. By way of example, but not by way of limitation, providing HCN2 protein to the subject may comprise one or more of transplanting modified cells, PEGylation, trogocytosis, the use of extracellular vesicles, the use of bacterial vessels, and the use of liposomes.

[0094] Protein or RNA overexpression strategies are well-known in the art include, but are not limited to gene editing methods, such as (CRISPR / Cas) and various vector systems well-known in the art, to provide additional gene copy numbers to the cells / tissues / cell collectives / organs of interest, and / or to modify regulatory elements. In some embodiments, the methods and compositions utilize a conditional gene expression system, to allow for spatial and / or temporal control over the protein or RNA expression or overexpression. Conditional gene expression systems are known in the art and include, without limitation the Cre-lox system.

[0095] In some embodiments, compounds that modulate HCN2 activity, or that target ion channels are provided. By way of example, but not by way of limitation, compounds that modulate HCN2 activity include Lamotrigine (activator), Gabapentin (activator), Erbstatin (inhibitor), ZD7288 (inhibitor), Ivabradine (inhibitor), miR-1 and miR-133 (expression inversely correlated), Zatebradine (inhibitor), MEL55a (inhibitor), Cilobradine (inhibitor), Fasudil (inhibitor), and TS-035 (inhibitor).

[0096] Additional non-limiting methods to increase the activity of ion channels such as HCN2 channels include but are not limited to activation of channel via cAMP (e.g., via use of allosteric modulators to increase affinity for cAMP, or engineering the cAMP binding), increasing the concentration of cAMP, and upregulating chaperonin proteins to improve trafficking to cells.

[0097] In embodiments of any of the above non-limiting methods, one or more of the HCN2 modulating agents described above may be administered with one or more additional active agents, e.g., such as in combination therapy. In some embodiments, the one or more additional active agents comprise electroceutical agents. In some embodiments, the one or more additional active agents comprise anti-aging / lifespan enhancing compositions. By way of example and not by way ofAtty. Dkt. No.166118.01474 limitation, such compounds include rapamycin, metformin, alpelisib, everolimus, rileminidine, 17-α- estradiol, acarbose, canagliflozin, captoril, glycine, metformin + rapamycin, and nordihydroguaiaretic acid (NDGA). Additionally or alternatively, in some embodiments, the additional active agent comprises one or more additional ion channel modulators. Additionally or alternatively, in some embodiments, the one or more additional active agents is a drug or therapeutic prescribed to treat or alleviate a disease or condition the subject currently has. In embodiments, the therapeutic compositions and methods of the present disclosure (e.g., comprising HCN2 modulators), enhance the effect of the drug or therapeutic.

[0098] Exemplary Subjects and Diseases

[0099] Disclosed herein are methods and composition for treating subjects suffering from various disease states or conditions, and / or suffering from the discomforts and characteristic of aging. By modulating the bioelectric patterns in organs, cell collectives, and tissues of such subjects (i.e., by modulating cellular Vmem), the discomfort, signs and symptoms of such diseases and conditions are reduced, slowed, or alleviated.

[0100] Regarding aging and increasing health and or lifespan and reducing or slowing the wear and tear of life, modulating the bioelectric pattern by administering the therapeutic compositions disclosed herein is anticipated to provide the subject with one or more of the following positive effects: greater resistance to carcinogens, greater resistance to teratogens, greater resistance to disease and / or infection, greater resistance to the normal wear-and-tear of life including but not limited to cardiovascular disease, vascular dysfunction, skin damage, muscle loss, bone loss, arthritis or other inflammatory disorders, anemia, immune dysfunction, cognitive decline, non-cancerous regeneration of cells instead of cell senescence, reducing one or more of: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, disabled macroautophagy, deregulated nutrient-sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis.

[0101] Aging subjects that will typically benefit from the compositions and methods disclosed herein are about 40 years old or older, 50 years old or older, 60 years old or older, 70 years old or older, 80 years old or older or 90 years old or older.

[0102] Regarding cardiovascular disease and vascular dysfunction, ionic channels modulate blood vessel dilation. Accordingly, modulating cellular Vmem and thereby modulating bioelectric patterns using the methods and compositions disclosed herein, is particularly suited to the treatment, amelioration, delay of onset, and / or prevention of cardiovascular disease and vascular dysfunction.Atty. Dkt. No.166118.01474

[0103] Regarding neurological diseases or disorders, modulating cellular Vmem, and thereby modulating the bioelectric pattern, by administering the therapeutic compositions disclosed herein, is anticipated to provide the subject with relief from the symptoms of, slow the progress of, or alleviate the discomfort associated with one or more of dyskinesia, seizure disorder, epilepsy, ataxia, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, depression, multiple sclerosis.

[0104] Regarding cardiovascular diseases or disorders, modulating cellular Vmem, and thereby modulating the bioelectric pattern, by administering the therapeutic compositions disclosed herein, is anticipated to provide the subject with relief from the symptoms of, slow the progress of, or alleviate the discomfort associated with cardiovascular disease.

[0105] Regarding metabolic, autoimmune, or inflammatory diseases or disorders, modulating cellular Vmem, and thereby modulating the bioelectric pattern, by administering the therapeutic compositions disclosed herein, is anticipated to provide the subject with relief from the symptoms of, slow the progress of, or alleviate the discomfort associated with the autoimmune or inflammatory disease or disorder. Exemplary disease or disorders include chronic inflammation, Type 2 diabetes, obesity, metabolic syndrome.

[0106] Subjects suffering from a disease or disorder may be of any age, and may also be aging subjects.

[0107] In some embodiments, the subject is suffering from the effects of exposure to a teratogen, carcinogen, or other toxin, and administration of a therapeutic composition according to the methods disclosed herein is anticipated to modulate cellular Vmem, and thereby modulate the bioelectric pattern, and allow for rescue or recovery of the damage due to the exposure. In some embodiments, the subject is in utero.

[0108] In some embodiments, Vmem, and the bioelectric pattern is modulated in one or more cells, in a cell collective, in a tissue, or in an organ in vivo, or ex vivo. By way of example but not by way of limitation, to prepare cells, a tissue, a cell collective, or an organ for transplant, the cells, tissue, cell collective, or organ is treated with a composition that modulates cellular Vmem, thereby modulating the bioelectric pattern (e.g., an ion channel modulator, such as a HCN2 channel modulator) to sharpen the electric filed, or to modulate the bioelectric pattern to more closely resemble a wild-type field, or to modulate the bioelectric pattern to deviate from wild-type. The age of the organ, cell collective, or tissue may be any age, and treatment according to the present methods may result in a “younger” organ, cell collective, or tissue after treatment, e.g., after gabapentin or lamotrigine treatment the organ, cell collective, or tissue may exhibit regeneration.Atty. Dkt. No.166118.01474

[0109] In any of the above subjects, treatment according to the present disclosure may result in “younger” organ(s), cell collective(s), or tissue after treatment, as compared to before treatment, e.g. the treated organ(s), cell collective(s), or tissue may exhibit organ, cell collective, or tissue regeneration. An organ, cell collective, or tissue may be characterized using RNAseq and immunohistochemistry, to verify that the cells display classical aging related changes in gene expression, cell senescence, and death prior to treatment.

[0110] In some embodiments, the cells, cell collective, tissue or organ are harvested from a first subject, treated as described above, and then transplanted into a second subject. In some embodiments, the first and second subject are the same. In some embodiments, the first and second subjects are different. EXEMPLARY EMBODIMENTS

[0111] Embodiment 1. A method for increasing the health and / or lifespan of a subject, the method comprising: increasing the expression and / or activity of HCN2 in the subject.

[0112] Embodiment 2. The method of embodiment 1, wherein increased health and / or lifespan comprises one or more of: greater resistance to carcinogens, greater resistance to teratogens, greater resistance to the normal wear-and-tear of life including but not limited to muscle loss, bone loss, anemia, immune dysfunction, cognitive decline, cardiovascular disease and vascular dysfunction.

[0113] Embodiment 3. The method of embodiment 1 or 2, wherein increasing the expression and / or activity of HCN2 comprises one or more of: (a) providing HCN2 nucleic acid (DNA and / or mRNA) to the subject, (b) providing HCN2 protein to the subject, (c) providing an HCN2 agonist / activator to the subject, (d) providing a compound that mimics the effect of HCN2 overexpression to the subject.

[0114] Embodiment 4. The method of embodiment 3, wherein providing HCN2 DNA and / or mRNA to the subject comprises administering a composition comprising HCN2 DNA and / or mRNA formulated (i) with lipid nanoparticles, (ii) lipid / polymer nanoparticles, or (iii) as a vector, optionally wherein the HCN2 DNA and / or mRNA is administered via microinjection.

[0115] Embodiment 5. The method of embodiment 3, wherein providing HCN2 protein to the subject comprises administering genetically modified cells (i) expressing or overexpressing HCN2 protein or (ii) comprising a conditional HCN2 expression or overexpression system.Atty. Dkt. No.166118.01474

[0116] Embodiment 6. The method of embodiment 3, wherein the HCN2 agonist is a direct agonist.

[0117] Embodiment 7. The method of embodiment 3, wherein the HCN2 agonist is an indirect agonist.

[0118] Embodiment 8. The method of embodiment 3, wherein (c) comprises one or more of lamotrigine and gabapentin, optionally, wherein the method further comprises administering one or more additional ion channel modulators, and / or one or more additional active agents.

[0119] Embodiment 9. The method of any one of embodiments 2-8, wherein greater resistance to the normal wear-and-tear of life comprises non-cancerous regeneration of cells instead of cell senescence.

[0120] Embodiment 10. The method of any one of embodiments 3-9, wherein the HCN2 DNA and / or mRNA, HCN2 protein, the HCN2 agonist and / or the compound that mimics the effect of HCN2 overexpression is provided to the subject systemically.

[0121] Embodiment 11. The method of any one of embodiments 3-9, wherein the HCN2 DNA and / or mRNA, HCN2 protein, the HCN2 agonist and / or the compound that mimics the effect of HCN2 overexpression is provided to an organ, cell collective, or tissue of the subject.

[0122] Embodiment 12. The method of embodiment 10 or 11, wherein the subject is a non-diseased animal subject.

[0123] Embodiment 13. The method of embodiment 10 or 11, wherein the subject is in utero, and optionally, wherein the HCN2 DNA and / or mRNA, HCN2 protein, the HCN2 agonist and / or the compound that mimics the effect of HCN2 overexpression is administered by microinjection.

[0124] Embodiment 14. The method of embodiment 13, wherein the subject is diagnosed with defect caused by teratogen exposure.

[0125] Embodiment 15. The method of embodiment 10, wherein the subject is diagnosed with cancer.

[0126] Embodiment 16. The method of embodiment 11, wherein the subject is diagnosed with cancer, and the expression and / or activity of HCN2 is increased in the cancer tissue or a tissue or cell collective adjacent to the cancer tissue.

[0127] Embodiment 17. The method of embodiment 10, wherein the subject has a wound.Atty. Dkt. No.166118.01474

[0128] Embodiment 18. The method of embodiment 11, wherein the subject has a wound and the expression and / or activity of HCN2 is increased in the wounded tissue or in a tissue or cell collective adjacent to the wound.

[0129] Embodiment 19. The method of embodiment 10, wherein the subject has a cardiovascular disease and / or vascular dysfunction.

[0130] Embodiment 20. The method of embodiment 11, wherein the subject has a cardiovascular disease and / or vascular dysfunction and the expression and / or activity of HCN2 is increased in the heart, and / or cardiovascular and / or vascular system.

[0131] Embodiment 21. The method of embodiment 10, wherein the subject has a neurological disease or disorder.

[0132] Embodiment 22. The method of embodiment 11, wherein the subject has a neurological disease or disorder and the expression and / or activity of HCN2 is increased in the brain and / or nervous system.

[0133] Embodiment 23. The method of embodiment 21 or 22, wherein the neurological disease or disorder comprises one or more of dyskinesia, seizure disorder, epilepsy, ataxia, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, multiple sclerosis.

[0134] Embodiment 24. The method of embodiment 10, wherein the subject is diagnosed with an inflammatory and / or autoimmune disease or condition.

[0135] Embodiment 25. The method of embodiment 11, wherein the subject is diagnosed with an inflammatory and / or autoimmune disease or condition and the expression and / or activity of HCN2 is increased in the tissue(s), cell collective(s), or organ(s) exhibiting inflammation.

[0136] Embodiment 26. The method of embodiment 24 or 25, wherein the inflammatory disease or condition comprises chronic inflammation.

[0137] Embodiment 27. The method of embodiment 10, wherein the subject is diagnosed with a metabolic disease or condition.

[0138] Embodiment 28. The method of embodiment 11, wherein the subject is diagnosed with a metabolic disease or condition.

[0139] Embodiment 27. The method of embodiment 27 or 28, wherein the metabolic disease or condition comprises Type 2 diabetes, obesity, metabolic syndrome.

[0140] Embodiment 30. The method of embodiment 1, wherein increasing health and / or lifespan comprises reducing one or more of: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, disabled macroautophagy, deregulated nutrient-sensing,Atty. Dkt. No.166118.01474 mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis.

[0141] Embodiment 31. The method of any one of the previous embodiments, wherein the composition is administered multiple times per day, once per day, every other day, weekly, monthly, as needed as determined by a subject’s physician.

[0142] Embodiment 32. The method of any one of the previous embodiments, wherein the subject is a human.

[0143] Embodiment 33. A composition for increasing health and / or lifespan of a subject, comprising one or more of: (a) HCN2 mRNA, (b) HCN2 protein, (c) HCN2 agonist / activator, (d) a compound that mimics the effect of HCN2 overexpression.

[0144] Embodiment 34. The composition of embodiment 33, comprising a pharmaceutical carrier, and optionally one or more additional active agents.

[0145] Embodiment 35. The composition of embodiment 33 or 34, wherein the composition is formulated for systemic administration.

[0146] Embodiment 36. The composition of embodiment 33 or 34, wherein the composition is formulated for local administration.

[0147] Embodiment 37. The composition of embodiment 33 or 34, wherein the composition is formulated for administration orally, by inhalation, injection, or transdermally.

[0148] Embodiment 38. A genetically engineered cell comprising increased expression and / or activity of HCN2.

[0149] Embodiment 39. The genetically engineered cell of embodiment 38 comprising a mammalian cell.

[0150] Embodiment 40. The genetically engineered cell of embodiment 38 or 39, comprising an exogenous copy of an HCN2 gene operably linked to a promoter, optionally wherein the cell comprises a conditional HCN2 expression system.

[0151] Embodiment 41. The cell of any one of embodiments 38-40, wherein the engineered cell is in an organ, cell collective, or a tissue.

[0152] Embodiment 42. The cell of embodiment 41, wherein the organ, cell collective, or tissue is ex vivo, and is intended for transplant to a subject.Atty. Dkt. No.166118.01474

[0153] Embodiment 43. A method to modulate bioelectric signaling and a bioelectric pattern, optionally, to restore and / or establish endogenous (wild-type) bioelectric patterns, in a subject in need thereof, the method comprising: modulating the expression and / or activity of HCN2 in the subject.

[0154] Embodiment 44. The method of embodiment 43, wherein increasing the expression and / or activity of HCN2 comprises one or more of: (a) providing HCN2 mRNA to the subject, (b) providing HCN2 protein to the subject, (c) providing an HCN2 agonist / activator to the subject, (d) providing a compound that mimics the effect of HCN2 overexpression to the subject.

[0155] Embodiment 45. The method of embodiment 43 or 44, wherein the bioelectric patterns to be restored comprise a specific tissue, cell collective, or organ of the subject.

[0156] Embodiment 46. The method of embodiment 45, wherein the tissue, cell collective, or organ comprises neural tissue (including but not limited to the brain, spinal cord, eyes), epidermis (skin), heart, lungs, muscle tissue, stomach, esophagus, adrenal gland, colon, pituitary gland, testis, and vascular tissue.

[0157] Embodiment 47. The method of any one of embodiments 44-46, wherein the one or more of (a)-(d) is provided to the subject as a pharmaceutical composition.

[0158] Embodiment 48. The method of embodiment 47, wherein the pharmaceutical composition is administered systemically.

[0159] Embodiment 49. The method of embodiment 47, wherein the pharmaceutical composition is administered locally (e.g., to a specific region of the body, organ, cell collective, or tissue).

[0160] Embodiment 50. The method of any one of the previous embodiments, wherein the subject is a human.

[0161] Embodiment 51. The method or composition of any one of the previous embodiments further comprising an additional active agent.

[0162] Embodiment 52. The method or composition of embodiment 51, wherein the additional active agent comprises an anti-aging agent, and / or an additional ion channel modulator.

[0163] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. NoAtty. Dkt. No.166118.01474 language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0164] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0165] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0166] The following examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter. EXAMPLES Example 1: Effect of HCN2 overexpression on lifespan

[0167] The effect of HCN2 overexpression on aging was tested in model organism Drosophila melanogaster. Flies were engineered to systemically overexpress HCN2, and fly lifespan was measured. In female individuals overexpressing the HCN2 protein, greater mean longevity was exhibited (FIG.5). Only 50% of the wild-type females were alive after 50 days, while more than 80% of the HCN2 overexpressing females were alive at the same time point. More than 75% of the HCN2 overexpressing females were alive at 57 days as compared to less than 50% of the wild-type females. However, overexpression in male fruit flies had no apparent effect (FIG 6). Example 2: Effect of HCN2 overexpression on bioelectric pattern

[0168] The effect of tissue-specific HCN2 overexpression was also assessed in Drosophila melanogaster, using flies engineered to overexpress HCN2 in intestinal tissue (FIGS.14A and 14B). Intestinal tissue was removed from young and old flies and analyzed by voltage imaging to calculate a measure of bioelectric sharpness (Moran’s I). In control flies, the Moran’s I was significantly lower in the tissue harvested from old flies as compared to young flies (P-value: 0.01), indicating a disruption / blurring of bioelectric patterns. Interestingly, the old HCN2-overexpressing fliesAtty. Dkt. No.166118.01474 demonstrated a restoration in bioelectric sharpness (P-value: 0.008 vs. old control flies). Additionally, the bioelectric sharpness in old HCN2-overexpressing flies was significantly higher than that in young HCN2-overexpressing flies (P-value: 0.01). Without wishing to be bound by a particular theory, this finding suggests that HCN2 expression may have a varied effect on bioelectric sharpness at different stages of an organism’s lifespan. Example 3: Creation of Tissue Specific, Drug Inducible Expression of HCN2 in Transgenic Mice that will be Crossed to Existing Transgenic Mice

[0169] To create a transgenic mouse with tissue specific, drug inducible expression of HCN2, CRISPR mice with floxed HCN2 and CRISPR / Cas9 gene editing will be generated. This mouse will be mated with tissue-specific Cre transgene mice with a mutation Estrogen receptor (ER) that binds to Tamoxifen. The progeny of the mated mice will carry the Cre transgene along with unactivated HCN2. Cre expression will be induced by Tamoxifen binding to mutated ER, resulting in removal of a stop codon and expression of HCN2 in the organ of interest (e.g., liver, brain, or ovaries). Mice with tissue-specific Cre are commercially available (e.g., Charles River).

[0170] Induction of tissue-specific expression will occur once treated with Tamoxifen, allowing for observation of the effects of HCN2 at various aging stages, from young pups to older mice that more closely reflect old age. Assessment will include, (1) induction of HCN2 expression at about 6 months, to observe the effect on older mice, (2) induction immediately after birth to assess the impact on young mice, and (3) induction of organ-specific expression in one mouse, followed by surgical removal of the HCN2-expressing organ for transplantation into a second mouse. The HCN2 transgenic mice will also be mated with mice used in models of aging, obesity, Alzheimer’s, Parkinson’s, and / or diabetes.

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Claims

Atty. Dkt. No.166118.01474 CLAIMS 1. A method for increasing the health and / or lifespan of a subject, the method comprising: increasing the expression and / or activity of HCN2 in the subject.

2. The method of claim 1, wherein increased health and / or lifespan comprises one or more of: greater resistance to carcinogens, greater resistance to teratogens, greater resistance to the normal wear-and-tear of life including but not limited to muscle loss, bone loss, anemia, immune dysfunction, cognitive decline, cardiovascular disease and vascular dysfunction.

3. The method of claim 2, wherein increasing the expression and / or activity of HCN2 comprises one or more of: (a) providing HCN2 nucleic acid (DNA and / or mRNA) to the subject, (b) providing HCN2 protein to the subject, (c) providing an HCN2 agonist / activator to the subject, (d) providing a compound that mimics the effect of HCN2 overexpression to the subject.

4. The method of claim 3, wherein providing HCN2 DNA and / or mRNA to the subject comprises administering a composition comprising HCN2 DNA and / or mRNA formulated (i) with lipid nanoparticles, (ii) lipid / polymer nanoparticles, or (iii) as a vector, optionally wherein the HCN2 DNA and / or mRNA is administered via microinjection.

5. The method of claim 3, wherein providing HCN2 protein to the subject comprises administering genetically modified cells (i) expressing or overexpressing HCN2 protein or (ii) comprising a conditional HCN2 expression or overexpression system.

6. The method of claim 3, wherein the HCN2 agonist is a direct agonist.

7. The method of claim 3, wherein the HCN2 agonist is an indirect agonist.

8. The method of claim 3, further comprising administering one or more additional ion channel modulators and / or one or more additional active agents.

9. The method of claim 2, wherein greater resistance to the normal wear-and-tear of life comprises non-cancerous regeneration of cells instead of cell senescence.Atty. Dkt. No.166118.01474 10. The method of claim 3, wherein the HCN2 DNA and / or mRNA, HCN2 protein, the HCN2 agonist and / or the compound that mimics the effect of HCN2 overexpression is provided to the subject systemically.

11. The method of claim 3, wherein the HCN2 DNA and / or mRNA, HCN2 protein, the HCN2 agonist and / or the compound that mimics the effect of HCN2 overexpression is provided to an organ, cell collective, or tissue of the subject.

12. The method of claim 10 or 11, wherein the subject is a non-diseased animal subject.

13. The method of claim 10, wherein the subject is diagnosed with cancer.

14. The method of claim 11, wherein the subject is diagnosed with cancer, and the expression and / or activity of HCN2 is increased in the cancer tissue or a tissue or cell collective adjacent to the cancer tissue.

15. The method of claim 10, wherein the subject has a wound.

16. The method of claim 11, wherein the subject has a wound and the expression and / or activity of HCN2 is increased in the wounded tissue or in a tissue or cell collective adjacent to the wound.

17. The method of claim 10, wherein the subject has a cardiovascular disease and / or vascular dysfunction.

18. The method of claim 11, wherein the subject has a cardiovascular disease and / or vascular dysfunction and the expression and / or activity of HCN2 is increased in the heart, and / or cardiovascular and / or vascular system.

19. The method of claim 10, wherein the subject has a neurological disease or disorder.

20. The method of claim 11, wherein the subject has a neurological disease or disorder and the expression and / or activity of HCN2 is increased in the brain and / or nervous system.

21. The method of claim 19 or 20, wherein the neurological disease or disorder comprises one or more of dyskinesia, seizure disorder, epilepsy, ataxia, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, multiple sclerosis.

22. The method of claim 10, wherein the subject is diagnosed with an inflammatory and / or autoimmune disease or condition.Atty. Dkt. No.166118.01474 23. The method of claim 11, wherein the subject is diagnosed with an inflammatory and / or autoimmune disease or condition and the expression and / or activity of HCN2 is increased in the tissue(s), cell collective(s), or organ(s) exhibiting inflammation.

24. The method of claim 22 or 23, wherein the inflammatory disease or condition comprises chronic inflammation.

25. The method of claim 10, wherein the subject is diagnosed with a metabolic disease or condition.

26. The method of claim 11, wherein the subject is diagnosed with a metabolic disease or condition.

27. The method of claim 25 or 26, wherein the metabolic disease or condition comprises Type 2 diabetes, obesity, metabolic syndrome.

28. The method of claim 1, wherein increasing health and / or lifespan comprises reducing one or more of: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, disabled macroautophagy, deregulated nutrient-sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis.

29. The method of any one of the previous claims, wherein the composition is administered multiple times per day, once per day, every other day, weekly, monthly, as needed as determined by a subject’s physician.

30. The method of any one of the previous claims, wherein the subject is a human.

31. A composition for increasing health and / or lifespan of a subject, comprising one or more of: (a) HCN2 mRNA, (b) HCN2 protein, (c) HCN2 agonist / activator, (d) a compound that mimics the effect of HCN2 overexpression.

32. The composition of claim 31, comprising a pharmaceutical carrier, and optionally one or more additional active agents.Atty. Dkt. No.166118.01474 33. The composition of claim 31 or 32, wherein the composition is formulated for systemic administration.

34. The composition of claim 31 or 32, wherein the composition is formulated for local administration.

35. The composition of claim 31 or 32, wherein the composition is formulated for administration orally, by inhalation, injection, or transdermally.

36. A genetically engineered cell comprising increased expression and / or activity of HCN2.

37. The genetically engineered cell of claim 36 comprising a mammalian cell.

38. The genetically engineered cell of claim 36 or 37, comprising an exogenous copy of an HCN2 gene operably linked to a promoter, optionally wherein the cell comprises a conditional HCN2 expression system.

39. The cell of any one of claims 36-38, wherein the engineered cell is in an organ, a cell collective, or a tissue.

40. The cell of claim 39, wherein the organ, cell collective, or tissue is ex vivo, and is intended for transplant to a subject.

41. A method to modulate bioelectric signaling and a bioelectric pattern, optionally, to restore and / or establish endogenous (wild-type) bioelectric patterns, in a subject in need thereof, the method comprising: modulating the expression and / or activity of HCN2 in the subject.

42. The method of claim 41, wherein increasing the expression and / or activity of HCN2 comprises one or more of: (a) providing HCN2 mRNA to the subject, (b) providing HCN2 protein to the subject, (c) providing an HCN2 agonist / activator to the subject, (d) providing a compound that mimics the effect of HCN2 overexpression to the subject.

43. The method of claim 41 or 42, wherein the bioelectric patterns to be restored comprise a specific tissue, cell collective, or organ of the subject.Atty. Dkt. No.166118.01474 44. The method of claim 43, wherein the tissue, cell collective, or organ comprises neural tissue (including but not limited to the brain, spinal cord, eyes), epidermis (skin), heart, lungs, muscle tissue, stomach, esophagus, adrenal gland, colon, pituitary gland, testis, and vascular tissue.

45. The method of claim 44, wherein the one or more of (a)-(d) is provided to the subject as a pharmaceutical composition.

46. The method of claim 45, wherein the pharmaceutical composition is administered systemically.

47. The method of claim 45, wherein the pharmaceutical composition is administered locally (e.g., to a specific region of the body, organ, cell collective, or tissue).

48. The method of any one of claims 41-47, wherein the subject is a human.

49. The method or composition of any one of the previous claims further comprising an additional active agent.

50. The method or composition of claim 49, wherein the additional active agent comprises an anti-aging agent, and / or an additional ion channel modulator.

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