Improving immune function with enhanced NAD+ compositions

Abstract

Provided are methods of improving immune function that include administration of an enhanced NAD+ composition to a subject. The method can include treatment of a chronic viral infection. The administration of the enhanced NAD+ composition can increase a number of immune cells in the subject.

Description

Docket No.: 2967100.2.W01Customer No 190456IMPROVING IMMUNE FUNCTION WITH ENHANCED NAD+ COMPOSITIONS

[0001] All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety. Such patent applications include but are not limited to PCT / US23 / 35557, entitled “Enhanced NAD+ Compositions and Methods of Making and Using the Same.”

[0002] The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights.CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Application No. 63 / 687,107, entitled “IMPROVING IMMUNE FUNCTION WITH ENHANCED NAD+ COMPOSITIONS,” filed August 26, 2024, the contents of which are incorporated herein by reference in its entirety.BACKGROUND

[0005] Chronic diseases such as human immunodeficiency virus (HIV) can result in immune dysfunction, even with treatment. Improved treatments for immune dysfunction are needed.SUMMARY

[0006] Nicotinamide adenine dinucleotide (NAD) is an important coenzyme involved in cellular metabolism, energy production, and various signaling pathways. Chronic infections such as human immunodeficiency virus (HIV) can lead to NAD depletion, contributing to metabolic dysfunction, oxidative stress, and immune system impairment. Enhanced NAD+ supplementation is useful for addressing immune depletion and exhaustion in chronic infections, particularly HIV, and combatting cellular and immune senescence. Enhanced NAD+ supplementation can offer a multi-faceted approach to improving immune function in individuals with chronic infections byDocket No.: 2967100.2.W01Customer No 190456 restoring mitochondrial function, enhancing antioxidant defenses, modulating immune cell function through sirtuin activation, and potentially exerting direct antiviral effects. Enhanced NAD+ supplementation can be useful in situations where CD4+ T cell counts or immune function are compromised, such as autoimmune diseases or chronic viral infections.

[0007] Provided herein, in some embodiments, are methods that include administering enhanced NAD+ to a subject in need thereof. Some embodiments include a method of treatment, comprising: administering a therapeutically effective amount of an enhanced NAD+ composition to a subject having immune dysfunction. Some embodiments relate to a method of treatment, comprising: identifying a subject having immune dysfunction; and administering to the subject a therapeutically effective amount of a composition comprising an oxidized form of nicotinamide adenine dinucleotide (NAD+) and polyethylene glycol (PEG). In some embodiments, the composition is about 10% NAD+, about 20% NAD+, about 30% NAD+, about 40% NAD+, about 50% NAD+, about 60% NAD+, about 70% NAD+, about 80% NAD+, or about 90% NAD+. In some embodiments, the composition is about 10% PEG, about 20% PEG, about 30% PEG, about 40% PEG, about 50% PEG, about 60% PEG, about 70% PEG, about 80% PEG, or about 90% PEG. Some embodiments include PEG and NAD+ in about a 1 : 1 ratio (e.g. about 50% NAD+ and about 50% PEG). In some embodiments, the administration is oral. The oral administration can include buccal or sublingual administration. In some embodiments, the administration comprises doses over a period of about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, about 20 years, about 25 years, or longer. In some embodiments, the administration comprises daily doses. In some embodiments, the administration comprises doses of about 1 mg NAD+ per day, about 2.5 mg NAD+ per day, about 5 mg NAD+ per day, about 10 mg NAD+ per day, about 15 mg NAD+ per day, about 25 mg NAD+ per day, about 50 mg NAD+ per day, about 75 mg NAD+ per day, about 100 mg NAD+ per day, about 200 mg NAD+ per day, about 300 mg NAD+ per day, about 400 mg NAD+ per day, about 500 mg NAD+ per day, about 600 mg NAD+ per day, about 700 mg NAD+ per day, about 800 mg NAD+ per day, about 900 mg NAD+ per day, about 1000 mg NAD+ per day, about 1250 mg NAD+ per day, about 1500 mg NAD+ per day, about 1750 mg NAD+ per day, or about 2000 mg NAD+ perDocket No.: 2967100.2.W01Customer No 190456 day. In some embodiments, the immune dysfunction comprises T cell depletion. In some embodiments, the subject has a low CD4+ T cell count. In some embodiments, the low CD4+ T cell count comprises a CD4+ T cell measurement below 359 units / pL in a blood sample. In some embodiments, the immune dysfunction results from or corresponds with the subject having an infection. In some embodiments, the infection comprises influenza, pneumonia, hepatitis B, a cytomegalovirus infection, or coronavirus disease 2019. In some embodiments, the infection is chronic. In some embodiments, the infection comprises a retrovirus infection. In some embodiments, the infection comprises a lentivirus infection. In some embodiments, the infection comprises a human immunodeficiency virus (HIV) infection. In some embodiments, the infection comprises a simian immunodeficiency virus (SIV) infection. In some embodiments, the administration increases an NAD level in the subject. In some embodiments, the NAD level is increased by at least 5% relative to a baseline measurement. In some embodiments, the administration increases an immune cell measurement in the subject. In some embodiments, the immune cell measurement comprises a CD4+ T cell measurement. In some embodiments, the immune cell measurement is increased by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30% by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, or by at least 75%, relative to a baseline measurement. In some embodiments, the administration alleviates lymphodepletion in the subject within about 28 days or 1 month of initiation of treatment. In some embodiments, the subject is human.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Hereinafter, various exemplary embodiments of the disclosure are illustrated in more detail with reference to the drawings.

[0009] FIG. 1A is a table of immune cell-related measurements obtained before and 14 days after treatment of a human subject with an enhanced NAD+ composition under one embodiment of the present disclosure.

[0010] FIG. IB provides tables of measurements as part of a metabolic panel before and 14 days after treatment of a human subject with an enhanced NAD+ composition under one embodiment of the present disclosure.Docket No.: 2967100.2.W01Customer No 190456

[0011] FTG. 2A is a table of immune cell-related measurements obtained after 28 days of treatment of a human subject with an enhanced NAD+ composition under one embodiment of the present disclosure.

[0012] FIG. 2B provides tables of measurements as part of a metabolic panel after 28 days of treatment of a human subject with an enhanced NAD+ composition under one embodiment of the present disclosure.

[0013] FIG. 2C provides a plot showing CD4+ T cell concentrations in samples of a subject before and after treatment with an enhanced NAD+ composition under one embodiment of the present disclosure. The y-axis is CD4+ cells per pL of whole blood.

[0014] FIG. 3 includes a table of immune cell-related measurements during treatment of a human subject with an enhanced NAD+ composition for over one year under one embodiment of the present disclosure.DETAILED DESCRIPTION

[0015] Reference will now be made in detail to embodiments of the present disclosure, one or more drawings of which are set forth herein. Each drawing is provided by way of explanation of the present disclosure and is not a limitation. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the teachings of the present disclosure without departing from the scope of the disclosure. For instance, features illustrated or described as part of one embodiment can be used with another embodiment to yield a still further embodiment.

[0016] While the making and using of various embodiments of the present disclosure are discussed in detail below, it should be appreciated that the present disclosure provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the disclosure.

[0017] Thus, it is intended that the present disclosure covers such modifications and variations as come within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the present disclosure are disclosed in, or are obvious from, the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is aDocket No.: 2967100.2.W01Customer No 190456 description of exemplary embodiments only and is not intended as limiting the broader aspects of the present disclosure.Introduction

[0018] Nicotinamide adenine dinucleotide (NAD) is a ubiquitous coenzyme found in living cells, playing a role in numerous biological processes. It serves as an electron carrier in redox reactions, facilitating energy production through cellular respiration and other metabolic pathways. Beyond its role in metabolism, NAD functions as a substrate for various enzymes, including sirtuins and poly(ADP-ribose) polymerases (PARPs), which are involved in DNA repair, gene expression regulation, and stress responses. The cellular pool of NAD is maintained through de novo biosynthesis from tryptophan, as well as salvage pathways utilizing nicotinamide and other NAD precursors. NAD levels naturally decline with age and in various pathological conditions, including chronic viral infections like human immunodeficiency virus (HIV).

[0019] Some chronic infections, particularly HIV, are characterized by persistent inflammation, leading to a state of immune depletion (due to direct attack of the virus on the lymphoid and myeloid cells) and exhaustion (due to the downstream consequences of the persistent immune activation). Immune exhaustion can include progressive loss of T cell function, such as a depletion in CD4+ T cells and CD8+ T cells. Immune exhaustion can be associated with decreased proliferative capacity of T cells, reduced cytokine production, impaired effector functions, increased expression of inhibitory receptors (e.g., PD-1, CTLA-4), and altered metabolic profiles in immune cells.

[0020] In chronic infection, despite effective treatment suppressing pathogen replication, many patients experience incomplete immune recovery, characterized by persistently low CD4+ T cell counts. This phenomenon, which can be referred to as “immunological non-response,” is often associated with increased morbidity and mortality in vulnerable populations such as patients infected with HIV. Similar immune dysfunction can be observed in other chronic conditions, highlighting the potential broader applicability of strategies aimed at restoring immune function, including in cancer, autoimmune disease, and metabolic disorders of aging.Docket No.: 2967100.2.W01Customer No 190456Metabolic and mitochondrial dysfunction in chronic inflammation

[0021] Chronic infections, particularly HIV, can lead to a significant depletion of intracellular NAD levels, a phenomenon which can be referred to as “intracellular pellagra.” This depletion can occur through multiple mechanisms, including increased activity of CD38 (an NAD glycohydrolase upregulated in HIV infection), activation of poly(ADP-ribose) polymerases (PARPs) due to infection-induced oxidative stress, or interference with NAD biosynthesis pathways.

[0022] NAD depletion can affect mitochondrial function and cellular energy production. As an electron carrier in the electron transport chain, reduced NAD availability can lead to stunted ATP production and impaired oxidative phosphorylation. Insufficient NAD levels can in turn impair the activity of sirtuins, particularly SIRT1 and SIRT3, which play roles in maintaining mitochondrial function. NAD depletion can also affect mitochondrial fusion and fission processes, potentially leading to accumulation of dysfunctional mitochondria. T cell metabolism adapts to different phases of the immune response. Naive T cells primarily rely on oxidative phosphorylation. Upon activation during an acute infection, T cells switch to glycolysis to meet their increased energy demands. After an infection is cleared, memory T cells return to a resting state, utilizing oxidative phosphorylation and performing fatty acid oxidation.Oxidative stress and inflammation

[0023] Chronic infections can be associated with increased oxidative stress, characterized by elevated production of reactive oxygen species (ROS) and depletion of antioxidant defenses. In HIV, viral proteins can directly induce ROS production in infected and bystander cells. Chronic immune activation can lead to increased ROS production by activated immune cells, while impaired mitochondrial function due to NAD depletion can amplify ROS generation. Additionally, chronic infections can be associated with reduced levels of antioxidants, including glutathione, superoxide dismutase, and catalase.

[0024] Persistent inflammation is a found in certain chronic infections, even in individuals on effective treatment regimens. Ongoing pathogen replication, microbial translocation, and exposure to antigens can drive chronic immune activation and elevated levels of pro-inflammatory cytokines. NAD depletion and oxidative stress can promote activation of NF-KB, a keyDocket No.: 2967100.2.W01Customer No 190456 transcription factor involved in inflammatory responses. The chronic inflammatory state accelerates age-related changes in the immune system, a process termed "inflammaging".Immune exhaustion and senescence

[0025] T cell depletion, exhaustion, and senescence can impact immune function in chronic infections and other persistent inflammatory conditions. Understanding and addressing these phenomena are useful for developing effective therapies to restore immune competence and improve patient outcomes.

[0026] T cell exhaustion can include a state of dysfunction that occurs in the setting of persistent antigen exposure and inflammation, commonly observed in chronic viral infections, cancer, and some autoimmune diseases. This state can be characterized by progressive loss of effector functions, including reduced proliferative capacity, diminished cytokine production, and impaired cytotoxicity. Exhausted T cells can upregulate inhibitory receptors such as PD-1, CTLA- 4, and TIM-3, which can act as immune checkpoints to dampen T cell responses. Exhausted T cells can exhibit altered expression of transcription factors, impaired mitochondrial function, and increased reliance on glycolysis for energy production. Persistent stimulation can lead to epigenetic modifications that reinforce the exhausted state, making it challenging to reverse.

[0027] T cell exhaustion can impact on the body's ability to control an infection or malignancy. In HIV infection, T cell exhaustion contributes to the progressive loss of virus-specific CD4+ and CD8+ T cells, interfering with the immune system's capacity to suppress viral replication. In cancer, exhaustion of tumor-infiltrating lymphocytes impairs the immune system's ability to recognize and eliminate malignant cells. Addressing T cell exhaustion, therefore, represents a useful strategy for enhancing immune responses against persistent pathogens and tumors.

[0028] Cellular senescence, a state of permanent cell cycle arrest where the cell no longer divides, further compounds immune dysfunction in chronic infections and aging. Infections and chronic inflammation can accelerate the accumulation of senescent T cells, which can be characterized by shortened telomeres and expression of senescence-associated markers. These cells lose their functional capacity and actively contribute to tissue dysfunction through the senescence-associated secretory phenotype (SASP). The SASP involves the secretion of pro- inflammatory cytokines, chemokines, and matrix-remodeling enzymes, which can propagate chronic inflammation and promote disease progression. The impact of cellular senescence canDocket No.: 2967100.2.W01Customer No 190456 extend beyond individual T cells. Senescence of hematopoietic stem and progenitor cells can limit the regenerative capacity of the immune system, impairing the production of new, functional immune cells. This can lead to a gradual decline in the diversity and functionality of the T cell repertoire. Cellular senescence can be associated with decreased NAD levels, creating a vicious cycle of metabolic dysfunction and accelerated aging. NAD depletion can further impair the activity of sirtuins, regulators of cellular metabolism and stress responses, further exacerbating the senescent phenotype and associated immune dysfunction.NAD restoration and immune modulation

[0029] Targeting T cell depletion, exhaustion, or cellular senescence can be useful for therapeutic intervention in cases of immune dysfunction, such as in chronic infections or age- related immune dysfunction. Such therapeutic intervention can include enhanced NAD+ supplementation or administration. By addressing the underlying metabolic and epigenetic alterations, it can be possible to restore immune competence and improve long-term health outcomes in a wide range of conditions characterized by persistent immune activation and inflammation.

[0030] In certain embodiments, enhanced NAD+ supplementation can be used for treating immune depletion, exhaustion, and senescence in chronic infections.

[0031] In embodiments, the present disclosure relates to the use of enhanced NAD+ administration to treat immune dysfunction in chronic infections, particularly HIV, with potential applications in other conditions. Enhanced NAD+ supplementation can be useful in improving CD4+ T cell counts, reducing markers of immune exhaustion and cellular senescence, and enhancing overall immune function in individuals with chronic infections or immune dysfunction. By targeting fundamental aspects of cellular metabolism and function, enhanced NAD+ supplementation can complement existing therapies and improve long-term outcomes for affected individuals living with chronic conditions characterized by mitochondrial dysfunction, oxidative stress, and immune dysregulation.SIRT1 modulation

[0032] Enhanced NAD+ administration is associated with increased plasma levels of SIRT1, a master regulator of several cellular processes of direct relevance to mitochondrial and immuneDocket No.: 2967100.2.W01Customer No 190456 functioning, as well as cellular signaling and metabolism. Without being bound by theory, modulation of SIRT1 represents an exemplary mechanism through which NAD+ repletion restores immune function.

[0033] SIRT1 (Silent Information Regulator 1) includes a highly conserved NAD-dependent deacetylase that plays a role in regulating various cellular processes. As a member of the sirtuin family, SIRT1 requires NAD as a co-substrate for full enzymatic activity. SIRT1 modulates glucose and lipid metabolism, enhances cellular stress resistance, suppresses inflammation by inhibiting NF-KB signaling, promotes anti-inflammatory polarization of macrophages, promotes genomic stability, and stimulates mitochondrial biogenesis. In the context of chronic infections like HIV, SIRT1 can play a multifaceted role in both modulating pathogen replication and affecting immune function. For example, SIRT1 can deacetylate the HIV-1 Tat protein, directly suppressing viral transcription and potentially contributing to viral latency. SIRT1 also can regulate T cell activation, differentiation, and memory formation, processes that are dysregulated in chronic infections. SIRT1 activation can delay cellular senescence and promote longevity of immune cells, counteracting accelerated aging observed in chronically infected individuals. By modulating cellular metabolism in immune cells, SIRT1 can help reverse the metabolic dysfunction associated with T cell exhaustion in chronic infections.

[0034] As a SIRT1 stimulation strategy, enhanced NAD+ administration can be useful for improving mitochondrial function. This can be done by enhancing oxidative phosphorylation, activating SIRT1, promoting mitophagy, or stimulating mitochondrial biogenesis, or by bolstering overall antioxidant defenses by supporting glutathione synthesis, increasing NADPH production, or enhancing antioxidant gene expression. Enhanced NAD+ supplementation can stimulate mitochondrial biogenesis. In certain embodiments, enhanced NAD+ supplementation improves immune cell function by any one or more of the following:• promoting a metabolic shift towards oxidative phosphorylation in T cells, reversing the exhaustion-associated glycolytic phenotype;• enhancing stability and suppressive function of regulatory T cells, which can help control chronic immune activation;• promoting the development and maintenance of memory T cells, improving long-term immune responses.Docket No.: 2967100.2.W01Customer No 190456• slowing or reversing aspects of cellular senescence in immune cells, improving their overall function and longevity.Effects of enhanced NAD+ administration on T-cell response proteins

[0035] In some embodiments, enhanced NAD+ administration modulates proteins involved in immune regulation and T cell function. Enhanced NAD+ administration can be associated with decreased levels of MMP9, CXCL10, and CXCL12. These proteins can play a role in T cell migration, activation, and inflammatory responses. Without being bound by theory, certain nonlimiting, exemplary roles of such proteins are summarized below.• MMP9 (Matrix Metallopeptidase 9): This enzyme is involved in the breakdown of extracellular matrix and plays a role in T cell migration and tissue remodeling. Elevated MMP9 levels can be associated with chronic inflammation and tissue damage. In embodiments, enhanced NAD+ administration’s reduces MMP9 levels and helps mitigate tissue remodeling, dampens pathological inflammatory responses, or any combination thereof.• CXCL10 (C-X-C Motif Chemokine Ligand 10): This chemokine is a potent attractor of activated T cells and can be upregulated in chronic inflammatory conditions. Without being bound by theory, in embodiments, administration of a therapeutically effective amount of enhanced NAD+ reduces CXCL10 levels and modulates excessive T cell recruitment to reduce ongoing inflammation in conditions of chronic immune activation.• CXCL12 (C-X-C Motif Chemokine Ligand 12): Also known as SDF-1 (Stromal Cell- Derived Factor 1), CXCL12 plays a role in T cell trafficking and homing. Without being bound by theory, in embodiments, administration of enhanced NAD+ modulates CXCL12 to impact T cell distribution and localization, which is beneficial in conditions where altered T cell trafficking contributes to pathology.

[0036] Joint modulation of these exemplary, non-limiting proteins by enhanced NAD+ administration offers a multi-faceted approach to addressing immune dysfunction. By reducing excessive inflammation, modulating T cell migration, influencing the overall immune microenvironment, or any combination thereof, enhanced NAD+ administration can help create conditions favorable for the restoration of proper T cell function and the reversal of T cell exhaustion.Docket No.: 2967100.2.W01Customer No 190456Additional non-limiting, exemplary effects of enhanced NAD+ administration

[0037] In embodiments, NAD+ repletion (such as via administration of enhanced NAD+) prevents or ameliorates T-cell dysfunction to promote antiviral defense. In embodiments, NAD+ precursor supplementation with nicotinamide mononucleotide (NMN) prevents STING-induced senescence in CD8+ T cells. In certain embodiments and without wishing to be bound by theory, STING-induced senescence in CD8+ T cells following NAD+ precursor supplementation with NMN is accomplished by improving mitochondrial homeostasis and promoting mitophagy. NMN treatment can suppress senescence and neuroinflammation and improve the survival cycle of mice, suggesting that promotion of mitophagy can be a useful strategy to prevent CD8+ T cells from senescence due to mitochondrial dysfunction. Without being bound by theory, similar or better effects can be achieved upon enhanced NAD+ administration according to any of the various embodiments disclosed herein.

[0038] HIV-1 infection can reduce NAD+ capping of host cell snRNA and snoRNA. NAD+ can serve as an alternative 5' cap on various RNAs, including small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs). The presence of NAD cap on U1 snRNA can directly decrease the stability of the Ul / HIV-1 pre-mRNA duplex. Reducing the quantity of NAD-capped RNA by overexpressing the NAD RNA decapping enzyme DXO can increase HIV- 1 infectivity, suggesting that NAD capping can play an important role in antiviral defense.

[0039] NAD can have antiviral effects. In certain embodiments, NAD capping influences interactions between host cell RNAs and viral RNAs, and interferes with viral replication processes. In embodiments, changes in NAD levels and NAD capping affect the expression of viral genes, either directly or through modulation of host cell transcription factors. NAD and its metabolites can be involved in regulation of innate immune responses, which can contribute to antiviral defense mechanisms.

[0040] In various embodiments, in the context of chronic infection or inflammation, administration of an enhanced NAD+ prevents T cell depletion, exhaustion, senescence, or a combination thereof. In certain embodiments, such prevention of T cell depletion, exhaustion, senescence or a combination thereof, occurs via the modulation of certain identifiable molecular mechanisms and biological pathways. Without wishing to be bound by theory, enhanced NAD+ administration increases intracellular NAD levels to combat immune depletion, exhaustion,Docket No.: 2967100.2.W01Customer No 190456 senescence, or a any combination thereof in chronic infections and other conditions characterized by persistent immune activation, mitochondrial, and metabolic dysfunction.Immunometabolism and Antiviral Therapies

[0041] In the context of antiviral therapies, understanding connections between cellular metabolism and immune function can help in developing effective treatments and mitigating side effects. Antiviral therapies themselves can have significant impacts on cellular metabolism, which in turn can affect immune function. For example, some nucleoside reverse transcriptase inhibitors (NRTIs) used in HIV treatment can impair mitochondrial function, contributing to metabolic complications and accelerated immune aging. Conversely, certain antiviral treatments can have beneficial effects on immune metabolism. For instance, some antiretroviral drugs modulate cellular NAD levels, potentially influencing T cell function and senescence.

[0042] In embodiments and without wishing to be bound by theory, administration of a therapeutically effective amount of enhanced NAD+ restores intracellular NAD levels. In embodiments, administration of a therapeutically effective amount of enhanced NAD+ can be used to complement existing antiviral therapies by:• Mitigating metabolic side effects. Without being bound by theory, supporting mitochondrial function and cellular energy production, enhanced NAD+ administration can counteract metabolic disturbances induced by some antiviral medications.• Enhancing immune responses. Without being bound by theory, increased NAD levels via enhanced NAD+ administration can boost T cell function and longevity, potentially enhancing the overall efficacy of antiviral treatments.• Reducing inflammation. By modulating the inflammatory environment through effects on proteins like MMP9, CXCL10, and CXCL12, NAD+ administration via any of the various embodiments disclosed herein creates a favorable immune milieu for antiviral therapies to act.• Combating drug-induced senescence. In embodiments, enhanced NAD+ administration treats or prevents senescence through improved mitochondrial homeostasis.

[0043] Enhanced NAD+ administration can have synergistic effects in improving overall immune health and treatment outcomes when combined with administration of antiviral or immunometabolic therapies.Docket No.: 2967100.2.W01Customer No 190456Methods of treatment

[0044] Disclosed herein, in some embodiments, are methods of treatment. The method can include administering nicotinamide adenine dinucleotide (NAD) to a subject in need thereof. The NAD can be or include NAD+. The NAD can be or include NADH. The NAD can be administered as part of an NAD composition. Administering NAD can include administering an NAD composition. The NAD composition can include an NAD+ composition such as an enhanced NAD+ composition. Some embodiments relate to administering NAD to a subject in need thereof. Some embodiments relate to administering NAD to a subject in need thereof.

[0045] Some embodiments include identifying the subject as having immune dysfunction, and administering the treatment. Provided herein, in some embodiments, are methods of treatment, comprising: identifying a subject having immune dysfunction; and administering to the subject a therapeutically effective amount of a composition comprising enhanced NAD+. Some embodiments include identifying a subject having immune dysfunction; and administering to the subject a therapeutically effective amount of a composition comprising NAD+, PEG, or a combination thereof. In some embodiments, the immune dysfunction comprises T cell depletion.

[0046] In some embodiments, the subject has an infection. In some embodiments, the immune dysfunction results from the infection. In some embodiments, the immune dysfunction results from or corresponds with the subject having an infection. In some embodiments, the infection comprises influenza, pneumonia, hepatitis B, a cytomegalovirus infection, or coronavirus disease 2019.

[0047] In some embodiments, the infection is chronic. In some embodiments, the infection comprises a retrovirus infection. In some embodiments, the infection comprises a lentivirus infection. In some embodiments, the infection comprises a human immunodeficiency virus (HIV) infection. In some embodiments, the infection comprises a simian immunodeficiency virus (SIV) infection. In some embodiments, the subject has received an antiretroviral therapy.

[0048] Some embodiments include obtaining an NAD level of a subject. The NAD level can be obtained following the NAD+ administration. Some embodiments include obtaining a baseline NAD level of a subject. The baseline NAD level can be obtained before the NAD+ administration. The NAD level can be or include an intracellular NAD (icNAD) measurement. The icNAD can be obtained from a blood or tissue sample. The NAD level can be or include a circulating NAD (CirNAD) measurement. The CirNAD level can be obtained from a blood, serum, or plasmaDocket No.: 2967100.2.W01Customer No 190456 sample. The icNAD level can be or include an NAD+ measurement. The NAD level can be or include an NADH measurement. The NAD level can be or include a combined NAD+ and NADH measurement.

[0049] The NAD level can fall within a range considered severely deficient, deficient, suboptimal, optimal, or too high. By way of non-limiting example, a severely deficient range for icNAD levels can be less than about 20 pM, or up to about 20 pM (e.g. 0 to about 20 pM). An exemplary deficient range for icNAD levels can between about 20 pM and up to about 30 pM. In exemplary embodiments, a suboptimal range for icNAD levels is between about 30 pM and about 40 pM, inclusive. An exemplary optimal range for icNAD levels is over about 40 pM. The upper end of an optimum range for icNAD levels can be over 100 pM. The upper end can be considered too high. For example, the optimal range for icNAD levels can be between about 40 pM and up to about 100 pM, and a level over about 100 pM can be considered too high.

[0050] In some embodiments, the administration increases an NAD level in the subject. In some embodiments, the administration increases the NAD level relative to the baseline NAD level. In some embodiments, the NAD level is increased by about 2.5% or more, about 5% or more, or about 7.5% or more, relative to the baseline NAD level. In some embodiments, the NAD level is increased by at least 5% relative to a baseline measurement. In some embodiments, the NAD level is increased by about 10% or more, relative to the baseline NAD level. In some embodiments, the NAD level is increased by no more than about 2.5%, no more than about 5%, or no more than about 7.5%, in each case, relative to the baseline NAD level. In some embodiments, the NAD level is increased by no more than about 10%, relative to the baseline NAD level. Some embodiments include performing an assay to determine the NAD level following NAD+ administration (e.g. following administration of an enhanced NAD+ composition).

[0051] Some embodiments include obtaining an immune cell measurement of a subject. The immune cell measurement can be obtained following NAD+ administration. Some embodiments include obtaining a baseline immune cell measurement of a subject. The baseline immune cell measurement can be obtained before NAD+ administration. The immune cell measurement can be or include a circulating immune cell measurement. The immune cell measurement can be obtained from whole blood. The immune cell measurement can be obtained from a buffy coat layer. The immune cell measurement can include a lymphocyte measurement. The immune cell measurement can include a T cell measurement. The immune cell measurement can include a CD4+ cellDocket No.: 2967100.2.W01Customer No 190456 measurement. The immune cell measurement can include a helper T-lymphocyte CD4+ cell measurement. The immune cell measurement can include a number of immune cells. The immune cell measurement can be indicated as a number or units per volume. The immune cell measurement can be indicated as units / pL. A normal reference interval for CD4+ cell measurement can be 359- 1519 units / pL, where a value below 359 can be considered low. In some embodiments, the subject has a low CD4+ T cell count. In some embodiments, the low CD4+ T cell count comprises a CD4+ T cell measurement below 359 units / pL in a blood sample. The immune cell measurement can include a percentage, such as a percentage of blood cells or white blood cells.

[0052] In some embodiments, the administration of an enhanced NAD+ increases an immune cell measurement in the subject. In some embodiments, the administration increases the immune cell measurement relative to the baseline immune cell measurement. In some embodiments, the immune cell measurement is increased by about 2.5% or more, about 5% or more, or about 7.5% or more, relative to the baseline immune cell measurement. In some embodiments, the immune cell measurement is increased by about 10% or more, relative to the baseline immune cell measurement. In some embodiments, the immune cell measurement is increased by about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 32% or more, or about 35%, relative to the baseline immune cell measurement. In some embodiments, the immune cell measurement is increased by about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, or about 75% or more, relative to the baseline immune cell measurement. In some embodiments, the immune cell measurement is increased by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, or by at least 75%, relative to a baseline measurement. In some embodiments, the immune cell measurement is increased by no more than about 2.5%, no more than about 5%, or no more than about 7.5%, relative to the baseline immune cell measurement. In some embodiments, the immune cell measurement is increased by no more than about 10%, relative to the baseline immune cell measurement. In some embodiments, the immune cell measurement is increased by no more than about 15%, no more than about 20%, no more than about 25%, no more than about 30%, no more than about 32%, no more than about 33%, no more than about 34%, no more than about 35%, no more than about 40%, no more than about 45%, no more than about 50%, no more than about 55%, no more than about 60%, no moreDocket No.: 2967100.2.W01Customer No 190456 than about 65%, no more than about 70%, no more than about 75%, or no more than about 80%, relative to the baseline immune cell measurement. In some embodiments, the immune cell measurement comprises a CD4+ T cell measurement. Some embodiments include performing an assay to determine the increased immune cell measurement following NAD+ administration (e.g. following administration of an enhanced NAD+ composition).

[0053] In some embodiments, the administration comprises daily doses. In some embodiments, the administration comprises doses of about 1 mg NAD+ per day, about 2.5 mg NAD+ per day, about 5 mg NAD+ per day, about 10 mg NAD+ per day, about 15 mg NAD+ per day, about 25 mg NAD+ per day, about 50 mg NAD+ per day, about 75 mg NAD+ per day, about 100 mg NAD+ per day, about 200 mg NAD+ per day, about 300 mg NAD+ per day, about 400 mg NAD+ per day, about 500 mg NAD+ per day, about 600 mg NAD+ per day, about 700 mg NAD+ per day, about 800 mg NAD+ per day, about 900 mg NAD+ per day, about 1000 mg NAD+ per day, about 1250 mg NAD+ per day, about 1500 mg NAD+ per day, about 1750 mg NAD+ per day, or about 2000 mg NAD+ per day Any range of doses therebetween can be used.

[0054] Embodiments as described herein can be administered to a subject one time (e.g., as a single injection, bolus, or deposition, or oral exposurejin some embodiments, the administration includes multiple doses. For example, multiple doses can be given daily. Alternatively, administration can be once or twice daily to a subject for a period of time, such as from about 2 weeks to about 28 days. Administration can continue for up to one year. In embodiments, administration can continue for the life of the subject. It can also be administered once or twice daily to a subject for period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof. In some embodiments, 1, 2, 3, 4, or 5 doses are administered daily. In some embodiments, doses are provided for a number of days. For example, doses can be provided for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or 31 days, or a range of days defined by any of the aforementioned numbers of days. In some embodiments, the composition is administered for acute treatment or for a short term. For example, the subject can receive doses over a period of about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks, or a range therebetween. In embodiments, compositions as described herein can be administered to a subject chronically. “Chronic administration” can refer to administration in a continuous manner, such as to maintain theDocket No.: 2967100.2.W01Customer No 190456 therapeutic effect (activity) over a prolonged period of time. Tn some embodiments, the composition is administered for chronic treatment or for a long term. For example, the subject can receive doses over a period of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, about 20 years, about 25 years, a range therebetween, or longer. In some embodiments, the administration comprises doses over a period of about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, about 20 years, about 25 years, or longer. In some embodiments, a subject is administered the composition for the rest of their life after treatment begins. In some embodiments, the administration includes doses for a range of time defined by any two of the numbers of days or weeks provided herein.

[0055] The administration can be through a route of administration. The administration can include enteral administration. Examples of enteral administration can include oral, buccal or sublingual administration. In some embodiments, the administration is oral. In some embodiments, the administration is sublingual. In some embodiments, the administration is buccal. The administration can include parenteral administration. In some embodiments, the administration is intravenous. In some embodiments, the administration includes an injection. Some examples of routes of administration can include intramuscular, subcutaneous, intraarterial, rectal, vaginal, nasal, inhalation, or transdermal. In some embodiments, the administration is through an implant. The implant can be or include a rod.

[0056] Oral compositions can include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Oral formula of the drug can be administered once a day, twice a day, three times a day, or four times a day, for example, depending on the half-life of the drug.Docket No.: 2967100.2.W01Customer No 190456

[0057] In some embodiments, the subject is a vertebrate. In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal. In some embodiments, the subject is a primate. In some embodiments, the subject is human.NAD compositions

[0058] As used herein, the term “NAD” refers to “nicotinamide adenine dinucleotide” and encompasses NAD+ and / or NADH. The term “NAD+” as used in this disclosure describes compositions consisting ofNAD+. NAD (e.g. NADH or NAD+) can be included in a composition. The composition can include NAD+. The composition can be an enhanced NAD+ composition. In some embodiments, the NAD comprises NAD+. The composition can include NADH. The composition can include NAD+ and NADH. The enhanced NAD+ composition can be a pharmaceutical composition. An enhanced NAD+ composition can include a bioavailable formulation. In embodiments, enhanced NAD+ composition comprises any of the various compositions disclosed WO2024086312, which is incorporated by reference in its entirety. Some embodiments relate to an orally bioavailable NAD+ formulation. There has been a longstanding challenge of direct NAD replenishment, which has been limited by poor oral bioavailability and compound stability which has been addressed, for example, through the use of compounds, methods, and compositions described in WO2024086312. Such formulations and compositions can allow for efficient absorption and cellular uptake, offering advantages over NAD precursor supplements such as NMN in rapidly restoring intracellular NAD levels. Results from feasibility studies have revealed that orally bioavailable enhanced NAD+ formulations are useful in modulating proteins or biological pathways involved in cellular metabolism, transcriptional regulation, mitochondrial biogenesis, immune regulation, and T cell function. The formulation can include an oral formulation. The bioavailable formulation can be an orally bioavailable formulation. An example of an enhanced NAD+ composition is an NAD+ composition that includes a compound that increases oral bioavailability such as polyethylene glycol. The enhanced NAD+ composition can be useful in any of the methods described herein. For example, the enhanced NAD+ composition can be administered to a subject in need thereof. In some embodiments, the enhanced NAD+ composition is for use in a method described herein.

[0059] The NAD+ of an enhanced NAD+ composition can be present in an amount from about 5 mg to about 4000 mg. In some embodiments, the amount of NAD+ in the composition is aboutDocket No.: 2967100.2.W01Customer No 190456100 mg to about 3500 mg. In some embodiments, the amount of NAD+ is about 20 mg to about 2500 mg. In some embodiments, the amount of NAD+ is about 50 mg to about 2000 mg. In some embodiments, the amount of NAD+ is about 100 mg to about 1500 mg. In some embodiments, the amount of NAD+ is about 200 mg to about 1000 mg. In some embodiments, the amount of NAD+ is about 300 mg to about 900 mg. In some embodiments, the amount of NAD+ is about 400 mg to about 800 mg. In some embodiments, the amount of NAD+ is about 500 mg to about 700 mg. In some embodiments, the amount of NAD+ is about 550 mg to about 650 mg. In some embodiments, the amount of NAD+ is about 500 mg. In some embodiments, the amount of NAD+ is about 600 mg. In some embodiments, the amount of NAD+ is about 700 mg. In some embodiments, the amount of NAD+ is about 800 mg. In some embodiments, the amount of NAD+ is about 900 mg. In some embodiments, the amount of NAD+ is about 1000 mg. In some embodiments, the amount of NAD+ is about 500 mg. In some embodiments, the amount of NAD+ is about 400 mg. In some embodiments, the amount of NAD+ is about 300 mg. In some embodiments, the amount of NAD+ is about 200 mg. In some embodiments, the amount of NAD+ is about 250 mg. Any range therebetween can be used.

[0060] In some embodiments, the composition includes polyethylene glycol (PEG). Some examples of PEG include PEG of various sizes or variations, such as PEG300, PEG400 PEGMME (Polyethylene glycol monomethyl ether) 550, PEG600, PEG1000, PEGMME 2000, PEG335O, and / or PEG4000. The composition can include PEG at a concentration of about 10% to about 75% In some embodiments, the composition comprises PEG at a concentration of about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 60%, about 70%, or about 75%, or a range therebetween. Some embodiments include about 50% PEG. Some embodiments include about 50% PEG and 50% NAD+. In some embodiments, the composition is about 10% NAD+, about 20% NAD+, about 30% NAD+, about 40% NAD+, about 50% NAD+, about 60% NAD+, about 70% NAD+, about 80% NAD+, or about 90% NAD+. Any range of percentages of NAD+ therebetween can be used. In some embodiments, the composition is about 10% PEG, about 20% PEG, about 30% PEG, about 40% PEG, about 50% PEG, about 60% PEG, about 70% PEG, about 80% PEG, or about 90% PEG. Any range of PEG percentages therebetween can be used. Some embodiments include PEG and NAD+ in about a 1 : 1 ratio (e.g. about 50% NAD+ and about 50% PEG). In some embodiments, the composition is about 40%-60% NAD+ and about 40%-60% PEG. In some embodiments, the composition is about 45%-55% NAD+ and about 45%-55% PEG.Docket No.: 2967100.2.W01Customer No 190456In some embodiments, the composition is about 50% NAD+ and about 50% PEG. Such embodiments can be examples of an enhanced bioavailable NAD+ supplement.

[0061] The composition can be formulated in solid or liquid forms. Some examples of formulations can include tablets, capsules, powders, lozenges, suppositories, syrups, elixirs, sterile solutions, suspensions or emulsions, pastes, ointments, jellies, waxes, oils, or lipids. The formulation can include vesicles such as liposomes, microparticles, or microcapsules. Some additional examples of formulations can include DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbowax, semi-solid gels, or semi-solid mixtures containing carbowax. In some embodiments, the composition is administered through an implant (e.g. an implantable rod). In some embodiments, the composition is in a powder form.Definitions

[0062] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0063] Throughout this application, various embodiments can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0064] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof.Docket No.: 2967100.2.W01Customer No 190456

[0065] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0066] Some embodiments relate to compositions and methods that include nicotinamide adenine dinucleotide (NAD). NAD can be in an oxidized form (NAD+) or in a reduced form (NADH). NAD+ is shown in Formula I below:(Formula I)

[0067] In some embodiments, the chemical name of Formula I is: [[(2R,3S,4R,5R)-5-(6- aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2R,3S,4R,5R)-5-(3- carbamoylpyridin-l-ium-l-yl)-3,4-dihydroxyoxolan-2-yl]methyl hydrogen phosphate. In some embodiments, the chemical formula of Formula I is C2iH28N?Oi4P2+. In some embodiments, the molecular weight of protonated NAD+ (positively charged, anhydrate) is 664.44 g / mol. In some embodiments, the CASRN of Formula I is 53-84-9 and the source is PubChem (CID 5893). In some embodiments, the molecular weight of NAD+ (zwitterion, anhydrate) is 663.43 g / mol.

[0068] The term “subject,” “individual,” or “patient” can refer to any organism to which aspects of the invention can be performed, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. A “subject” can be a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria,Docket No.: 2967100.2.W01Customer No 190456 viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. Subjects to which methods as described herein are performed comprise mammals, such as primates, for example humans. For veterinary applications, a wide variety of subjects are suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals and pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals are suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like. The term “living subject” can refer to a subject noted herein or another organism that is alive. The term “living subject” can refer to the entire subject or organism and not just a part excised (e.g., a liver or other organ) from the living subject. The subject can be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease. The term “normal subject” can refer to a subject that is not afflicted with a disease or condition, such as a subject that is not afflicted with a chronic infection. A “subject” can be an induvial who is afflicted with an immune dysfunction resulting from chronic infections.

[0069] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting.

[0070] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.

[0071] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.Docket No.: 2967100.2.W01Customer No 190456

[0072] As used herein, the term “about” a number refers to that number plus or minus 15% of that number. The term “about” a range refers to that range minus 15% of its lowest value and plus 15% of its greatest value.

[0073] As used herein, the phrase “therapeutic agent” can refer to any agent that elicits a desired pharmacological effect when administered to a subject. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, the appropriate population may be a population of model organisms. In some embodiments, an appropriate population may be defined by various criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, etc. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition.

[0074] The term “therapeutically effective amount”, as used herein, can refer to an amount of a therapeutic agent whose administration, when viewed in a relevant population, correlates with or is reasonably expected to correlate with achievement of a particular therapeutic effect. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect). In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay and / or alleviate one or more symptoms of the disease, disorder, and / or condition. Disease progression can be monitored by clinical observations, laboratory and imaging investigations apparent to a person skilled in the art. A therapeutically effective amount is administered in a dosing regimen that can comprise multiple unit doses. For a therapeutic agent, a therapeutically effective amount (and / or an appropriate unit dose within an effective dosing regimen) can vary, for example, depending on route of administration, on combination with other pharmaceutical agents. Also, the specific therapeutically effective amount (and / or unit dose) for a patient can depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and / or rate of excretion or metabolism of the specific fusion protein employed; the duration of the treatment; and like factors as is well known in theDocket No.: 2967100.2.W01Customer No 190456 medical arts. Furthermore, an effective amount may be administered via a single dose or via multiple doses within a treatment regimen. In some embodiments, individual doses or compositions are considered to contain a “therapeutically effective amount” when they contain an amount effective as a dose in the context of a treatment regimen. Those of ordinary skill in the art will appreciate that a dose or amount may be considered to be effective if it is or has been demonstrated to show statistically significant effectiveness when administered to a population of patients; a particular result need not be achieved in a particular individual patient in order for an amount to be considered to be therapeutically effective as described herein.

[0075] As used herein, the terms “treatment” or “treating” are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit or therapeutic effect can be used interchangeably to refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject can still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease can undergo treatment, even in the absence of diagnosis of this disease. The word “treating” can mean the medical management of a subject with the intent that a prevention, cure, stabilization, or amelioration of the symptoms or condition will result. This term includes active treatment, that is, treatment directed specifically toward improvement of the disorder; palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disorder; preventive treatment, that is, treatment directed to prevention of disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the disorder. The term “treatment” also includes symptomatic treatment, that is, treatment directed toward constitutional symptoms of the disorder. “Treating” a condition with the compounds of the invention involves administering such a compound, alone or in combination and by any appropriate means, to a patient. For example,Docket No.: 2967100.2.W01Customer No 190456“treating” an immune dysfunction by raising intracellular NAD+ levels in a patient or subject. Treatment can be administered to a subject who does not exhibit signs of immune dysfunction, chronic infection, and / or condition (e.g., prior to an identifiable disease, disorder, and / or condition), and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0076] A treatment can include administration, e.g. administration of NAD+. Administration can include oral administration. In some embodiments, an administration is intravenous. An administration can include an injection. Administration can include supplementation. Administration or supplementation can include repletion, such as repletion of NAD. An administration can result in repletion of the administered aspect in a tissue, cell, or fluid.

[0077] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0078] Embodiments as described herein can involve isolating, collecting, or obtaining a biological sample from a subject. As used herein, the term “collecting a sample” or “isolating a sample”, for example, can refer to any process for directly or indirectly acquiring a biological sample from a subject. For example, a biological sample may be obtained (e.g., at a point-of-care facility, e.g., a physician's office, a hospital, laboratory facility) by procuring a tissue sample (such as a skin biopsy) from a subject. Alternatively, a biological sample may be obtained by receiving the biological sample (e.g., at a laboratory facility) from one or more persons who procured the sample directly from the subject. The biological sample may be, for example, a tissue (e.g., biopsy), fluid (e.g., cerebrospinal fluid, plasma, blood, serum) or cell (e.g., skin fibroblast cells, peripheral blood cells) of a subject.

[0079] The term “sample” can refer to a biological sample obtained or derived from a source of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a biological sample comprises biological tissue or fluid. In some embodiments, a biological sample is or comprises bone marrow; spleen, blood; blood cells; blood mononuclear cells; serum; plasma; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductalDocket No.: 2967100.2.W01Customer No 190456 lavages or bronchoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and / or excretions; and / or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy {e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid {e.g., blood, lymph, feces etc.), etc. In some embodiments, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of certain components, etc.

[0080] EXAMPLESExample 1: Immune enhancement in a subject with AIDS

[0081] A 37-year-old adult, male, human subject having a human immunodeficiency virus (HIV) infection was treated with an orally administered enhanced NAD+ formulation that included doses of 500 mg NAD+ and 500 mg PEG. Thus, each dose included 1G of the composition. A 1G dose was provided to the subject each day for 39 days from 7 / 22 / 2024 to 8 / 29 / 2024. A 500 mg dose was then provided to the subject each day for the next 62 days from 8 / 30 / 2024 to 10 / 30 / 2024. A 1G dose was then provided to the subject each day for the next 27 days from 10 / 31 / 2024 to 11 / 26 / 2024. A 500 mg dose was then provided to the subject each day for the next 119 days from 11 / 27 / 2024 to 3 / 25 / 2025. A 1G dose was then provided to the subject each day for the next 122 days from 3 / 26 / 2025 to 7 / 25 / 2025. The subject had been receiving antiretroviral therapy before the orally administered enhanced NAD+ composition. Upon administration of the enhanced NAD+ composition, the subject had been receiving the following: dovato (dolutegravir 50 mg, lamivudine 300 mg), enclomiphene 12.5mg, DHEA 25mg, vitamin D3 / K2 (5,000 IUS), methyl B12 5000 mcg, and a multivitamin derived from desiccated beef organs - 600 mg each of heart, liver, kidney, pancreas, and spleen (Heart & Soil Beef Organs Complex).Docket No.: 2967100.2.W01Customer No 190456

[0082] As evident from Figs. 1 -3, the treatment resulted in an increase in an NAD level relative to baseline NAD level. Before treatment, a blood sample was obtained from the subject, and intracellular NAD (icNAD) was measured in the subject to obtain a baseline NAD level. The icNAD level included a combined intracellular NADH and NAD+ measurement obtained by Jinfiniti Precision Medicine. The baseline NAD level was 23.2 pM, which was considered as falling within the deficient range. Treatment commenced on 7 / 22 / 2024. A second icNAD level was obtained 14 days after initiating treatment, on 8 / 5 / 2024. The second icNAD level had a value of 25.0 pM. A third icNAD level was obtained 29 days after initiating treatment, on 8 / 20 / 2024. The third icNAD level had a value of 36.9 pM. A fourth icNAD level was obtained 39 days after initiating treatment, on 8 / 30 / 2024. The fourth icNAD level had a value of 33.7 pM. A fifth icNAD level was obtained 57 days after initiating treatment, on 9 / 17 / 2024. The fifth icNAD level had a value of 40.0 pM. A sixth icNAD level was obtained 81 days after initiating treatment, on 10 / 11 / 2024. The sixth icNAD level had a value of 43.2 pM. A seventh icNAD level was obtained 102 days after initiating treatment, on 11 / 1 / 2024. The seventh icNAD level had a value of 43.2 pM. An eighth icNAD level was obtained 116 days after initiating treatment, on 11 / 15 / 2024. The eighth icNAD level had a value of 60.8 pM. A ninth icNAD level was obtained 134 days after initiating treatment, on 12 / 3 / 2024. The ninth icNAD level had a value of 43.4 pM. A tenth icNAD level was obtained 169 days after initiating treatment, on 1 / 7 / 2025. The tenth icNAD level had a value of 44.0 pM. An eleventh icNAD level was obtained 197 days after initiating treatment, on 2 / 4 / 2025. The eleventh icNAD level had a value of 48.8 pM. A twelfth icNAD level was obtained 247 days after initiating treatment, on 3 / 26 / 2025. The twelfth icNAD level had a value of 37.3 pM. A thirteenth icNAD level was obtained 277 days after initiating treatment, on 4 / 25 / 2025. The thirteenth icNAD level had a value of 62.0 pM. A fourteenth icNAD level was obtained 296 days after initiating treatment, on 5 / 14 / 2025. The fourteenth icNAD level had a value of 40.5 pM. A fifteenth icNAD level was obtained 368 days after initiating treatment, on 7 / 25 / 2025. The fifteenth icNAD level had a value of 43.6 pM.

[0083] The treatment resulted in about a 31.6% increase in a circulating CD4+ T cell measurement relative to baseline measurement after 20 days. The baseline circulating CD4+ T cell measurement obtained before treatment had a value of 263 CD4+ T cells per microliter of blood. Fourteen days after initiating the treatment, the circulating CD4+ T cell measurement was 346 cells / pL. After another 15 days (after 29 days total) of treatment with the enhanced NAD+Docket No.: 2967100.2.W01Customer No 190456 composition, an additional CD4+ T cell measurement was obtained, and the number rose to 467 cells / pL for a total increase of 77.6% after the 29 days of treatment. After another 10 days (after 39 days total) of treatment with the enhanced NAD+ composition, an additional CD4+ T cell measurement was obtained, and the number dropped to 435 cells / pL for a total increase of 65.4% after the 39 days of treatment. After another 18 days (after 57 days total) of treatment with the enhanced NAD+ composition, an additional CD4+ T cell measurement was obtained, and the number dropped to 428 cells / pL for a total increase of 62.7% after the 57 days of treatment. After another 24 days (after 81 days total) of treatment with the enhanced NAD+ composition, an additional CD4+ T cell measurement was obtained, and the number maintained at 428 cells / pL for a total increase of 62.7% after the 81 days of treatment. After another 20 days (after 101 days total) of treatment with the enhanced NAD+ composition, an additional CD4+ T cell measurement was obtained, and the number rose to 498 cells / pL for a total increase of 89.4% after the 101 days of treatment. After another 15 days (after 116 days total) of treatment with the enhanced NAD+ composition, an additional CD4+ T cell measurement was obtained, and the number rose to 528 cells / pL for a total increase of 100.8% after the 116 days of treatment. After another 18 days (after 134 days total) of treatment with the enhanced NAD+ composition, an additional CD4+ T cell measurement was obtained, and the number dropped to 440 cells / pL for a total increase of 67.3% after the 134 days of treatment. After another 35 days (after 169 days total) of treatment with the enhanced NAD+ composition, an additional CD4+ T cell measurement was obtained, and the number rose to 474 cells / pL for a total increase of 80.2% after the 169 days of treatment. After another 28 days (after 197 days total) of treatment with the enhanced NAD+ composition, an additional CD4+ T cell measurement was obtained, and the number dropped to 468 cells / pL for a total increase of 77.9% after the 197 days of treatment. After another 50 days (after 247 days total) of treatment with the enhanced NAD+ composition, an additional CD4+ T cell measurement was obtained, and the number dropped to 423 cells / pL for a total increase of 60.8% after the 247 days of treatment. After another 30 days (after 277 days total) of treatment with the enhanced NAD+ composition, an additional CD4+ T cell measurement was obtained, and the number rose to 513 cells / pL for a total increase of 95.1% after the 277 days of treatment. After another 19 days (after 296 days total) of treatment with the enhanced NAD+ composition, an additional CD4+ T cell measurement was obtained, and the number dropped to 502 cells / pL for a total increase of 90.9% after the 296 days of treatment. After another 72 days (after 368 days total) of treatment with theDocket No.: 2967100.2.W01Customer No 190456 enhanced NAD+ composition, an additional CD4+ T cell measurement was obtained, and the number dropped to 460 cells / pL for a total increase of 74.9% after the 368 days of treatment. This put the patient’s CD4+ T cell range out of the “Low” category. The measurements were obtained in whole blood samples obtained by venipuncture in a non-fasted state. Data from samples obtained before treatment, 14 days after treatment, and 29 days after treatment are provided in FIG. 1A-2C. Data from all samples obtained are provided in FIG. 3.

[0084] These data indicate that enhanced NAD+ administration can result in a significant increase in CD4+ T cells, an indicator of immune function. The administration can improve a subject who has a low CD4+ T cell count, and can even alleviate lymphodepletion within 1 month of treatment to be non-existent. These data also indicate that enhanced NAD+ administration can increase intracellular NAD concentrations.

[0085] While non-limiting, exemplary embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

Docket No.: 2967100.2.W01Customer No 190456CLAIMSWhat is claimed is:

1. A method of treatment, comprising: administering a therapeutically effective amount of an enhanced NAD+ composition to a subject having immune dysfunction.

2. The method claim 1, wherein the composition is about 10% NAD+, about 20% NAD+, about 30% NAD+, about 40% NAD+, about 50% NAD+, about 60% NAD+, about 70% NAD+, about 80% NATH, or about 90% NATH.

3. The method of any one of the previous claims, wherein the composition is about 10% PEG, about 20% PEG, about 30% PEG, about 40% PEG, about 50% PEG, about 60% PEG, about 70% PEG, about 80% PEG, or about 90% PEG.

4. The method of claim 1, wherein, the enhanced NAD+ composition comprises NAD+ and PEG at about a 1 : 1 ratio.

5. The method of claim 1, wherein the enhanced NATH composition is about 50% NAD+ and about 50% PEG.

6. The method of any one of the previous claims, wherein the administration is oral.

7. The method of any one of the previous claims, wherein the administration is buccal or sublingual.

8. The method of any one of the previous claims, wherein the administration comprises at least one dose over a period of about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, about 20 years, about 25 years, or longer.

9. The method of any one of the previous claims, wherein the administration comprises daily doses.

10. The method of any one of the previous claims, wherein the administration comprises doses of about 1 mg NAD+ per day, about 2.5 mg NATH per day, about 5 mg NAD+ per day, about 10 mg NATH per day, about 15 mg NATH per day, about 25 mg NAD+ per day, about 50 mg NAD+ per day, about 75 mg NAD+ per day, about 100 mg NAD+ per day, about 200Docket No.: 2967100.2.W01Customer No 190456 mg NAD+ per day, about 300 mg NAD+ per day, about 400 mg NAD+ per day, about 500 mg NAD+ per day, about 600 mg NAD+ per day, about 700 mg NAD+ per day, about 800 mg NAD+ per day, about 900 mg NAD+ per day, about 1000 mg NAD+ per day, about 1250 mg NAD+ per day, about 1500 mg NAD+ per day, about 1750 mg NAD+ per day, or about 2000 mg NAD+ per day.

11. The method of any one of the previous claims, wherein the immune dysfunction comprises T cell depletion.

12. The method of any one of the previous claims, wherein the subject has a low CD4+ T cell count.

13. The method of any one of the previous claims, wherein the low CD4+ T cell count comprises a CD4+ T cell measurement below 359 units / pL in a blood sample.

14. The method of any one of the previous claims, wherein the immune dysfunction results from or corresponds with the subject having an infection.

15. The method of any one of the previous claims, wherein the infection comprises influenza, pneumonia, hepatitis B, a cytomegalovirus infection, or coronavirus disease 2019.

16. The method of any one of the previous claims, wherein the infection is chronic.

17. The method of any one of the previous claims, wherein the infection comprises a retrovirus infection.

18. The method of any one of the previous claims, wherein the infection comprises a lentivirus infection.

19. The method of any one of the previous claims, wherein the infection comprises a human immunodeficiency virus (HIV) infection.

20. The method of any one of the previous claims, wherein the infection comprises a simian immunodeficiency virus (SIV) infection.

21. The method of any one of the previous claims, wherein the administration increases an NAD+ measurement in the subject.

22. The method of any one of the previous claims, wherein the NAD+ measurement is increased by at least 5% relative to a baseline measurement.

23. The method of any one of the previous claims, wherein the administration increases an immune cell measurement in the subject.Docket No.: 2967100.2.W01Customer No 19045624. The method of any one of the previous claims, wherein the immune cell measurement comprises a CD4+ T cell measurement.

25. The method of any one of the previous claims, wherein the immune cell measurement is increased by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, or by at least 75%, relative to a baseline measurement.

26. The method of any one of the previous claims, wherein the administration alleviates lymphodepletion in the subject within about 28 days or 1 month of initiation of treatment.

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

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