GPX3 as a blood-based calorie restriction mimetic to treat age-related cognitive decline

Abstract

Methods and compositions for improving cognitive function by increasing GPX3 in a subject are provided.

Description

PATENT Atorney Docket No. 081906-1521136-255910PC Client Ref. No. SF2024-206GPX3 AS A BLOOD-BASED CALORIE RESTRICTION MIMETIC TO TREAT AGE-RELATED COGNITIVE DECLINECROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application No. 63 / 693,067, filed September 10, 2024, which is incorporated by reference.BACKGROUND OF THE INVENTION

[0002] Aging leads to a decline in cognitive function and is the primary risk factor for age- related neurodegenerative diseases, such as Alzheimer’s disease. Calorie Restriction can be effective and is a well-studied non-genetic intervention to improve cognitive decline. However, adherence to dietary interventions is especially difficult in the elderly.

[0003] Glutathione Peroxidases (sometimes abbreviated as GPxs or GPXs are enzymes that play a role in eliminating forms of hydrogen peroxide generated in the body and that catalyze the conversion of glutathione (GSH) to oxidized glutathione (GSSG). The GPX family has at least 8 different GPX members, which are phylogenetically related but antigenically and structurally different. Different family members localize to different cell compartments, such as the nucleus, cytosol, mitochondria, and endoplasmic reticulum (ER). GPX3 is present in the extracellular fluid. Among all reported GPXs, natural GPx3, which is active as a homotetramer, can eliminate all complex hydroperoxides. GPX3 circulates in plasma. See, Nirgude et al., Biochemical Pharmacology Volume 184, February 2021, p. 114365.BRIEF SUMMARY OF THE INVENTION

[0004] In some embodiments, methods of treating or preventing age-related cognitive dysfunction in an individual in need thereof is provided, e.g., by increasing GPX3 in the individual. In some embodiments, the method comprises administering to the individual an effective amount of a Glutathione Peroxidase 3 (GPX3) polypeptide that is administered1KILPATRICK TOWNSEND 78793839 1systemically or locally to the brain, thereby treating or preventing age-related cognitive dysfunction.

[0005] In some embodiments, the GPX3 polypeptide is administered systemically. In some embodiments, the GPX3 polypeptide is administered by intravenous, intraperitoneal, subcutaneous, or intramuscular injection. In some embodiments, the GPX3 polypeptide is administered orally or mucosally.

[0006] In some embodiments, the individual is a human. In some embodiments, the human is at least 50 years old. In some embodiments, the individual has mild cognitive impairment. In some embodiments, the individual has dementia. In some embodiments, the individual has Alzheimer’s disease.

[0007] In some embodiments, the GPX3 polypeptide has catalytic activity and comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO: 1.

[0008] In some embodiments, the effective amount is 1 pg to 1000 pg per kg body weight of the individual.

[0009] In some embodiments, the GPX3 polypeptide or a functional fragment thereof is administered more than once as part of a course of treatment.

[0010] In some embodiments, the method further comprises testing the cognitive function of the individual after the administering. In some embodiments, the method further comprises testing the cognitive function of the individual prior to administering, and comparing the cognitive function of the individual prior to and after administering. In some embodiments, cognitive function is determined by testing the individual for semantic, episodic, procedural, priming, and / or working memory. In some embodiments, cognitive function is determined by testing the individual for language ability, executive function, visuospatial function, or dementia.

[0011] Also provided is a method of treating or preventing age-related cognitive dysfunction in an individual in need thereof, the method comprising introducing a nucleic acid construct encoding a GPX3 polypeptide into the individual such that the GPX3 polypeptide is expressed in the individual.2KILPATRICK TOWNSEND 78793839 1

[0012] Also provided is a method of treating or preventing age-related cognitive dysfunction in an individual in need thereof, the method comprising genetically modifying cells of an individual to enhance expression of a GPX3 polypeptide into the individual.

[0013] In some embodiments, cells are obtained from the individual, the cells are modified ex vivo, and then administered to the individual.

[0014] In some embodiments of the methods of introducing the nucleic acid construct or genetically-modifying cells, the individual has mild cognitive impairment. In some embodiments of the methods of introducing the nucleic acid construct or genetically-modifying cells, the individual has dementia. In some embodiments of the methods of introducing the nucleic acid construct or genetically-modifying cells, the individual has Alzheimer’s disease.DEFINITIONS

[0015] “Glutathione peroxidase 3,” GPX3,” or “GPX3 polypeptide” or similar terms refer to polypeptides comprising a selenocysteine (Sec) and being capable of reduction of peroxides using GSH. In some embodiments, the polypeptides comprise an amino acid sequence at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 1 or a catalytically-active fragment. Catalytic activity can include reduction of different types of peroxides using GSH as the reducing agent in vitro. See, e.g., Esworthy, et al., Arch Biochem Biophys 307, 29-34 (1993); Takebe, J Biol Chem 277, 41254-41258 (2002), describing exemplary assays for measuring catalytic activity. GPX3 is involved in the detoxification of reactive oxygen species (e.g., hydrogen peroxide) using glutathione. GPX3 is a selenoprotein where the selenocysteine is located in the active site of the enzyme and is involved in reducing hydrogen peroxide by acquiring and displacing the hydroxyl group (Zhang et al. 2020 Antioxidants (Basel). 2020 May 5;9(5):383).

[0016] The terms “cognition,” “cognitive ability,” “cognitive function,” and like terms refer to a collection of mental tasks and functions, including but not limited to: memory (e.g., semantic, episodic, procedural, priming, or working); orientation; language; problem solving; visual perception, construction, and integration; planning; organizational skills; selective attention; inhibitory control; and ability to mentally manipulate information.3KILPATRICK TOWNSEND 78793839 1

[0017] The terms “improved cognition,” “increased cognitive ability,” “improved cognitive function,” and like terms refer to an improvement in cognition under a given condition (e.g. treatment with GPX3) compared to cognition absent that particular condition (e.g., absent treatment with GPX3). For an individual experiencing cognitive decline, an improvement in cognition might be a reduction in the rate of cognitive decline (i.e., an improvement compared to the absence of treatment), but not an actual improvement in cognitive ability. An increase in cognitive ability can also be an increase in brain activity in a specified area, e.g., as determined by MRI, or an inhibition of brain activity that results in better overall brain function. An increase in cognitive ability can also be improvement in a cognitive performance test as described in more detail herein. An improvement or increase in cognitive ability can be in any one cognitive aspect or function, or any combination of individual cognitive functions.

[0018] An individual in need of improved cognitive function refers to individuals with age- related cognitive decline; a neurodegenerative disease; a mental or mood disorder; traumatic brain injury; developmental delay; genetic disorder resulting in reduced cognitive ability; brain injury due to stroke, brain cancer, MS, epilepsy, radiation or chemotherapy; etc. An individual in need of improved cognitive function can also include individuals that desire increased mental function to fight the effects of stress, sleep deprivation, jet lag, or pain, or to heighten ability for a particular task. A more complete and specific list of such individuals in included in the “Cognitive conditions and disorders” section herein.

[0019] A “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a polypeptide or antibody specifically reactive with a target polypeptide. Any method known for conjugating a protein to the label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

[0020] A “labeled” molecule (e.g., GPX3 polypeptide) is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals,4KILPATRICK TOWNSEND 78793839 1electrostatic, or hydrogen bonds to a label such that the presence of the molecule may be detected by detecting the presence of the label bound to the molecule.

[0021] The term “diagnosis” refers to a relative probability that a disorder is present in an individual. Similarly, the term “prognosis” refers to a relative probability that a certain future outcome may occur in the individual. For example, in the context of the present disclosure, prognosis can refer to the likelihood that an individual suffer cognitive decline, or the likely severity of the disease (e.g., severity of symptoms, rate of functional decline, etc.). The terms are not intended to be absolute, as will be appreciated by any one of skill in the field of medical diagnostics.

[0022] The terms “therapy,” “treatment,” and “amelioration” refer to any reduction in the severity of symptoms (cognitive decline), or improvement in cognitive function, or where motor function is affected, an improvement in motor function. As used herein, the terms “treat” and “prevent” are not intended to be absolute terms. Treatment and prevention can refer to any delay in cognitive decline, amelioration of symptoms (e.g., confusion), etc. Treatment and prevention can be complete or partial, such that cognition is better than would be expected without treatment (e.g., compared to cognition in the same individual before treatment or compared to cognition in similar non-treated individuals). The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment. In some aspects, cognition is improved by at least 1%, as compared, e.g., to the individual before administration or to a control individual not undergoing treatment. In some embodiments, cognition is improved by at least 2, 3, 5, 7, 10, 15, 20, 25%, 50%, 75%, 80%, or 90%, or more, determined using tests of cognition, molecular proxies, or structural changes associated with brain function.

[0023] The terms “effective amount,” “effective dose,” “therapeutically effective amount,” etc. refer to that amount of the therapeutic agent sufficient to ameliorate a disorder, as described above. For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of therapeutic effect at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2- fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.5KILPATRICK TOWNSEND 78793839 1

[0024] As used herein, the term “pharmaceutically acceptable” is used synonymously with physiologically acceptable and pharmacologically acceptable. A pharmaceutical composition will generally comprise agents for buffering and preservation in storage, and can include buffers and carriers for appropriate delivery, depending on the route of administration.

[0025] The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of active ingredient given to an individual at each administration. For the present invention, the dose refers to the amount of GPX3 polypeptide. The dose will vary depending on a number of factors, including frequency of administration; size and tolerance of the individual; type and severity of the condition; risk of side effects; and the route of administration. One of skill in the art will recognize that the dose can be modified depending on the above factors or based on therapeutic progress. The term “dosage form” refers to the particular format of the pharmaceutical, and depends on the route of administration. For example, a dosage form can be in a liquid, e.g., a saline solution for injection.

[0026] “Subject,” “patient,” “individual” and like terms are used interchangeably and refer to, except where indicated, mammals such as humans and non-human primates, as well as dogs, horses, pigs, mice, rats, and other mammalian species. The term does not necessarily indicate that the subject has been diagnosed with a particular disease, but typically refers to an individual under medical supervision. A patient can be an individual that is seeking treatment, monitoring, adjustment or modification of an existing therapeutic regimen, etc.

[0027] The words "protein", "peptide", and "polypeptide" are used interchangeably to denote an amino acid polymer or a set of two or more interacting or bound amino acid polymers. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymer.

[0028] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O- phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical6KILPATRICK TOWNSEND 78793839 1structure as a naturally occurring amino acid, e.g., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs may have modified R groups (e.g, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.

[0029] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. The following amino acids are typically conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0030] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or doublestranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, polypeptide-nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also encompasses “conservatively modified variants” thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be7KILPATRICK TOWNSEND 78793839 1achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al. , J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). The term nucleic acid can include for example, a gene, cDNA, mRNA, oligonucleotide, or polynucleotide.

[0031] The terms "identical" or “percent identity,” in the context two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides, or amino acids, that are the same (i.e., about 60% identity, preferably at least 65%, 70%, 75%, 80%, or 85% identity; and often at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithm (e.g., a BLASTP algorithm with default parameters for comparison of two polypeptide sequences. See, e.g., the NCBI web site at ncbi.nlm.nih.gov / BLAST. Such sequences are then said to be "substantially identical." As described below, the algorithms can account for gaps and the like. GPX3 polypeptides can have at least that is at least 70%, 75%, 80%, or 85% identity; and often at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, e.g. 200, 400, 500, 600, 700, or 800 amino acids, or greater in length, or over the entire length of the reference sequence unless indicated otherwise.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1A-K. Late-onset, short-term calorie restriction promotes synaptic plasticity, neurogenesis, and rejuvenates cognitive function in aged mice. A, Schematic illustrating Novel Object Recognition (NOR) and Radial Arm Water Maze (RAWM) used to assess hippocampal-dependent learning and memory in aged (22 months) mice following shortterm (4 weeks) caloric restriction (CR, 30% decrease in food intake) initiated late in life (21 months) or ad libitum (AL) feeding. B, Object recognition memory was assessed using the Novel object recognition (NOR) task and quantified as percent time spent exploring the novel versus familiar object (n=14-16 mice / group). C, Hippocampal-dependent spatial learning and memory was evaluated by Radial arm water maze (RAWM). Changes in cognition were quantified as number of errors while attempting to find the goal with a hidden escape platform (n=14-168KILPATRICK TOWNSEND 78793839 1mice / group). D, UMAP projection of identified cell types from single nucleus RNA sequencing of hippocampal nuclei isolated from late-onset, short-term CR mice and AL mice. E, Venn diagrams (top) and bar graphs (bottom) of number of differentially expressed genes (DEGs) in different cell types for CR versus AL control groups. F, UpSet plot displaying shared and unique DEGs per cell type for the CR (CR versus AL) comparison. G, Volcano plot of DEGs in CAI neurons for the CR comparison. H, Top GO terms of biological processes associated with DEGs in CAI neurons for the CR comparison. I, Volcano plot of DEGs in the dentate gyrus (DG) neurons for the CR comparison. J, Top GO terms of biological processes associated with DEGs in DG neurons for the CR comparison. K, Image (left) and quantification (right) of MCM2+ DAPI+ cells in the hilus of DG of CR and AL mice (n=8-9 mice / group). Arrows pointing to MCM2+ DAPI+ cells in the hilus of the DG. Data shown as mean+ / - s.e.m. Statistical analysis was performed using t-test (K), one-sample t-test versus 50% (B), two-way ANOVA with Sidak’s post hoc test (C); *,p<0.05, **,p<0.01,***,p0.001.

[0033] Figure 2A-N. Calorie restriction blood plasma administration promotes synaptic plasticity, neurogenesis, and rejuvenates cognitive function in aged mice. A, Schematic illustrating Novel Object Recognition (NOR) and Radial Arm Water Maze (RAWM) used to assess hippocampal-dependent learning and memory in aged (22 months) mice following systemic administration of CR or AL blood plasma 8 times over 24 days (100 pl per intravenous injection). B, Object recognition memory was assessed using the Novel object recognition (NOR) task and quantified as percent time spent exploring the novel versus familiar object (n=13-14 mice / group). C, Hippocampal-dependent spatial learning and memory was evaluated by Radial arm water maze (RAWM). Changes in cognition were quantified as number of errors while attempting to find the goal with a hidden escape platform (n=13-14 mice / group). D, UMAP projection of identified cell types from single nucleus RNA sequencing of hippocampal nuclei isolated from mice that received systemic administration of CR or AL blood plasma. E, Venn diagrams (top) and bar graphs (bottom) of number of differentially expressed genes (DEGs) in different cell types for CR versus AL control groups with comparison to Direct CR. F, UpSet plot displaying shared and unique DEGs per cell type for the CR plasma (CR blood plasma versus AL blood plasma) comparison. G, Volcano plot of DEGs in inhibitory neurons for the CR plasma comparison. H, Top GO terms of biological processes associated with DEGs in inhibitory neurons for the CR plasma comparison. I, Volcano plot of DEGs in CAI neurons for9KILPATRICK TOWNSEND 78793839 1the CR plasma comparison. J, Top GO terms of biological processes associated with DEGs in CAI neurons for the CR plasma comparison. K, Pie chart of 338 overlapping DEGs between Direct CR and CR plasma comparisons (top) separated by unidirectional (green) versus bidirectional changes. Overlapping unidirectionally changing DEGs between Direct CR and comparisons referred to as the conserved signature. Bar graph (bottom) of conserved DEGs by cell type. L, UpSet plot displaying shared and unique conserved DEGs between both Direct CR and CR plasma comparisons, by cell type. M, Top GO terms of biological processes associated with conserved DEGs. N, Image (top) and quantification (bottom) of MCM2+ DAPI+ cells in the hilus of DG of CR and AL mice (n=12-14 mice / group). Arrows pointing to MCM2+ DAPI+ cells in the hilus of the DG. Scale bar 100 um. Data shown as mean+ / - s.e.m. Statistical analysis was performed using t-test (N), one-sample t-test versus 50% (B), two-way ANOVA with Sidak’s post hoc test (C); *,p<0.05.

[0034] Figure 3A-C. Circulating Glutathione Peroxidase 3 (GPX3) is elevated in blood plasma following late-onset, short-term calorie restriction relative to ad libitum diet in aged mice. A, Schematic illustrating blood plasma isolation from mice that underwent late-onset, short-term CR or maintained on AL diet (n= 8 mice / group). B, Volcano plot of proteins that are significantly increased in blood plasma of CR mice compared to AL mice C, Image (left) and quantification (right) of western blot analysis of GPX3 in blood plasma of CR and AL mice. Data shown as mean+ / - s.e.m. Statistical analysis was performed using t-test (C); *,p<0.05.

[0035] Figure 4A-M. Systemic Gpx3 treatment promotes RNA processing, neurogenesis, and rejuvenates cognitive function in aged mice. A, Schematic illustrating Novel Object Recognition (NOR), Radial Arm Water Maze (RAWM), Y-Maze used to assess hippocampal- dependent learning and memory in aged (22 months) mice following hydrodynamic tail vein injection (HDTVI) of GPX3 or GFP control constructs. B, Representative image (left) and quantification of western blot analysis of GPX3 protein levels in blood plasma 24 hours following HDTVI (n=3-4 mice / group). C, Object recognition memory was assessed using the Novel object recognition (NOR) task and quantified as percent time spent exploring the novel versus familiar object (n=10-13 mice / group). D, Hippocampal-dependent spatial learning and memory was evaluated by Radial arm water maze (RAWM). Changes in cognition were quantified as number of errors while attempting to find the goal with a hidden escape platform10KILPATRICK TOWNSEND 78793839 1(n=l 0- 13 mice / group). E, Spatial working memory was assessed using the Y-maze as the discrimination index for the novel arm (n=12-14 mice / group). F, UMAP projection of identified cell types from single nucleus RNA sequencing of hippocampal nuclei isolated from GPX3 or GFP control mice. G, Venn diagrams (top) and bar graphs (bottom) of number of differentially expressed genes (DEGs) in different cell types for GPX3 versus GFP control groups. H, UpSet plot displaying shared and unique DEGs per cell type for the GPX3 (HDTVI of GPX3 versus GFP control) comparison. I, Volcano plot of DEGs in the DG neurons for the GPX3 comparison. J, Top GO terms of biological processes associated with DEGs in DG neurons for the GPX3 comparison. K, Volcano plot of DEGs in CAI neurons for the GPX3 comparison. L, Top GO terms of biological processes associated with DEGs in CAI neurons for the GPX3 comparison. M, Image (left) and quantification (right) of MCM2+ DAPI+ cells in the hilus of DG of CR and AL mice (n=8-9 mice / group). Arrows pointing to MCM2+ DAPI+ cells in the hilus of the DG. Scale bar 100 um. Data shown as mean+ / - s.e.m. Statistical analysis was performed using t-test (B, M), one-sample t-test versus 50% (C) and versus 0 (E), two-way ANOVA with Sidak’s post hoc test (D); *,p<0.05, **,p<0.01,***,p0.001.DETAILED DESCRIPTION OF THE INVENTION

[0036] The inventors have identified Glutathione Peroxidase 3 (GPX3) - an enzyme that protects against oxidative stress through detoxification of hydroperoxides - as a calorie restriction-induced rejuvenating blood factor that can in part recapitulate the rejuvenating effects of calorie restriction on the aged hippocampus when increased systemically in the blood. Accordingly, systemically increasing GPX3 in an individual can mitigate, prevent, and / or counteract cognitive, neurological, and regenerative dysfunction associated with aging. GPX3 can be delivered, for example, but without limitation, as a recombinant protein, via viral-or other vector-mediated gene delivery, via molecular approaches (such as via CRISPRa-Cas9 or another gene-targeted gene modification system), and lipid nanoparticle-based mRNA approaches for over expression of GPX3, systemically in the blood.

[0037] GPX3 is a glutathione peroxidase and has been identified in a number of species, including humans. In general, when introduced into a human as a therapeutic, it can be desirable11KILPATRICK TOWNSEND 78793839 1that the GPX3 polypeptide be identical to, or substantially similar to human GPX3 so as to avoid potential immune reactions from the human receiving the GPX3 protein. Accordingly, in some embodiments, the polypeptide introduced into the human to mitigate, prevent, and / or counteract cognitive, neurological, and regenerative dysfunction comprises SEQ ID NO: 1 (human GPX3) or a catalytically-active fragment thereof. In some embodiments, the polypeptide introduced into the human is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 1 or a catalytically-active fragment.

[0038] In some embodiments, the polypeptide introduced into the human comprises a GPX3 protein or catalytically-active fragment as detailed herein fused to a heterologous amino acid sequence. Exemplary heterologous amino acid sequences can include, but are not limited to, amino acid sequences that extend the blood half-life of the polypeptide (e.g., a human Fc domain or human serum albumin) or a detectable label (e.g., a FLAG tag or other amino acid tag sequence). Fc domains can be human Fc domain or can include one or more mutations that for example increases half-life or other desired functions. See, e.g., Ko et al., Experimental & Molecular Medicine volume 54, pages 1850-1861 (2022); Lee et al., Nature Communications volume 10, Article number: 5031 (2019); Booth et al., MAbs. 2018 Oct; 10(7): 1098-1110; U.S. Patent Publication No. US20190048078.

[0039] In some embodiments, the polypeptide introduced into the human comprises a GPX3 protein or catalytically-active fragment as detailed herein is PEGylated and / or comprises a secondary modification such as but not limited to phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, or lipidation. There is data to suggest that an unglycosylated mutant form human GPX3 has reduced catalytic activity (Song et al. 2014, Sci Rep. 2014 Oct 21 :4:6698).

[0040] GPX catalytic activity includes glutathione peroxidase activity. For example, GPX3 can reduce different types of peroxides using GSH as the reducing agent in vitro. See, e.g., Esworthy, et al. , Arch Biochem Biophys 307, 29-34 (1993); Takebe, J Biol Chem 277, 41254- 41258 (2002), describing exemplary assays for measuring catalytic activity. The active center of GPX3 is selenocysteine (Sec), which is incorporated into proteins by a specific mechanism. See, e.g., Song, et al., Scientific Reports volume 4, Article number: 6698 (2014). Natural GPX3 is believed to form a functional enzyme as a glycosylated tetramer, however functional variants12KILPATRICK TOWNSEND 78793839 1have been constructed, for example changing all cysteines to serines, that function as monomers. See, e.g., Song, et al., Scientific Reports volume 4, Article number: 6698 (2014). The selenocysteine is required for GPX3’s catalytic activity. Selenocysteine can be introduced to the protein by the UGA codon. Although the UGA serves as a stop codon, inclusion of a SECIS (Selenocysteine insertion sequence) element in the conserved stem-loop structure of the 3' UTR allows for the recognition of UGA as a Selenocysteine codon.Methods of Improving Cognitive Function

[0041] GPX3 polypeptides, or functional variants and fragments thereof, can be used to improve cognition for a number of conditions and situations. This includes treatment of individuals with lower than normal or declining cognitive ability, or prophylactic treatment of individuals in need of improved or increased cognitive ability. GPX3 polypeptide levels in an individual can be increased by administering a functional recombinant GPX3 polypeptide, by administering a nucleic acid that encodes a functional GPX3 polypeptide, e.g., as a viral or plasmid vector, or by genetic manipulation of cells in an individual, e.g., using CRISPR / CAS genetic modification techniques, or techniques comprising use of transcription activator-like effector nucleases (TALENS), CRISPRa or zinc finger nucleases.

[0042] GPX3 polypeptides (and functional variants and fragments thereof) and genetic manipulation to increase the levels of GPX3 polypeptide can be used to prevent or reduce cognitive decline associated with aging, e.g. in individuals 50 years of age or older, or 60 years of age or older, or upon initial signs of cognitive decline.

[0043] GPX3 polypeptides (and functional variants and fragments thereof) can also be used to treat individuals with age-related, non-age related, or disease related conditions including, but not limited to dementia and neurodegenerative diseases that may include impaired cognition, e.g., Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, frontotemporal dementia, progressive supranuclear palsy, corticobasalar degeneration, mild cognitive impairment, vascular dementia, Lewy body dementia, amyotropic lateral sclerosis, prion disorder, or HIV-related dementia.

[0044] In some embodiments, an individual treated in accordance with the invention has Alzheimer’s Disease or Mild Cognitive Impairment, frontotemporal dementia; or vascular dementia.13KILPATRICK TOWNSEND 78793839 1

[0045] As used herein, "Alzheimer’s disease" refers to senile dementia as diagnosed using commonly accepted criteria in the art, such as the criteria set forth by The National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s disease and Related Disorders Association and / or the criteria as listed in the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR) published by the American Psychiatric Association. The Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition, revised in 2000), also known as the DSM-IV-TR, outlines a detailed set of criteria for the diagnosis of Alzheimer’s disease.

[0046] In some embodiments, an individual may have mild to moderate dementia, or early- stage Alzheimer’s disease, which can be identified using neurological testing and other clinical endpoints. For example, a subject with mild to moderate dementia, e.g., Alzheimer’s disease, can be identified using the Mini-Mental State Examination (MMSE). Typically, a score of 16 to 26 (both inclusive) is indicative of mild to moderate Alzheimer’s disease. Patients with advanced Alzheimer’s disease can also be identified based on clinical parameters. Subjects with this form of Alzheimer’s disease may no longer respond to therapy with acetylcholinesterase inhibitors, and may have a markedly reduced acetylcholine level.

[0047] In some embodiments, a patient treated with a polypeptide of the invention may have Mild Cognitive Impairment. Such patients are at risk for development of Alzheimer’s disease. Mild Cognitive Impairment can be diagnosed and evaluated using any of the many objective tests or criteria well-known and accepted in the fields of psychology or psychiatry.

[0048] “Frontotemporal dementia” is a neurodegenerative disease characterized by progressive neuronal loss predominantly involving the frontal and / or temporal lobes. It is distinguished from Alzheimer's disease and Lewy body dementia based on the fact that it does not manifest with amyloid plaques, neurofibrillary tangles, or Lewy bodies. The term "frontotemporal lobar degeneration” or “FTLD” is used to describe the specific pathological diseases that result in frontotemporal dementia syndromes. These are united by their impact on frontal and temporal brain structures. Subtyping is based on the specific proteins found within neuronal inclusions. Most degeneration subtypes are either FTLD-tau, which includes Pick’s disease, CBD and PSP, all of which show tau-containing inclusions or FTLD-TDP, which includes several subtypes in which TDP-43 containing inclusions are seen.14KILPATRICK TOWNSEND 78793839 1

[0049] In some embodiments, a subject treated with a GPX3 polypeptide as described herein has a mental or mood disorder, e.g., depression, schizophrenia, attention deficit / hyperactivity disorder, autism spectrum disorder, intellectual disability, a mood disorder, and a psychotic disorder.

[0050] In some embodiments, a subject treated with a GPX3 polypeptide as described herein has a childhood neurodevelopmental syndrome or brain tumors, e.g., X-linked mental disability or retardation, astrocytoma, ependymoma, medulloblastoma, oligodendroglioma, Down’s syndrome, Angelman’s syndrome, Rett’s syndrome; phenylketonuria, Lesch-Nyhan, galactosemia, or adrenoleukodystrophy.

[0051] In some embodiments, a subject treated with a GPX3 polypeptide as described herein has a cognitive decline associated with chemotherapy and / or radiation therapy.

[0052] Additional conditions and disorders that can be treated with a GPX3 polypeptide include: pain-associated cognitive effects, traumatic brain injury, stroke, multiple sclerosis, neuroautoimmune disease, epilepsy, delirium, paraneoplastic disorder, developmental delay, and leukodystrophies.

[0053] GPX3 polypeptides (and functional variants and fragments thereof) can be also be administered to provide increased cognition for individuals desiring improved cognition, e.g., individuals exposed to stress, sleep deprivation, or jet lag, or for individuals requiring superior cognitive function, such as surgeons, air-traffic controllers, and military personal.

[0054] Cognitive ability can be measured using any method known in the art, e.g., for testing memory, language ability, executive functions, visuospatial function, dementia, or multiparameter neuropsychological abilities. In some embodiments, GPX3 administration (as a polypeptide or by genetic manipulation) results in at least a 1%, 2%, 5%, 7%, 10%, 15%, 20%, 30%, 50%, or greater improvement in score on a standard cognitive ability test (e.g., measured 1- 3 days after administration). In some embodiments, the testing is carried out more than once for an individual, e.g., one or more time over the course of treatment with GPX3.

[0055] For example, standard tests for memory and learning can be applied, e.g., to determine semantic, episodic, procedural, priming, and / or working (i.e., short term) memory. Common tests include Cambridge prospective memory test (CAMPROMPT), memory assessment scales15KILPATRICK TOWNSEND 78793839 1(MAS), Rey auditory verbal learning test, Rivermead behavioral memory test, Test of memory and learning (TOMAL), Wechsler memory scale (WMS), and Test of memory malingering (TOMM). Tests for language functions include, e.g., Boston Diagnostic Aphasia Examination (BDAE), Comprehensive aphasia test (CAT), and Multilingual aphasia examination (MAE).

[0056] Executive function (e.g., problem solving, planning, organization, inhibitory control) can be tested using Behavioral assessment of dysexecutive syndrome (BADS), CNS vital signs (Brief Core Battery), Controlled oral word association test (COW AT), Delis-Kaplan Executive Function System (D-KEFS), Digit vigilance test, Kaplan Baycrest neurocognitive assessment (KBNA), Hayling and Brixton tests, Tests of variables of attention (TOVA), Wisconsin card sorting test (WCST), or Test of everyday attention (TEA). Visuospatial ability (e.g., visual perception, construction and integration) can be tested using the Clock Test, Hooper visual organization task (VOT), or Rey-Osterrieth complex figure tests. Dementia can be quantified using the clinical dementia rating or dementia rating scale.

[0057] Multi-parameter tests for neuropsychological function (e.g., cognitive function) include but are not limited to the Barcelona neuropsychological test (BNT), Cambridge neuropsychological test automated battery (CANTAB), Cognistat, Cognitive assessment screening instrument (CASI), Cognitive function scanner (CFS), Dean-Woodcock neuropsychology assessment system (DWNAS), General practitional assessment of cognition (GPCOG) Mini mental state examination (MMSE), NEPSY, or the CDR computerized assessment system.

[0058] Alternatively, cognition can be determined using structural or molecular proxies for cognitive activity, e.g., compared over time to detect changes. Cognitive changes can be detected, e.g., by observing changes to brain structure, connectivity, activation, inhibition, or synaptic plasticity, e.g., by MRI, fMRI, EEG, TMS and TES, and / or any combination of these. In some embodiments, brain activity is observed. In some embodiments, GPX3 administration results in a 1.5-fold, 2-fold, 5-fold, 7-fold, 10-fold, or greater increase in brain activity (e.g., measured 24-30 days after administration). Molecular proxies for improved cognition include, but are not limited to: increased levels of GluN2B, increased GluN2B synaptic localization, increased NMDA receptor activation, and / or increased c-fos activation in the brain. These measures are particularly relevant to cognition. Such method can include, e.g., obtaining a16KILPATRICK TOWNSEND 78793839 1sample of neuronal tissue or CSF from an individual and using standard assays to determine gene expression or activation.

[0059] Similarly, in mice and other non-human animals, cognitive ability can be tested with measures of executive function (working memory, attention, processing speed, set shifting), visiospatial learning and memory, object memory, pattern recognition, fear memory, passive avoidance memory, habituation, and novel object recognition, for example. Common tests include but are not limited to the Morris water maze, Barnes maze, radial arm water maze, y- maze, T-maze, and open field habituation. Brain imaging techniques are similarly applicable.

[0060] In some embodiments, a GPX3 polypeptide is administered to an individual to improve motor function. Accordingly, in some embodiments, a GPX3 polypeptide composition of the present invention is administered to a subject, e.g., a human subject, having impaired motor function for the treatment of the impaired motor function. For example, in some embodiments, the subject has stroke to the brain or spinal cord (ischemic or hemorrhagic), neurodegenerative disease (Parkinson’s disease, Lewy body dementia, multiple system atrophy, amyotropic lateral sclerosis, prion disorder, Huntington’s disease, supranuclear palsy), Parkinsonism, traumatic brain injury, neuroinfectious brain lesions, multiple sclerosis and related autoimmune and demyelinating disease, spinal cord lesions (compressive, infectious, toxic or metabolic, autoimmune , oncologic), brain tumor, epilepsy, paraneoplastic disorder, neurodevel opmental disorder (mitochondrial, autosomal genetic), muscle disease (polymyositis, dermatomyositis, inclusion body myositis, infectious, endocrine, metabolic, toxic, congenital myopathy, congential muscular dystrophy, hereditary), neuropathies (Guillain-Barre syndrome, axonl and demyelinating, diabetic, toxic, metabolic, infectious, critical illness, entrapment), tick paralysis, myasthenia gravis, and spinal muscular atrophy. Changes in motor function can be assayed as known in the art. Illustrative motor function assays include but are not limited to electromyogram and nerve conduction studies, direct or device-assisted clinical testing of strength, tone, and muscle bulk, reflex examination, coordination examination, and gait analysis. Assays for testing etiologies causing deficits of motor function include but are not limited to magnetic resonance imaging of the central nervous system, muscle biopsy, nerve biopsy, and laboratory studies.Administration of GPX3 polypeptides17KILPATRICK TOWNSEND 78793839 1

[0061] Provided herein are methods of improving cognitive function and / or improving impaired motor function in an individual comprising administering an effective amount of a GPX3 polypeptide to the individual. In some embodiments, the method of treatment comprises administering to an individual an effective amount of a GPX3 polypeptide or a catalytically- functional variant or catalytically-functional fragment thereof. In some embodiments, the treatment is prophylactic, e.g., to prevent cognitive decline associated with aging. In some embodiments, the individual has been diagnosed with a cognitive disorder. In some embodiments, the individual is receiving or has received therapy for a cognitive disorder or for a condition that is related to cognitive function e.g., cognitive decline in response to chemotherapy). In some embodiments, a GPX3 polypeptide is administered to an individual that has impaired motor function.

[0062] In some embodiments, the method further comprises monitoring the individual for cognitive ability, either through a molecular proxy (e.g., changes in NMDA receptor or c-fos activation, or GluN2B levels in the brain, changes in MRI brain scans (e.g., functional MRI), changes in EEG, changes in TMS and TES, changes in neuropsychologic test scores, or tests of cognitive ability (e.g., for learning, short or long term memory, executive functions, language ability, and visuospatial function). In some embodiments, the individual is monitored using more than one of the above tests in any combination. In some embodiments, the dose of the GPX3 polypeptide for each administration is determined based on the therapeutic progress of the individual, e.g., where a higher dose is administered if the individual is not responding sufficiently to therapy.

[0063] In some embodiments, the GPX3 polypeptide is administered in a pharmaceutical composition with a physiologically (i.e., pharmaceutically) acceptable carrier. The term “carrier” refers to a typically inert substance used as a diluent or vehicle for a diagnostic or therapeutic agent. The term also encompasses a typically inert substance that imparts cohesive qualities to the composition. Physiologically acceptable carriers can be liquid, e.g., physiological saline, phosphate buffer, normal buffered saline (135-150 mM NaCl), water, buffered water, 0.4% saline, 0.3% glycine, glycoproteins to provide enhanced stability (e.g., albumin, lipoprotein, globulin, etc.), and the like. Since physiologically acceptable carriers are determined in part by the particular composition being administered as well as by the particular18KILPATRICK TOWNSEND 78793839 1method used to administer the composition, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (See, e.g., Remington's Pharmaceutical Sciences, 17thed., 1989).

[0064] The presently described compositions can be sterilized by conventional, well-known sterilization techniques or may be produced under sterile conditions. Aqueous solutions can be packaged for use or fdtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. Sugars can also be included for stabilizing the compositions, such as a stabilizer for lyophilized antibody compositions.

[0065] Dosage forms can be prepared for mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g., subcutaneous, intravenous, intramuscular, or intraarterial injection, either bolus or infusion), oral, or transdermal administration to a patient. Examples of dosage forms include, but are not limited to: dispersions; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.

[0066] Injectable compositions can comprise a solution of the GPX3 polypeptide suspended in an acceptable carrier, such as an aqueous carrier. Any of a variety of aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.9% isotonic saline, 0.3% glycine, 5% dextrose, and the like, and may include glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. In some embodiments, normal buffered saline (135-150 mM NaCl) is used. The compositions can contain pharmaceutically acceptable auxiliary substances to approximate19KILPATRICK TOWNSEND 78793839 1physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

[0067] Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Injection solutions and suspensions can also be prepared from sterile powders, granules, and tablets. In some embodiments, the composition is administered by intravenous infusion, topically, intraperitoneally, intravesically, or intrathecally. The GPX3 polypeptide formulation can be provided in unit-dose or multi-dose sealed containers, such as ampoules and vials.

[0068] The GPX3 polypeptide composition, alone or in combination with other suitable components, can be made into aerosol formulations (“nebulized”) to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, and nitrogen.

[0069] The pharmaceutical preparation can be packaged or prepared in unit dosage form. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component, e.g., according to the dose of GPX3 polypeptide. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation. The composition can, if desired, also contain other compatible therapeutic agents. In some embodiments, the GPX3 polypeptide composition can be formulated in a kit for administration.

[0070] In some embodiments, a pharmaceutical composition comprising a GPX3 polypeptide is administered orally. In some embodiments, a pharmaceutical composition comprising a GPX3 polypeptide is administered mucosally, e.g., nasally. In some embodiments, a pharmaceutical composition comprising a GPX3 polypeptide is administered by injection, e.g., subcutaneous, intraperitoneal, intravenous, or intramuscular. In some embodiments, a pharmaceutical20KILPATRICK TOWNSEND 78793839 1composition comprising a GPX3 polypeptide is administered by infusion, e.g., using a reservoir or osmotic minipump.

[0071] An example of administration of a pharmaceutical composition includes storing the GPX3 polypeptide at 10 mg / ml in sterile isotonic aqueous saline solution at 4°C, and diluting it in an appropriate solution for injection prior to administration to the patient. In some embodiments, the GPX3 polypeptide composition can be administered by intravenous infusion over the course of 0.25-2 hours. In some embodiments, the administration procedure is via bolus injection.

[0072] In therapeutic use, the GPX3 polypeptide can be administered at the initial dosage of about 0.1 pg / kg to about 1000 pg / kg daily and adjusted over time. A daily dose range of about 1 pg / kg to about 500 pg / kg, or about 10 pg / kg to about 100 pg / kg, or about 30 pg / kg to about 50 ug / kg can be used. The dosage is varied depending upon the requirements of the patient, the severity of the condition being treated, and the route of administration. For example, for injection of GPX3 polypeptide, the effective dose can be in the range of 10-100 pg / kg, while for direct delivery to the central nervous system (CNS), the effective dosage is lower, e.g., 5-30 pg / kg. For oral administration, the effective dose is higher, e.g., in the range of 50-10,000 pg / kg (e.g., 100pg / kg-2mg / kg). The dose is chosen in order to provide effective therapy for the patient. The dose may be repeated at an appropriate frequency which may be in the range of once or twice per day, once or twice per week to once every three months, depending on the pharmacokinetics of the GPX3 polypeptide composition (e.g., half-life in the circulation) and the pharmacodynamic response (e.g., the duration of the therapeutic effect).

[0073] Administration can be periodic. Depending on the route of administration, the dose can be administered, e.g., once every 1, 3, 5, 7, 10, 14, 21, or 28 days or longer (e.g., once every 2, 3, 4, or 6 months). In some cases, administration is more frequent, e.g., 2 or 3 times per day. The patient can be monitored to adjust the dosage and frequency of administration depending on therapeutic progress and any adverse side effects, as will be recognized by one of skill in the art.

[0074] Dosages can be empirically determined considering the type and severity of cognitive condition diagnosed in a particular patient. The dose administered to a patient, in the context of the present disclosure, should be sufficient to affect a beneficial therapeutic response in the21KILPATRICK TOWNSEND 78793839 1patient over time. The size of the dose will also be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of any particular composition in a particular patient, as will be recognized by the skilled practitioner.

[0075] Thus in some embodiments, additional administration is dependent on patient progress, e.g., the patient is monitored between administrations. For example, after the first administration or round of administrations, the patient can be monitored for cognitive ability or for side effects, e.g., weakness, dizziness, nausea, etc.

[0076] In some embodiments, the individual has a chronic condition, so that GPX3 is administered over an indefinite period, e.g., for the lifetime of the patient. In such cases, administration is typically periodic. Diseases that are considered long-term or chronic include, but are not limited to Alzheimer’s disease, and cognitive decline associated with other diseases, including Parkinson’s disease, Huntington’s disease, and chronic conditions such as with hypertension and heart disease.

[0077] In some embodiments, the GPX3 polypeptide is linked to a stabilizing moiety such as PEG, glycosylation, or a liposome or other nanocarrier. US Patent Nos. 4,732,863 and 7892554 and Chattopadhyay et al. (2010) Afo / Pharm 7:2194 describe methods for attaching a polypeptide to PEG, PEG derivatives, and nanoparticles (e.g., liposomes). Liposomes containing phosphatidyl-ethanolamine (PE) can be prepared by established procedures as described herein. The inclusion of PE provides an active functional site on the liposomal surface for attachment. In some embodiments, the GPX3 polypeptide is linked to an affinity tag, e.g., a histidine tag (e.g., 4-16 histidine residues), streptavidin, or an antibody target.

[0078] The GPX3 polypeptide can also be formulated as a sustained-release preparation (e.g., in a semi-permeable matrices of solid hydrophobic polymers (e.g., polyesters, hydrogels (for example, poly (2-hydroxyethyl-methacrylate), or poly (vinylalcohol)), polylactides. The GPX3 polypeptide can be entrapped in a nanoparticle prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions.22KILPATRICK TOWNSEND 78793839 1

[0079] In some embodiments, the GPX3 polypeptide is labeled, e.g., for tracking in the body or ex vivo. The GPX3 polypeptide can be labeled any diagnostic agent known in the art, as provided, for example, in the following references: Armstrong et aL, Diagnostic Imaging, 5thEd., Blackwell Publishing (2004); Torchilin, V. P., Ed., Targeted Delivery of Imaging Agents, CRC Press (1995); Vallabhajosula, S., Molecular Imaging: Radiopharmaceuticals for PET and SPECT, Springer (2009). The diagnostic agent can be detected by a variety of ways, including as an agent providing and / or enhancing a detectable signal. Detectable signals include, but are not limited to, gamma-emitting, radioactive, echogenic, optical, fluorescent, absorptive, magnetic, or tomography signals. Techniques for imaging the diagnostic agent can include, but are not limited to, single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, positron emission tomography (PET), computed tomography (CT), x-ray imaging, gamma ray imaging, and the like. The terms “detectable agent,” “detectable moiety,” “label,” “imaging agent,” and like terms are used synonymously herein.

[0080] In some embodiments, the label can include optical agents such as fluorescent agents, phosphorescent agents, chemiluminescent agents, and the like. Numerous agents (e.g., dyes, probes, labels, or indicators) are known in the art and can be used in the present invention. (See, e.g., Invitrogen, The Handbook — A Guide to Fluorescent Probes and Labeling Technologies, Tenth Edition (2005)). Fluorescent agents can include a variety of organic and / or inorganic small molecules or a variety of fluorescent proteins and derivatives thereof. For example, fluorescent agents can include but are not limited to cyanines, phthalocyanines, porphyrins, indocyanines, rhodamines, phenoxazines, phenylxanthenes, phenothiazines, phenoselenazines, fluoresceins, benzoporphyrins, squaraines, dipyrrolo pyrimidones, tetracenes, quinolines, pyrazines, corrins, croconiums, acridones, phenanthridines, rhodamines, acridines, anthraquinones, chalcogenopyrylium analogues, chlorins, naphthalocyanines, methine dyes, indolenium dyes, azo compounds, azulenes, azaazulenes, triphenyl methane dyes, indoles, benzoindoles, indocarbocyanines, benzoindocarbocyanines, and BODIPY™ derivatives.Fluorescent dyes are discussed, for example, in U.S. Pat. No. 4,452,720, U.S. Pat. No. 5,227,487, and U.S. Pat. No. 5,543,295.

[0081] The label can also be a radioisotope, e.g., radionuclides that emit gamma rays, positrons, beta and alpha particles, and X-rays. Suitable radionuclides include but are not limited23KILPATRICK TOWNSEND 78793839 1to225Ac,72As,211At, "B,128Ba,212Bi,75Br,77Br,14C,109Cd,62Cu,64Cu,67Cu,18F,67Ga,68Ga,3H,166HO,123I,124I,125I,130I,131I, In,177LU,13N,150,32P,33P,212Pb,103Pd,186Re,188Re,47Sc,153Sm,89Sr, "mTc,88Y and90Y. In some embodiments, radioactive agents can includemIn- DTPA,99mTc(CO)3-DTPA,99mTc(CO)3-ENPy2,62 / 64 / 67Cu-TETA,99raTc(CO)3-IDA, and "mTc(CO)3triamines (cyclic or linear). In some embodiments, the agents can include DOTA and its various analogs withi nIn,177Lu,153Sm,88 / 90Y,62 / 64 / 67CU, or67 / 68Ga. In some embodiments, a nanoparticle can be labeled by incorporation of lipids attached to chelates, such as DTPA-lipid, as provided in the following references: Phillips et al. , Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, 1(1): 69-83 (2008); Torchilin, V.P. & Weissig, V., Eds. Liposomes 2nd Ed. '. Oxford Univ. Press (2003); Elbayoumi, T.A. & Torchilin, V.P., Eur. J. Nucl. Med. Mol. Imaging 33 : 1196-1205 (2006); Mougin-Degraef, M. et al., Int ’l J. Pharmaceutics 344: 110-117 (2007).

[0082] In some embodiments, the diagnostic agent can be associated with a secondary binding ligand or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and glucose oxidase. Secondary binding ligands include, e.g., biotin and avidin or streptavidin compounds as known in the art.Administration of GPX3 nucleic acids / Genetic manipulation

[0083] In certain embodiments, nucleic acids encoding a GPX3 polypeptide is used for enhancing expression of GPX3 in vivo. These nucleic acids can be inserted into any of a number of well-known vectors, including viral expression vectors or plasmid-based vectors for the transfection of target cells and organisms. The nucleic acids may be transfected into cells, e.g., hepatocytes, ex vivo or in vivo or administered in vivo.

[0084] In some embodiments, the nucleic acid constructs encoding a GPX3 polypeptide is administered as a purified nucleic acid molecule, for example, as a DNA plasmid-based vectors (“naked” DNA). Alternatively, in some embodiments, a nucleic acid construct encoding a GPX3 polypeptide may be contained within a viral vector and administered as a virus. Viral delivery systems include adenovirus vectors (e.g., Ad2, Ad5, Ad7), adeno-associated viral vectors, herpes simplex viral vectors, retroviral vectors, pox viral vectors (such as vaccinia and avian poxvirus vectors, such as the fowlpox and canarypox vectors), lentiviral vectors, alphavirus vectors,24KILPATRICK TOWNSEND 78793839 1poliovirus vectors, and other positive and negative stranded RNA viruses, viroids, and virusoids, or portions thereof. Methods of constructing and using such vectors are well known in the art.

[0085] Nucleic acids for administration to a subject are formulated for pharmaceutical administration. While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. For parenteral administration, including intranasal, intradermal, subcutaneous or intramuscular injection or electroporation, the carrier may comprises water, saline, and optionally an alcohol, a fat, a polymer, a wax, one or more stabilizing amino acids or a buffer. General formulation technologies are known to those of skill in the art.

[0086] DNA immunogenic compositions can be administered once or multiple times as needed to induce the desired response (e.g., increased circulating levels of GPX3). Multiple administrations can be administered, for example, bi-weekly, weekly, bi-monthly, monthly, or more or less often, as needed, for a time period sufficient to achieve the desired response.

[0087] Nucleic acids are administered by methods well known in the art, e.g., injection, electroporation, and the like. Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular or subcutaneous routes. In some embodiments, nucleic acids are introduced into cells, e.g., ex vivo and the cells are then introduced into the individual.

[0088] Nucleic acids can be administered in solution (e.g., a phosphate-buffered saline solution) by injection, usually by an intravenous, subcutaneous or intramuscular route. Dosages depend on the route of administration and can be readily determined by one of skill in the art.

[0089] In some embodiments, cells of an individual are genetically modified using modification techniques, such as CRISPR / CAS to increase circulating levels of GPX3 polypeptides.

[0090] In some embodiments, the GPX3 polypeptide or a polynucleotide encoding the GPX3 polypeptide, or a vector comprising the polynucleotide is delivered as part of or within a cell delivery system. Various delivery systems are known and can be used to administer a composition of the present disclosure, for example, encapsulation in liposomes, microparticles, microcapsules, or receptor-mediated delivery.25KILPATRICK TOWNSEND 78793839 1

[0091] Exemplary liposomal delivery methodologies are described in Metselaar et al., Mini Rev. Med. Chem. 2(4):319-29 (2002); O'Hagen et al. , Expert Rev. Vaccines 2(2):269-83 (2003); O'Hagan, Curr. Drug Targets Infect. Disord. l(3):273-86 (2001); Zho et al., Biosci Rep. 22(2):355-69 (2002); Chikh el al., Biosci Rep. 22(2):339-53 (2002); Bungener et al., Biosci. Rep. 22(2):323-38 (2002); Park, Biosci Rep. 22(2):267-81 (2002); Ulrich, Biosci. Rep. 22(2): 129-50; Lofthouse, Adv. Drug Deliv. Rev. 54(6):863-70 (2002); Zhou et al., J. Inmunmunother . 25(4):289-303 (2002); Singh et al., Pharm Res. 19(6):715-28 (2002); Wong et al., Curr. Med. Chem. 8(9): 1123-36 (2001); and Zhou et al., Immunonmethods (3): 229-35 (1994).

[0092] Exemplary nanoparticle delivery methodologies, including gold, iron oxide, titanium, hydrogel, and calcium phosphate nanoparticle delivery methodologies, are described in Wagner and Bhaduri, Tissue Engineering 18(1): 1-14 (2012) (describing inorganic nanoparticles); Ding et al., Mol Ther e-pub (2014) (describing gold nanoparticles); Zhang et al., Langmuir 30(3): 839- 45 (2014) (describing titanium dioxide nanoparticles); Xie et al., Curr Pharm Biotechnol14( 10): 918-25 (2014) (describing biodegradable calcium phosphate nanoparticles); and Sizovs et al., J Am Chem Soc 136(l):234-40 (2014).EXAMPLES

[0093] Aging leads to a decline in cognitive function and is the primary risk factor for age- related neurodegenerative diseases, such as Alzheimer’s disease. Systemic interventions such as modifications in diet and nutrition can slow down and reverse signs of aging, including in the brain (Bieri et al. 2023, PMID:36646876). Calorie Restriction (CR) is one of the most effective and well-studied non-genetic, rejuvenating interventions. CR is a form of dietary restriction that requires a moderate reduction (10-40% without malnutrition) in overall daily calorie intake compared to ad libitum (AL) feeding. CR extends lifespan and healthspan in a wide range of organisms from yeasts to non-human primates. In the brain, chronic CR modifies neuronal function through increased synaptic plasticity and preventing loss of dendritic spine density. Due to the difficulty in long-term adherence to dietary interventions in the elderly, identifying factors in blood that confer the benefits of CR would provide a novel and more tenable alternative to reverse brain aging and treat age-related dementias, such as Alzheimer’s disease.26KILPATRICK TOWNSEND 78793839 1

[0094] We first assessed the direct effects of CR on reversing age-related cognitive decline. 22-month-old aged mice were subjected to a 30% reduction in daily caloric intake for 4 weeks and age-matched aged mice placed on AL diet served as control (Fig 1A). Hippocampal- dependent learning and memory was assessed using Novel Object Recognition (NOR) assay and Radial Arm Water Maze (RAWM). During NOR testing, aged mice that underwent late-onset, short-term CR spent significantly more time with a novel object relative to a familiar object, while AL mice showed no preference for the novel object (Fig. IB). Late-onset, short-term CR also improved RAWM performance with CR mice committing fewer errors than AL mice (Fig 1C.).

[0095] To assess transcriptional changes in the hippocampus, nuclei was isolated from the hippocampus of mice that underwent late-onset, short-term CR or maintained on AL diet to conduct single nucleus RNA-sequencing (snRNAseq). A total of 28 cell clusters were identified and populations were compared across treatment groups (Fig. ID). We detected prominent transcriptional changes in excitatory neuronal populations in the hippocampus of aged mice that underwent late-onset, short-term CR compared to control AL mice (Fig. IE). We found that transcriptional responses in the hippocampus following late-onset, short-term CR encoded in different cell types with a combination of upregulated and downregulated genes with DEGs largely unique to cell type (Fig. IF). Bioinformatics analysis in excitatory neuronal subpopulations (CAI and DG) and GO analysis of DEGs between late-onset, short-term CR and control AL groups identified neuronal and synaptic plasticity-related biological processes (Fig. 1G-J). We also conducted neurogenesis histology to validate changes in neuronal biological processes. Mice that underwent late onset, short-term CR had more MCM2+ DAPI+ cells in the hilus of the dentate gyrus than mice maintained on AL diet (Fig. IK).

[0096] To investigate the role of CR-induced blood factors in rejuvenating the aged brain, blood plasma was isolated from mice that underwent late-onset, short-term CR and AL diet. 22- month-old aged mice were then systemically administered blood plasma from CR mice or AL control mice eight times over 24 days and hippocampal-dependent learning and memory was assessed using NOR and RAWM (Fig. 2A). Mice that received blood plasma from mice that underwent late-onset, short-term CR spent significantly more time with the novel object than27KILPATRICK TOWNSEND 78793839 1familiar object for NOR and committed significantly fewer errors in RAWM compared to control mice that received blood plasma from aged mice maintained on AL diet (Fig 2B,C).

[0097] To assess transcriptional changes in the hippocampus, nuclei were isolated from the hippocampus of mice that that received CR or AL blood plasma to conduct snRNAseq. A total of 28 cell clusters were identified and populations were compared across treatment groups (Fig. 2D). We detected prominent transcriptional changes in inhibitory and excitatory neuronal populations in the hippocampus of aged mice that that received CR blood plasma compared to control mice that received AL blood plasma (Fig. 2E). We found that transcriptional responses in the hippocampus following systemic administration of late-onset, short-term CR blood plasma encoded in different cell types with a combination of upregulated and downregulated genes with DEGs largely unique to cell type (Fig. 2F). Bioinformatics analysis of inhibitory and excitatory neuronal subpopulations (CAI) and GO analysis of DEGs between late-onset, short-term CR and control AL groups identified neuronal, metabolomic, and synaptic plasticity-related biological processes (Fig. 2G-J). Focused analysis on conserved DEGs that change following both direct CR and CR blood plasma administration (Figure 2E) revealed that most of the conserved transcriptional responses were unique to cell type with inhibitory and excitatory neuronal populations (CAI) exhibiting largest changes (Figure 2K). We found that transcriptional responses in the hippocampus that are conserved between direct CR and CR blood plasma administrations encoded in different cell types with a combination of upregulated and downregulated genes with DEGs largely unique to cell type (Fig. 2F). GO analysis of DEGs identified synaptic plasticity- and neuronal-related biological processes (Figure 2M). We also conducted neurogenesis histology to validate changes in neuronal biological processes. Mice that received systemic administration of CR blood plasma had more MCM2+ DAPI+ cells in the hilus of the dentate gyrus than mice that received AL blood plasma (Fig. 2N). Together, these data indicate that CR blood plasma administration can partially recapitulate the cognitive benefits and transcriptomic signature observed with late-onset, short-term CR.

[0098] To begin identifying blood factors potentially responsible for the CR blood plasma rejuvenation of hippocampal function in aged mice we utilized a proteomic mass spectrometry approach to identify proteins elevated in the blood plasma of mice that underwent late-onset, short-term CR compared to mice maintained on AL diet (Fig. 3A). Among these potential CR-28KILPA TRICK TOWNSEND 78793839 1induced rejuvenating circulating factors is Glutathione Peroxidase 3 (GPX3). GPX3 is an enzyme that protects against oxidative stress through the detoxification of hydroperoxides - has been shown to decrease in abundance in the blood plasma of aged rats and humans with age (Razygraev et al. 2019, PMID:31228366). Western blot analysis revealed that GPX3 is elevated in the blood plasma of late-onset, short-term CR mice relative to mice maintained on AL diet.

[0099] To investigate the effects of circulating GPX3 on cognitive function, aged mice were given hydrodynamic tail vein injection (HDTVI) with expression constructs encoding GPX3 or GFP for control and hippocampal-dependent learning and memory was assessed using NOR, RAWM, and Y-Maze (Fig. 4A). Western blot analysis validated Increase in GPX3 in blood plasma of aged mice that received GPX3 construct compared to control GFP construct (Fig. 4B). Both GPX3 and control GFP mice spent significantly more time with the novel object compared to the familiar object for NOR (Fig. 4C); however, GPX3 mice committed fewer errors during the training and testing phase of RAWM and made more entries in the novel arm than trained arm for Y-Maze compared to GFP control mice.

[0100] To assess transcriptional changes in the hippocampus, nuclei were isolated from the hippocampus of mice that had systemic increase of GPX3 or GFP for control for snRNAseq. A total of 28 cell clusters were identified and populations were compared across treatment groups (Fig. 4F). We detected prominent transcriptional changes in excitatory neuronal populations in the hippocampus of aged mice that had systemic increase of GPX3 (Fig. 4G). We found that transcriptional responses in the hippocampus following systemic increase of GPX3 encoded in different cell types with a combination of upregulated and downregulated genes with DEGs largely unique to cell type (Fig. 4H). Bioinformatics analysis in excitatory neuronal subpopulations (CAI and DG) and GO analysis of DEGs between GPX3 and GFP control aged mice identified translation biological processes (Fig. 4I-L). We also conducted histology to assess changes in neurogenesis. Mice that had systemic increase of GPX3 had more MCM2+ DAPI+ cells in the hilus of the dentate gyrus than GFP control diet (Fig. 4M). Collectively, these data identify Gpx3 as a circulating blood-based CR mimetic sufficient to rejuvenate hippocampal and cognitive function in aged animals.

[0101] We analyzed human plasma samples from participants of the NIH CALERIE™ trial at the 12-month timepoint. Participants were stratified based on actual percent CR into isocaloric29KILPATRICK TOWNSEND 78793839 1(0% CR), moderate CR (7-12% CR), and high CR (13-35% CR) groups, as previously described (Bareja, A., et al. (2024). Cell Rep. 43, 113881-113881). We detected an increase in GPX3 levels in plasma from individuals in the high CR group compared to the isocaloric and moderate CR groups. These data identify GPX3 as a CR induced circulating blood factor in aged mice and humans with potential relevance to cognitive function across species.

[0102] CALERIE™ Subjects. The Comprehensive Assessment of the Long-term Effects of Reducing Intake of Energy (CALERIE)™ trial (clinical trial registry NCT00427193) randomized 220 healthy, non-obese (BMI 22.0 %BMI <28.0 kg / m2), adults aged 21-50 years to either a 25% caloric restriction (CR) intervention condition or ad libitum (AL) control at a 2: 1 (CR:AL) ratio across three sites (Pennington Biomedical Research Center, Washington University, and Tufts University)(Rickman et al., 2011; Rochon et al., 2011). Participants were excluded from the study if they had significant medical conditions, abnormal laboratory markers, present or potential psychiatric or behavioral problems, regular use of medications (except oral contraceptives), currently smoked, were highly physically active, or were pregnant or breastfeeding. Randomization was stratified by study site, sex and BMI. The trial duration was 24 months. Further information about the CALERIE trial can be found at calerie.duke.edu.Throughout this study, a subset of participants was used based on their tissue availability and / or percentage caloric restriction.

[0103] Antioxidant enzymatic activity is necessary for the cognitive benefits of GPX3 on the aged hippocampus. GPX3 protects against oxidative stress through the detoxification of hydroperoxides via a selenocysteine residue at its active site. Therefore, we next tested whether disrupting the enzymatic active site of systemic GPX3, and subsequent antioxidant activity, mitigated its cognitive benefits on the aged hippocampus. We generated expression constructs encoding mutant GPX3 with site-directed mutations in the active site and screened for decreased enzymatic activity in vivo following HDTVI. Previous reports have described Tryptophan at position 181 in the enzymatic active site of GPX3, and we observed that mutations converting Tryptophan to Alanine at this position abrogated the enzymatic activity of systemic GPX3 in plasma. Subsequently, old (23 months) mice were given HDTVI with expression constructs encoding GPX3, catalytically inactive mutant GPX3 (W181A) or GFP control and hippocampal- dependent learning and memory was assessed using RAWM, NOR and forced alternation Y30KILPATRICK TOWNSEND 78793839 1maze. Elevated levels of HiBiT tagged GPX3 and W181 A GPX3, as well as abrogation of enzymatic activity for W181 A GPX3, were confirmed in plasma following HDTVI. No difference in body weight or health metrics were observed between groups. Old mice with increased systemic GPX3 committed fewer errors locating a hidden platform and exhibited a bias for the novel object and the novel arm compared to control old mice. However, no cognitive benefits were observed in old mice with increased systemic catalytically dead W181A GPX3. These behavioral data indicate that the enzymatic activity of systemic GPX3 is necessary for its cognitive benefits on the aged hippocampus, and mechanistically highlight the detoxification of hydroperoxides as a mediator of these rejuvenating effects.

[0104] GPX Activity Assay. Activity of GPX3 and mutant forms of GPX3 (W181A, Q107A, U73C, U73A) were assessed using the Glutathione Peroxidase Assay Kit (Cat# 703102, Cayman Chemicals). When GPX reduces organic hydroperoxide, oxidized glutathione is produced. The oxidized glutathione is then reduced and recycled using NADPH. GPX activity is indirectly measured by the oxidation of NADPH to NADP+ following the treatment of hydroperoxide which is accompanied by a decrease in absorbance at 340nm.Gpx3 expression plasmid cloning strategy

[0105] The murine Gpx3 coding sequence (CDS) and ~700bp of the adjacent 3’ UTR (including the SECIS element required for the insertion of the seleno-Cystein at the amino acid position *73) were PCR amplified from an adult mouse kidney cDNA library. During a second round of PCR amplification, the restriction sites Nhel and EcoRI as well as a Kozak sequence were added to the CDS and 3 ’UTR. Subsequently, Gpx3 CDS and its 3 ’UTR were inserted into an expression plasmid containing a constitutive CMV promoter and an IRES eGFP reporter using traditional restriction enzyme digestion and ligation. Plasmid sequences were validated using whole plasmid sequencing and amplified using endotoxin free maxi prep kits.

[0106] A constitutive expression plasmid was generated containing a CMV promoter and an IRES eGFP reporter. The murine Gpx3 coding sequence as well as 700nt of its 3 ’UTR containing the SECIS element, were PCR amplified from a mouse kidney cDNA library and cloned into the plasmid backbone using traditional PCR amplification and restriction enzymebased ligation approaches.31KILPATRICK TOWNSEND 78793839 1

[0107] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.32KILPATRICK TOWNSEND 78793839 1SEQUENCES:SEQ ID NO: 1 Human GPX3 protein:MARLLQASCLLSLLLAGFVSQSRGQEKSKMDCHGGISGTIYEYGALTIDGEEYIPFKQYA GKYVLFVNVASYUGLTGQYIELNALQEELAPFGLVILGFPCNQFGKQEPGENSEILPTLK YVRPGGGFVPNFQLFEKGDVNGEKEQKFYTFLKNSCPPTSELLGTSDRLFWEPMKVHDI RWNFEKFLVGPDGIPIMRWHHRTTVSNVKMDILSYMRRQAALGVKRK, where the underlined “U” is selenocysteine.33KILPATRICK TOWNSEND 78793839 1

Claims

WHAT IS CLAIMED IS:

1. A method of treating or preventing age-related cognitive dysfunction in an individual in need thereof, the method comprising administering to the individual an effective amount of a Glutathione Peroxidase 3 (GPX3) polypeptide that is administered systemically or locally to the brain, thereby treating or preventing age-related cognitive dysfunction.

2. The method of claim 1, wherein the GPX3 polypeptide is administered systemically.

3. The method of claim 1, wherein the GPX3 polypeptide is administered by intravenous, intraperitoneal, subcutaneous, or intramuscular injection.

4. The method of claim 1, wherein the GPX3 polypeptide is administered orally or mucosally.

5. The method of any one of claims 1 to 4, wherein the individual is a human.

6. The method of claim 5, wherein the human is at least 50 years old.

7. The method of claim 5, wherein the individual has mild cognitive impairment.

8. The method of claim 5, wherein the individual has dementia.

9. The method of claim 5, wherein the individual has Alzheimer’s disease.

10. The method of any one of claims 1 to 9, wherein the GPX3 polypeptide has catalytic activity and comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO: 1.

11. The method of any one of the foregoing claims, wherein the effective amount is 1 pg to 1000 pg per kg body weight of the individual.34KILPATRICK TOWNSEND 78793839 112. The method of any one of the foregoing claims, wherein the GPX3 polypeptide or a functional fragment thereof is administered more than once as part of a course of treatment.

13. The method of any one of the foregoing claims, further comprising testing the cognitive function of the individual after administering.

14. The method of claim 13, further comprising testing the cognitive function of the individual prior to administering, and comparing the cognitive function of the individual prior to and after administering.

15. The method of claim 13 or 14, wherein cognitive function is determined by testing the individual for semantic, episodic, procedural, priming, and / or working memory.

16. The method of claim 13 or 14, wherein cognitive function is determined by testing the individual for language ability, executive function, visuospatial function, or dementia.

17. A method of treating or preventing age-related cognitive dysfunction in an individual in need thereof, the method comprising introducing a nucleic acid construct encoding a GPX3 polypeptide into the individual such that the GPX3 polypeptide is expressed in the individual.

18. A method of treating or preventing age-related cognitive dysfunction in an individual in need thereof, the method comprising genetically modifying cells of an individual to enhance expression of a GPX3 polypeptide into the individual.

19. The method of claim 17 or 18, wherein cells are obtained from the individual, the cells are modified ex vivo, and then administered to the individual.

20. The method of any one of claims 17 to 19, wherein the individual has mild cognitive impairment.

21. The method of any one of claims 17 to 19, wherein the individual has dementia.35KILPATRICK TOWNSEND 78793839 122. The method of any one of claims 17 to 19, wherein the individual hasAlzheimer’s disease.36KILPATRICK TOWNSEND 78793839 1

Images