WO2019164876A1 - Therapeutics that invoke epigenetic changes for use in treating neurological conditions such as cognitive impairment - Google Patents

Abstract

It has been discovered that increasing TET activity to catalyze demethylation of 5 methylcytosine rescues age-related regenerative decline and enhances cognitive function. TET activity can be increased in neurological tissue using an expression vector that encodes a TET enzyme. Alternatively or in addition, oligonucleotides exemplified by decoys, antagomirs, and blockmirs, or small molecule drugs can be administered to increase expression or activity of endogenous TET. This technology can be used for improving neurological function or preventing neurological decline in a subject, for example, in the treatment of cognitive disorders and other neurological conditions.

Description

THERAPEUTICS THAT INVOKE EPIGENETIC CHANGES

FOR USE IN TREATING NEUROLOGICAL CONDITIONS SUCH AS COGNITIVE IMPAIRMENT

REFERENCE TO PREVIOUS APPLICATION

[0001] This application claims the priority benefit of U.S. provisional patent application 62/633,041, filed February 20, 2018. Said priority application is hereby incorporated herein by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[0002] This invention draws and synthesizes information from two different fields: the treatment of neurological conditions such as cognitive impairment, and the role of certain enzymes in epigenetic control and reprogramming of DNA expression. Gene products that affect epigenetics such as TET2 can help prevent neurological decline.

BACKGROUND

[0003] DNA methylation is an epigenetic mechanism that regulates gene expression by recruiting proteins involved in gene repression or by inhibiting the binding of transcription factors to DNA. DNA methylation occurs via the transfer of a methyl group onto the C5 position of cytosine to form 5-methylcytosine. During development, the pattern of DNA methylation in the genome changes as a result of a dynamic process involving both de novo DNA methylation and demethylation. As a consequence, differentiated cells develop a stable and unique DNA methylation pattern that regulates tissue-specific gene transcription. DNA methylation in general tends to inhibit gene expression, and mounting evidence has correlated alterations in DNA methylation state with aging and age-related diseases. Post mitotic neurons express DNA methyltransferases and components involved in DNA demethylation. Neuronal activity can modulate DNA methylation patterns in response to physiological and environmental stimuli.

[0004] Precise regulation of DNA methylation is essential for normal function of the nervous system. SUMMARY OF THE INVENTION

[0005] It has been discovered that increasing enzymatic activity that catalyzes demethylation of 5-methylcytosine rescues age-related regenerative decline and enhances cognitive function. TET (tet me thy Icy to sine dioxygenase) activity can be increased in neurological tissue using an expression vector that encodes an exogenous TET enzyme. Alternatively or in addition, oligonucleotides exemplified by decoys, antagomirs, and blockmirs, or small molecule drugs can be administered to increase expression or activity of endogenous TET. This technology can be used for improving neurological function or preventing neurological decline in a subject, for example, in the treatment of cognitive disorders and other neurological conditions.

[0006] Aspects of the invention are put forth in the following description, the accompanying drawings, and the appended claims.

DRAWINGS

[0007] FIG. 1 illustrates several new observations that provide a mechanistic framework that stands behind this invention. First, loss of TET2 and 5-hydroxymethylcytosine (5hmC) activity in the aged hippocampus associates with regenerative decline. Second, decreasing TET2 activity in the young mouse hippocampus impairs neurogenesis and cognition. Third, heterochronic parabiosis restores TET2 activity in the aged hippocampus. Fourth, restoring TET2 activity in the mature hippocampus rescues neurogenesis and enhances cognition.

[0008] FIG.2A is a sketch showing that the epigenetic clock correlates strongly with the chronological age of a person and the level of DNA methylation of his or her genome. FIG. 2B shows the effect of induced pluripotent stem cell (iPSC) reprogramming (circled dots). The data suggest that the epigenetic clock can be reset to a pre-birth state regardless of the age of the cell’ s donor.

[0009] FIG. 3 shows that the gain, loss and maintenance of the methyl marks on cytosine in a CpG context is the result of the relative contribution of three interconnected pathways.

[0010] FIGS. 4 A to 41 show that TET2 expression and 5hmC levels decline in the aged hippocampus and are associated with neurogenic processes.

[0011] FIGS. 5A to 51 provide a characterization of 5mC levels, hMeDIP-sequencing, and adult neurogenesis in the young and aged hippocampus.

[0012] FIGS. 6 A to 6F show abrogation of TET2 by lentiviral-mediated shRNA and 5hmC expression during neuronal differentiation.

[0013] FIG. 7A to 7D show that abrogation of TET2 in the adult dentate gyrus, or loss of TET2 in adult NPCs, impairs hippocampal neurogenesis.

[0014] FIG. 8 A to 8G show that abrogation of TET2 in the adult dentate gyrus, or loss of TET2 in adult NPCs, impairs hippocampal-dependent cognitive function. [0015] FIGS. 9 A to 9F show that abrogation of TET2 in the adult dentate gyrus, or loss of TET2 in adult NPCs, selectively impairs hippocampal-dependent cognitive function.

[0016] FIGS. 10A to 101 show that increasing TET2 in the mature adult dentate gyms rescues age-related regenerative decline.

[0017] FIG. 11 A to 1 IK provide a characterization of hMeDIP-sequencing in the mature hippocampus, and adult neurogenesis in the young hippocampus, following TET2 overexpression.

[0018] FIGS. 12 A to 12C provide data from an experiment in which lentivirus (FV) encoding TET2 was injected into the dentate gyms. Cells were labeled for expression of Doublecortin (DCX) and with DAPI. TET2 overexpression resulted in a significant increase in DCX positive cells per dentate gyms, compared with control tissue.

DETAILED DESCRIPTION

[0019] The data provided in this disclosure show that the epigenetic regulator ten eleven translocation methylcytosine dioxygenase 2 (TET2), which catalyzes the production of

5-hydroxymethylcytosine (5hmC), rescues age-related decline in adult neurogenesis and enhances cognition. Mimicking an aged condition in young adults by abrogating TET2 expression within the hippocampal neurogenic niche, or in adult neural stem cells, decreased neurogenesis and impaired learning and memory.

[0020] The data further show in a heterochronic parabiosis model of neurogenic rejuvenation hippocampal TET2 expression was restored. Overexpressing TET2 in the hippocampal neurogenic niche of mature adults increased 5hmC associated with neurogenic processes, offset the precipitous age-related decline in neurogenesis, and enhanced learning and memory. The data identify TET2 as a novel epigenetic mechanism of neurogenic rejuvenation.

[0021] This invention provides various ways to achieve targeted DNA demethylation in a temporally and spatially restricted manner suitable for therapeutic use. These include viral vector mediated and non-viral delivery of polynucleotides encoding one or more members of the TET protein family; and/or oligonucleotides that target sites that sequester or block endogenous miRNA negative regulators of TET gene expression. Using this technology, the clinician can temporally and spatially control DNA demethylation using cell specific promotors/enhancers, inducible expression systems, destabilized domain fusion proteins, and miRNA target sites incorporated in the UTR of target proteins.

[0022] The polynucleotides of this invention can be packaged into viral vectors containing specific capsid mutations for enhanced transduction of target cells as well as non-viral based gene delivery vehicles for in vivo expression in the central and peripheral nervous system. Role of epigenetic changes in modulating gene expression

[0023] In the mammalian genome, DNA methylation is an epigenetic mechanism involving the transfer of a methyl group onto the C5 position of the cytosine to form 5-methylcytosine. DNA methylation regulates gene expression by recruiting proteins involved in gene repression or by inhibiting the binding of transcription factor(s) to DNA. During development, the pattern of DNA methylation in the genome changes as a result of a dynamic process involving both de novo DNA methylation and demethylation. As a consequence, differentiated cells develop a stable and unique DNA methylation pattern that regulates tissue-specific gene transcription

[0024] Postmitotic neurons still express DNA methyltransferases and components involved in DNA demethylation. Moreover, neuronal activity can modulate their pattern of DNA methylation in response to physiological and environmental stimuli. The precise regulation of DNA methylation is essential for normal cognitive function. Indeed, when DNA methylation is altered as a result of developmental mutations or environmental risk factors, such as drug exposure and neural injury, mental impairment is a common side effect. See L.D. Moore et ah, Neuropsychopharmacology., 2013 Jan; 38(1): 23-38; K. Buiting, Am J Med Genet C Semin Med Genet. 2010 Aug

15;154C(3):365-76

[0025] Of the roughly 100 epigenetic enzymatic regulators that exist, most modify chromatin components (histones). Only a small subset of these epigenetic regulators is involved in direct DNA modification by adding, maintaining, or removing mC (methyl-cytosine). The gain, maintenance, or loss of the methyl marks on cytosine in a CpG context is the result of the relative contribution of three interconnected pathways (FIG. 2A): 1) acquisition of new methylation marks by de novo methylation {de novo DNA methyltransferases DNMT3A and DNMT3B in combination with DNMT3L establish a pattern of methylation); 2) maintenance of existing methylation patterns across DNA replication by the maintenance methyltransferase DNMT1; and 3) active replication-independent removal of DNA methylation by the DNA ten-eleven translocation (TET) proteins.

Ten-eleven translocation (TET) enzymes and DNA demethylation

[0026] Ten-eleven translocation (TET) enzymes are named for a common translocation in cancer where translocation can occur between chromosomes 10 and 11, creating a MLL-TET1 fusion protein (Lorsbach et ah, 2003). TET proteins catalyze the demethylation of 5-methylcytosine (5mC), which is key to both developmental and diseased states. Because methylation generally inhibits gene expression, TET demethylases oppose this effect by allowing the re-expression of silenced genes. Three TET enzymes (TET1, TET2, and TET3) exist in mammals which add a hydroxyl group onto the methyl group of 5mC to form 5-hydroxymethylcytosine (5hmC). [0027] TET proteins are iron(II) and a-ketoglutarate dependent dioxygenases, and their enzymatic activity involves hydroxylation of 5-methylcytosine to 5-hydroxymethylcytosine and further to 5-formylcytosine and 5-carboxylcytosine. These modified cytosines are then removed by enzymes involved in DNA repair. The expression levels and cell-type distribution differ for the TET enzymes. TET1/2 are found in embryonic stem cells, while TET3 is found in the germ line (Ito et al., 2010). The significance of these differences, and the mechanisms of TET regulation remains unknown. Active DNA demethylation of paternal chromosomes is an important part of the early development of the fertilized zygote, and TET-mediated DNA demethylation is likely to be critical for this process.

5hmC is highly enriched in neurons in the developed brain

[0028] The developed brain contains significant 5hmC levels in multiple regions, ranging from 0.3 to 0.7%, which is approximately tenfold lower than the average abundance of

5-hydroxymethylcytosine (5hmC). Once 5hmC is formed, two separate mechanisms can convert 5hmC back into cytosine in mammals. In the first, iterative oxidation by TET enzymes continues to oxidize 5hmC first to 5-formyl-cytosine and then to 5-carboxy-cytosine. In the second, 5hmC is deaminated by AID/APOBEC to form 5-hydroxymethyl-uracil. Consistent with the role of TET in converting 5mC into 5hmC in vivo, TET1 knockout mouse embryonic stem cells have reduced levels of 5hmC that is accompanied by a subtle increase in 5mC at a global level.

[0029] 5hmC not only serves as an intermediate of DNA demethylation, but can also perform as a stable epigenetic marker. 5hmC is ~ 10-fold more enriched in neurons than other cell types, and it is acquired globally and exhibits dynamic features and region-specific patterns during postnatal development and aging of the neuronal system. 5hmC can be enriched in distinct genomic regions, such as gene bodies, promoters, and distal regulatory regions, and the enrichment of 5hmC at gene bodies could be positively correlated with transcriptional level, which might be achieved via interaction with histone modifications.

[0030] The pattern of DNA methylation established during development can be modulated by neural activity in order to encode learning and memory. When the mechanisms that establish and recognize the DNA methylation pattern are dysfunctional, problems with learning and memory frequently result. Global 5hmC and differentially hydro xymethylated regions (DhMRs) are altered in several neurodevelopmental diseases, including Rett syndrome, autism, and neurodegenerative diseases like Huntington’s disease and fragile X-associated tremor/ataxia syndrome (FXTAS). Rewinding the epigenetic clock via DNA demethylation

[0031] As shown in FIG. 2B, a direct link between epigenetics and aging exists, referred to as the eclock (FIG. 2). The eclock correlates the chronological age of a human with the level of DNA methylation of the genome. This points to a set of possible interventions to slow, stop, and possibly reverse aging or diseases associated with the aging process. One potential avenue of therapeutic intervention is via modulation of TET expression. An increased frequency of somatic TET mutations occurring with age are associated with elevated risk for aging-associated disorders such as cancer, cardiovascular disease, and stroke. Furthermore, TET proteins and 5hmC are highly expressed in the brain and have been implicated in proper brain function. FIG. 3 shows the effect of IPSc reprogramming (circled dots). This suggests that the eclock can be reset to a pre-birth state regardless of the age of the cell’s donors.

[0032] For most of their life span, mammals have high levels of methylation in almost all cells and tissues. In dividing cells, DNA methylation has to be considered a dynamic epigenetic mark even if constant levels of genomic methylation are maintained over long time spans. In fact, every cell replication carries the potential to lose half of all methylated CpGs, and cells need to actively methylate newly synthesized DNA in order to maintain methylation homeostasis over several cell divisions. DNA demethylation is carried out by members of the TET family of enzymes, which oppose the actions of the DNMT family.

[0033] Non-dividing cells in the body will not lose ^mC (methyl-cytosine) passively as this only occurs during cell division. In theory, inhibiting ^mC writers (DNMTs) should phenocopy the activation of erasers (TETs). But low or noncycling cells will not get demethylated in the presence of DNMT inhibitors. Thus, TET overexpression is preferred over DNMTs inhibition when it comes to changing (rejuvenating) the epigenetic landscape of cells in vivo. The importance of maintaining DNA methylation is illustrated by the findings that conditional deletion of Dnmtl in the developing mouse brain causes severe genomic hypomethylation and postnatal lethality.

[0034] The adult hippocampus is particularly susceptible to the effects of aging, with the number of neural stem/progenitor cells (NPCs), and subsequently neurogenesis, precipitously declining with age in the subgranular zone of the dentate gyms (DG) (Fan et ah, 2017).

Experimental observations

[0035] This disclosure includes the discovery that TET2 offsets age-related neurogenic decline in the adult hippocampus. As shown in the data presented below, an age-dependent decrease in the levels of TET2 and 5hmC in the aging hippocampus coincident with decreased adult neurogenesis. Mimicking an age-related loss of TET2 in the adult hippocampus, or adult NPCs, of young mice impairs regenerative capacity and associated hippocampal-dependent learning and memory processes. Conversely, increasing TET2 in the hippocampus of mature animals increases 5hmC associated with neurogenic processes, restores adult neurogenesis to youthful levels, and enhances cognitive function.

[0036] Age-related loss of TET2 leads to decreased adult neurogenesis, with functional implications for cognitive impairment. The in vivo RNA interference (RNAi) and genetic data dissect the involvement of TET2 in regulating adult neurogenesis at the level of the neurogenic niche and adult NPCs— pointing to complementary cell autonomous and nonautonomous roles for TET2 in regulating NPC function versus neuronal differentiation processes. Moreover, increasing TET2 in the hippocampus is sufficient to rescue the precipitous age-related regenerative decline observed in the mature adult brain and enhance associated cognitive function.

[0037] Cumulatively, these findings indicate that TET2-mediated hydroxymethylation regulates age related regenerative decline in the aging hippocampus, with functional implications for neurogenic rejuvenation. These observations provide a basis for rejuvenating neurogenic processes in the brain by restoring TET2 levels.

Polynucleotides encoding TET for increasing hydroxymethylation

[0038] Reconstituting or enhancing the expression of TET enzymes in target tissues that no longer have adequate TET activity can be accomplished using an expression vector that encodes any one of the TET family members (TET1, TET2, and TET3), exemplified but not limited to TET2.

[0039] The nucleotide sequence for Homo sapiens Tet methylcytosine dioxygenase 1 (TET1), mRNA is provided in GenBank as NCBI Reference Sequence: NM_030625.2. The nucleotide sequence for Homo sapiens Tet methylcytosine dioxygenase 2 (TET2), transcript variant 1, mRNA is provided in GenBank as NCBI Reference Sequence: NM_001127208.2. The nucleotide sequence for Homo sapiens Tet methylcytosine dioxygenase 2 (TET2), transcript variant 2, mRNA is provided in GenBank as NCBI Reference Sequence: NM_017628.4. The nucleotide sequence for Homo sapiens Tet methylcytosine dioxygenase 3 (TET3), mRNA is provided in GenBank as NCBI Reference Sequence: NM_001287491.1. Suitable for use in an expression vector of this invention is any of the aforelisted sequences and transcript variants thereof. Also suitable are nucleotide sequences that encode a fragment of the respective enzyme that retains enzymatic activity of the parent molecule, including variants that include minor changes in amino acid sequence that do not adversely affect enzymatic activity.

Knockdown and interference strategies for epigenetic reprogramming

[0040] The terms“siRNA” or“short interfering RNA” refer to a short polynucleotide sequence that mediates a process of sequence-specific post-transcriptional gene silencing, translational inhibition, transcriptional inhibition, or epigenetic RNAi in animals. An siRNA can be made up of a first strand and a second strand that have the same number of nucleosides; however, the first and second strands are offset such that the two terminal nucleosides on the first and second strands are not paired with a residue on the complimentary strand. In certain instances, the two nucleosides that are not paired are thymidine resides. The siRNA should include a region of sufficient homology to the target gene, and be of sufficient length in terms of nucleotides, such that the siRNA, or a fragment thereof, can mediate down regulation of the target gene.

[0041] Thus, an siRNA includes a region which is at least partially complementary to the target RNA. It is not necessary that there be perfect complementarity between the siRNA and the target, but the correspondence must be sufficient to enable the siRNA, or a cleavage product thereof, to direct sequence specific silencing, such as by RNAi cleavage of the target RNA. Complementarity, or degree of homology with the target strand, is most critical in the antisense strand. Perfect complementarity in the antisense strand is often desired but not necessarily required. The mismatches are most tolerated in the terminal regions, and if present are preferably in a terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides of the 5’ and/or 3’ terminus. The sense strand need only be sufficiently complementary with the antisense strand to maintain the overall double-strand character of the molecule.

[0042] ID AX (also known as CXXC4), a reported inhibitor of Wnt signaling that has been implicated in malignant renal cell carcinoma and colonic villous adenoma, regulates TET2 protein expression. ID AX was originally encoded within an ancestral TET2 gene that underwent a chromosomal gene inversion during evolution, thus separating the TET2 CXXC domain from the catalytic domain. The ID AX CXXC domain binds DNA sequences containing unmethylated CpG dinucleotides, localizes to promoters and CpG islands in genomic DNA and interacts directly with the catalytic domain of TET2. ID AX expression results in caspase activation and TET2 protein downregulation, in a manner that depends on DNA binding through the ID AX CXXC domain, suggesting that ID AX recruits TET2 to DNA before degradation. ID AX depletion prevents TET2 downregulation in differentiating mouse embryonic stem cells, and short hairpin RNA against ID AX increases TET2 protein expression in the human monocytic cell line U937 (Ko et al., Nature, 2013). Notably, the expression and activity of TET3 is also regulated through its CXXC domain. The invention may be practiced as a siRNA against ID AX, resulting in increased TET2 expression levels.

[0043] The terms“miRNA” and“microRNA” refer to small non-coding RNAs of 20-22 nucleotides, typically excised from -70 nucleotide fold back RNA precursor structures known as pre- miRNAs. miRNAs negatively regulate their targets in one of two ways depending on the degree of complementarity between the miRNA and the target. First, miRNAs that bind with perfect or nearly perfect complementarity to protein- coding mRNA sequences induce the RNA-mediated interference (RNAi) pathway. miRNAs that exert their regulatory effects by binding to imperfect complementary sites within the 3’ untranslated regions (UTRs) of their mRNA targets, repress target-gene expression post-transcriptionally, apparently at the level of translation, through a RISC complex that is similar to, or possibly identical with, the one that is used for the RNAi pathway. Consistent with translational control, miRNAs that use this mechanism reduce the protein levels of their target genes, but the mRNA levels of these genes are only minimally affected. miRNAs encompass both naturally occurring miRNAs as well as artificially designed miRNAs that can specifically target any mRNA sequence. For example, the skilled artisan can design short hairpin RNA constructs expressed as human miRNA (e.g., miR-30 or miR-21) primary transcripts or“mishRNA.” This design adds a Drosha processing site to the hairpin construct and has been shown to greatly increase knockdown efficiency (Pusch et al, 2004). The hairpin stem consists of 22-nt of dsRNA {e.g., antisense has perfect complementarity to desired target) and a 15-19-nt loop from a human miR. Adding the miR loop and miR30 flanking sequences on either or both sides of the hairpin results in greater than 10- fold increase in Drosha and Dicer processing of the expressed hairpins when compared with conventional shRNA designs without microRNA. Increased Drosha and Dicer processing translates into greater siRNA/miRNA production and greater potency for expressed hairpins.

[0044] The terms“shRNA” and“short hairpin RNA” refer to double- stranded structure that is formed by a single self-complementary RNA strand. shRNA constructs containing a nucleotide sequence identical to a portion, of either coding or non- coding sequence, of the target gene are preferred for inhibition. RNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the portion of the target gene is preferred. The length of the duplex-forming portion of an shRNA can be at least 20, 21 or 22 nucleotides in length, e.g., corresponding in size to RNA products produced by Dicer-dependent cleavage. The shRNA construct can be at least 25, 50, 100, 200, 300 or 400 bases in length. The shRNA construct can be 400-800 bases in length. shRNA constructs are typically tolerant of variation in loop sequence and loop size.

[0045] The term“ribozyme” refers to a catalytically active RNA molecule capable of site- specific cleavage of target mRNA. Several subtypes have been described, e.g., hammerhead and hairpin ribozymes. Ribozyme catalytic activity and stability can be improved by substituting deoxyribonucleotides for ribonucleotides at noncatalytic bases. While ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy particular mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA has the following sequence of two bases: 5-UG-3’ .

[0046] An expression cassette can contain one or more of a crRNA, a tracrRNA, sgRNA, an siRNA, an miRNA, an shRNA, or a ribozyme and further comprises one or more regulatory sequences, such as, for example, a strong constitutive RNA pol PI promoter, e.g., human or mouse U6 snRNA promoter, the human and mouse HI RNA promoter, or the human tRNA-val promoter; an inducible RNA pol III promoter, e.g., U6- 6TetO promoter, HI -peroxide promoter; or a strong constitutive or inducible RNA pol P promoter.

[0047] The polynucleotides contemplated herein, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as expression control sequences, regulatory elements, promoters and/or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Art sites), guide RNA target sites, termination codons, transcriptional termination signals, and polynucleotides encoding self cleaving polypeptides, epitope tags, such that their overall length may vary considerably. It is therefore contemplated that a polynucleotide fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. miRNA sponges, decoys, antagomirs. and blockmirs for upregulation of DNA demethylases

[0048] Vectors expressing miRNA target sites can be used to saturate an endogenous miRNA and prevent it from regulating its natural targets. This technology, which has been described using the terms decoy, sponge, eraser, antagomir, blockmir, and knockdown, has used a variety of gene delivery systems, including plasmids, and vectors based on adenoviruses, retroviruses and lentiviruses.

Saturating a highly expressed miRNA using a vector-based approach requires high expression levels of the target-containing transcript in a cell. Studies that quantified the concentration of target transcripts produced in cells in which miRNA inhibition was accomplished found that the number of target copies per cell were in several-fold excess of the miRNA copies per cell. Supraphysiological target expression can be achieved with high vector copy, strong promoters and stable transcripts. The most commonly used vector design places multiple miRNA target sites downstream of a reporter, such as GFP or luciferase, expressed from a strong promoter, usually of viral origin. The reporter can provide a useful readout of miRNA saturation as its accumulation indicates that the miRNA activity has been overwhelmed by an effective decoy vector. RNA polymerase III promoters, which are commonly used for expressing short hairpin RNAs, have also been used to express transcripts bearing miRNA target sites, and were found to mediate effective miRNA inhibition.

[0049] It is possible to introduce vectors that express decoys into many different species, including human cells. In addition, because many miRNAs are multi-copy genes or part of a miRNA family, the effect of knocking out a single miRNA gene may be compensated by the other miRNA genes or family members. However, miRNA decoy vectors express target sites that will be recognized by any miRNA with the same sequence, regardless of the allele, and by any member of the miRNA family. Targeting of an entire miRNA family occurs because all family members have the same seed sequence (nucleotides 2-8 of the miRNA), and thus will have a common recognition site in the artificial target.

[0050] Blockmirs are designed to have a sequence that is complementary to an mRNA sequence that serves as a binding site for microRNA. Upon binding, blockmirs sterically block microRNA from binding to the same site, which prevents the degradation of the target mRNA via RNA-induced silencing complex (RISC). If a blockmir binds to a non-intended RNA, it will only cause an effect if it prevents binding of a microRNA or another cellular factor. This occurrence is highly unlikely, meaning off-target effects will rarely be an issue.

[0051] Hence, blockmirs enable modulation of microRNA-based gene regulation with exquisite specificity. Importantly, blockmirs are typically agonists of their target mRNA, i.e. they increase the synthesis of the protein encoded by the target mRNA. Blockmirs bind on the 3’ end of the untranslated region (UTR) of the mRNA strand, which adequately blocks microRNA from binding, as most microRNAs do not bind to the translated region. The invention can be practiced as a polynucleotide encoding a blockmir complementary to the TET2 3’ UTR.

[0052] MicroRNA decoys, sponges, antagomirs, or blockmirs may exert their effect either transiently or stable, depending on the delivery system utilized. MicroRNA antisense oligonucleotide decoys, which include cholesterol-conjugated antagomirs, provide an effective way to transiently inhibit the activity of a miRNA and can be used in human cells. Because antisense oligonucleotides provide transient inhibition of miRNAs, this approach may be useful for providing pulsatile bursts of TET expression. When based on an integrating system such as a lentiviral vector, decoy vectors can stably antagonize miRNAs without requiring multiple administrations. When based on a non integrating system such as AAV, antagonism would occur transiently as the episomal payload is gradually lost during cell proliferation. Transient bursts of TET2 expression (whether via overexpression of TET2 or miRNA strategies) may be preferred from a safety standpoint given that overexpression of TET2 has been observed in B-cell lymphocytes from Chronic Lymphocytic Leukemia patients compared with healthy donors (Hernandez-Sanchez et al., BRS, 2014). In addition, in CLL patients, an overexpression of TET2 was also observed in the clonal B cells compared with the nontumoral cells. Because TET enzymes are implicated in multiple forms of hematological malignancies, using existing vector technology it is possible to obtain cell type-specific and inducible inhibition of miRNA function.

Expression control

[0053] Polynucleotide sequences encoding a TET enzyme, decoy, antagomir, blockmir, or other effector nucleic acid of this invention can be introduced into a target tissue by way of an expression system, wherein the encoding sequence is placed under control of regulatory elements needed for transcription, and if necessary, translation of the sequence. The encoding sequence is placed under control of a promoter that directs the binding of RNA polymerase and thereby promotes RNA synthesis. The promoter may operate constitutively at a high level of expression, or it may selectively cause expression in particular cells or tissues.

[0054] Ubiquitous expression control sequences that may be tested for use in the invention include a cytomegalovirus (CMV) immediate early promoter, chicken beta-actin (CAG), a viral simian virus 40 (SV40) (e.g., early or late), a Moloney murine leukemia vims (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, and a herpes simplex vims (HSV) (thymidine kinase) promoter. Promoters that cause selective expression on neurological tissues of one kind or another include a glial fibrillary acidic protein (GFAP) promoter (astrocyte expression), a synapsin-1 (Syn-1) promoter (neuron expression), and calcium/calmodulin-dependent protein kinase P (neuron expression), tubulin alpha I (neuron expression), neuron-specific enolase (neuron expression), platelet-derived growth factor beta chain (neuron expression), or an Advillin promoter (neuron expression).

[0055] The cell type specific promoter may be specific for cell types found in the brain (e.g., neurons, glial cells). The invention can be practiced using a neural progenitor cell promoter such as the early neuronal promoter for Talphal tubulin Talpha (Roy et ah, J Neurosci, 2000). Furthermore, the proteolipid protein (PLP) is expressed in NPCs and may also be practiced in the invention.

Promoter regions derived from adult neural stem cell (NSC) expressed genes may also be practiced including Doublecortin (DCX), Glioma-Associated Oncogene Family Zinc Finger 1 (Glil), Translocation-Associated Notch Protein TAN-1 (Notchl), SRY-Related HMG-Box Gene 1 (Soxl), Sex Determining Region Y-Box 2 (Sox2), Neuroectodermal stem cell marker (Nestin), Musashi RNA-binding protein 1 (Musahil), LewisX (LeX), Prominin 1 (CD133), Platelet Derived Growth Factor Receptor Alpha (PDGF-R), fibroblast growth factor receptor (FGF-R), Nuclear Receptor Subfamily 2 Group E Member 1 (NR2E1) as reviewed by Basak and Taylor (Basak and Taylor, Cell Mol Life Sci, 2009).

Conditional and inducible gene expression control systems

[0056] Expression vectors of this invention can place a nucleic acid sequence encoding a therapeutic agent in a context that requires a particular event or condition for the sequence to be expressed. In this disclosure, the term“conditional expression” refers to any type of conditional expression including, but not limited to, inducible expression; repressible expression; expression in cells or tissues having a particular physiological, biological, or disease state. This definition is not intended to exclude cell-type or tissue-specific expression. [0057] Illustrative examples of inducible promoters or systems include steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible by interferon), the“GeneS witch” mifepristone - regulatable system (Sirin et al, 2003, Gene, 323:67), the cumate inducible gene switch (WO

2002/088346), and tetracycline-dependent regulatory systems.

[0058] The invention may be practiced using a tetracycline-responsive element promoter (TRE2 or TRE3G) regulated by a class of transcription factors (e.g. tTA, rtTA, tTS, Tet3G) whose activities are dependent on tetracycline or its analogs (e.g. doxycycline) (Proc Natl Acad Sci U S A. 89:5547 (1992); Science. 268:1766 (1995); J Gene Med. 1 :4 (1999), Gene Ther. 13:1382 (2006). Optionally, the iDimerize Regulated Transcription System enables A/C heterodimerization using AP20187 or AP21967 ligand (Pomerantz, J. L., Sharp, P. A., and Pabo, C. O. (1995) Science 267(5194):93-96). Optionally, a RheoSwitch may be used to control expression of a gene of interest by the RheoSwitch receptor (a heterodimer of an engineered ecdysone receptor ligand binding domain fused to a GAL4 DNA binding domain (GAL4:EcR) and an RXR protein fused to a VP16 activation domain

(VP16:RXR)) only in the presence of Activator Drug. One version of the RheoSwitch is comprised of an ecdysone receptor (EcR) fused to a GAL4 DNA binding domain, a chimeric retinoid X receptor fused to a VP16 activation domain (pVP16-Hs-LmRXR), a reporter gene, such as human secreted alkaline phosphatase (hSEAP) placed under the control of a 6xGAL4 response element, and an inducible minimal promoter (p6xGAL4RE- Inducible Promoter- hSEAP).

Inducible control of epigenetic reprogramming through destabilization domains

[0059] Destabilization domain (DD) technology is a means for regulation of gene product at the protein level. In the DD approach, the gene of interest is coupled to a DD, which leads to degradation of the fused protein by the proteasome (Banaszynski et al., 2006; Sellmyer et al., 2009; Egeler et al., 2011). In the presence of a stabilizing compound, the DD changes its structure, leading to a stable fusion protein (Banaszynski et al., 2006; Egeler et al., 2011). The DD is based on the mutated protein versions of FK506 binding protein (ddFKBP; Banaszynski et al., 2006) and dihydrofolate reductase (ddDHFR; Iwamoto et al., 2010; Muralidharan et al., 2011). The stabilizing compound can be Shield- 1, rapamycin or FK506 for the ddFKBP system, and Trimethoprim (TMP) for the ddDHFR system. Recently, a new DD system was established based on the estrogen receptor with one of two synthetic ligands, CMP8 or 4-hydroxytamoxifen, as a stabilizing compound (Miyazaki et al., 2012).

[0060] The invention may be practiced using a small destabilizing domain (DD) fused onto the 3’ end of the TET2 polynucleotide, enabling the conditional expression of TET2 and its destruction when a shielding ligand is absent. The Shieldl ligand therefore reversibly stabilizes and destabilizes a DD-tagged protein of interest in a predictable and dose-dependent manner.

MicroRNAs (miRNAsl regulate neural stem cell proliferation.

[0061] In the brain, vital processes like neurodevelopment and neuronal functions depend on expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death.

[0062] miR-124 is the most abundant miRNA in the adult brain (Lagos-Quintana et ab, 2002).

During neurogenesis, miR-124 expression is undetectable or expressed at low levels in progenitor cells and is upregulated in differentiating and mature neurons (Deo et al., 2006). Temporal control of TET2 can be accomplished via insertion of four copies of the miR-124 target site into the 3’ UTR of the TET2 cDNA. As the progenitor cell differentiates, miR-124 expression would then silence expression of TET2. The invention can be practiced as a polynucleotide encoding TET2 with or multiple copies of the miR-124 target sequence in the 3’ UTR of the TET sequence.

[0063] miR-137 promotes proliferation and represses differentiation of neural stem cells via translational repression of Ezh2, a histone H3 lysine 27 methyltransferase and a member of the Polycomb Group (PcG) protein family (Boyer et al., 2006). miR-137 expression is also up-regulated in MeCP2 -deficient immature neurons and miR-137 has a significant impact on the dendritic morphogenesis of young hippocampal neurons (Smrt et al., 2010). The neuronal maturation function of miR-137 is achieved by translational repression of Mind bomb-1, a ubiquitin ligase known to be important for neurogenesis and neurodevelopment (Choe et al., 2007; Ossipova et al., 2009), rather than Ezh2.

In adult neural stem cells, miR-184 is regulated by MBD1, but not by MeCP2. miR-184 promotes neural stem cell proliferation and inhibits differentiation by targeting Numblike (Liu et al., 2010). Therefore, MBD1, miR-184, and Numblike may form a regulatory network that controls the balance between the proliferation and differentiation of neural stem cells.

[0064] TET2 is under extensive microRNA regulation and such TET2-targeting is an important pathogenic mechanism in hematopoietic malignancies. Using a high-throughput 3 TR activity screen, >30 miRNAs were identified that inhibit TET2 expression and cellular 5hmC (Cheng et al., Cell Rep, 2013). Forced expression of TET2-targeting miRNAs in vivo disrupts normal hematopoiesis, leading to hematopoietic expansion and/or myeloid differentiation bias, whereas co expression of TET2 corrects these phenotypes. Several TET2-targeting miRNAs, including miR- 125b, miR-29b, miR-29c, miR-101, and miR-7, are preferentially overexpressed in TET2-wildtype acute myeloid leukemia. Expression of miR-29 and miR-26 family miRNAs has resulted in an inhibition of TET1 and TET3 3'UTR reporter activities (Cheng et al., Cell Rep, 2013). Polynucleotide delivery vehicles

[0065] Expression vectors according to this invention may be a recombinant vector capable of expression of a protein or polypeptide of interest or a fragment thereof, for example, an adeno- associated vims (AAV) vector, a lentivims vector, a retrovirus vector, a replication

competent adenovirus vector, a replication deficient adenovirus vector (e.g., a

gutless adenovirus vector), a herpes vims vector, a baculovims vector, or a nonviral plasmid.

[0066] For purposes of administration into the brain, it is typically preferable to use a viral or non-viral vector that does not result in the expression cassette being inserted into the genome of the cell. A non-limiting example of a non-integrative viral vector suitable for use with this technology is an Adeno-Associated Viral (AAV) vector.

[0067] The genome of AAV is a linear, single-stranded DNA molecule that is less than about 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for the non-stmctural replication (Rep) proteins and the structural (VP) proteins. The VP proteins (VP1, -2 and -3) form the capsid and contribute to the tropism of the vims. The terminal 145 nt ITRs are self-complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex. Following wild- type (wt) AAV infection in mammalian cells the Rep genes are expressed and function in the replication of the viral genome. Any combination of an AAV ITR and capsid may be used in the AAV vectors, including but not limited to ITRs and capsids from AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1, AAV 12, AAV13, AAV 14, AAV15, and AAV16.

[0068] A recombinant AAV vector is a vector comprising one or more therapeutic

polynucleotides that are flanked by one or more AAV ITRs. Such rAAV vectors can be replicated and packaged into infectious viral particles when present in an insect host cell that is expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). When an rAAV vector is incorporated into a larger nucleic acid construct (e.g., in a chromosome or in another vector such as a plasmid or baculovims used for cloning or transfection), then the rAAV vector is typically referred to as a“pro vector” which can be“rescued” by replication and encapsidation in the presence of AAV packaging functions and necessary helper functions.

[0069] rAAV vectors comprising two ITRs have a payload capacity of about 4.4 kB. Self complementary rAAV vectors contain a third ITR and package two strands of the recombinant portion of the vector leaving only about 2.1 kB. The AAV vector can be an scAAV vector.

[0070] AAV r3.45 is more selective for neural stem cells (NSCs) than mature neurons in a human embryonic stem cell-derived culture containing a mixture of cell types, including NSCs and neurons (Kotterman et al. Development, 2015). The invention can be practiced as a DNA demethylating polynucleotide packaged within an AAVr3.45 capsid mutant for selective transduction of NSCs.

[0071] Extended packaging capacities that are roughly double the packaging capacity of an rAAV(about 9kB) have been achieved using dual rAAV vector strategies. Dual vector strategies useful in producing rAAV according to this invention include, but are not limited to splicing (trans splicing), homologous recombination (overlapping), or a combination of the two (hybrid). In the dual AAV trans-splicing strategy, a splice donor (SD) signal is placed at the 3’ end of the 5’ half vector and a splice acceptor (SA) signal is placed at the 5’ end of the 3’ half vector. Upon co-infection of the same cell by the dual AAV vectors and inverted terminal repeat (ITR) -mediated head-to-tail concatemerization of the two halves, trans-splicing results in the production of a mature mRNA and full-size protein (Yan et al, 2000). Trans-splicing has been successfully used to express large genes in muscle and retina (Reich et al, 2003; Lai et al, 2005). Alternatively, the two halves of a large transgene expression cassette contained in dual AAV vectors may contain homologous overlapping sequences (at the 3’ end of the 5’ half vector and at the 5’ end of the 3’ half vector, dual AAV overlapping), which will mediate reconstitution of a single large genome by homologous recombination (Duan et al, 2001). This strategy depends on the recombinogenic properties of the transgene overlapping sequences (Ghosh et al., 2006). A third dual AAV strategy (hybrid) is based on adding a highly recombinogenic region from an exogenous gene (i.e., alkaline phosphatase; Ghosh et al, 2008, Ghosh et al, 2011)) to the trans-splicing vectors. The added region is placed downstream of the SD signal in the 5’ half vector and upstream of the S A signal in the 3’ half vector in order to increase recombination between the dual AAVs. The invention can be practiced as multiple DNA demethylases packaged within a dual vector AAV.

[0072] As an alternative to an AAV vector, the vector can be a retroviral vector or a lentiviral (LV) vector, in part since lentiviral vectors are capable of providing efficient delivery, integration and long term expression of transgenes into non-dividing cells both in vitro and in vivo. The vectors may have one or more LTRs, wherein either LTR comprises one or more modifications, such as one or more nucleotide substitutions, additions, or deletions. The vectors may further comprise one of more accessory elements to increase transduction efficiency (e.g., a cPPT/FLAP), viral packaging (e.g., a Psi (Y) packaging signal, RRE), and/or other elements that increase therapeutic gene expression (e.g., poly (A) sequences), and a WPRE or HPRE.

[0073] As an alternative to viral vectors, a gene payload can be transfected into a target cell using a non- viral vector, such as liposomes, nanocapsules, nanoparticles, exosomes, microparticles, microspheres, and lipid particles containing an expression cassette with the encoding sequence to be expressed operably linked to a suitable promoter. [0074] For further information on standard techniques useful for the practice of this invention, the reader is referred to standard reference information and suppliers. Aspects of gene therapy are described in the following: Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, Fourth Edition 2015, Nancy Smyth Templeton Ed. Translating Gene Therapy to the Clinic: Techniques and Approaches, 2014, Jeffrey Laurence and Michael Franklin Eds. Adeno-Associated Virus: Methods and Protocols (Methods in Molecular Biology), 2016, Richard O. Snyder Ed., Philippe Moullier Ed. Advanced Textbook on Gene Transfer, Gene Therapy and Genetic Pharmacology: Principles, Delivery and Pharmacological and Biomedical Applications, 2014, Daniel Scherman Ed. Gene Transfer Vectors for Clinical Application, Volume 507, Methods in Enzymology, 1st Edition, Theodore C. Friedman Ed. Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools (Methods in Molecular Biology), 2016, Maurizio Federico Ed. Non-viral Gene Therapy: Gene Design and Delivery, 2005, Kazunari Taira and Kazunori Kataoka Eds. SiRNA Delivery Methods: Methods and Protocols (Methods in Molecular Biology), 2017, Kato Shum and John Rossi Eds.

[0075] Biochemical and biological aspects of the TET2 system are described, for example, in Le role du gene TET2 dans Thematopoiese normale et pathologique, C. Quivoron, Editions universitaires europeennes, 2016; Epigenetics (International Review of Neurobiology Book 115),

S.C. Pandey, 2014, and The Tet Offensive: A Concise History, J. Willbanks, 2008. General methods in cell biology, protein chemistry, and antibody techniques can be found in Current Protocols in Protein Science (J. E. Colligan et al. eds., Wiley & Sons); Current Protocols in Cell Biology (J. S. Bonifacino et al., Wiley & Sons) and Current protocols in Immunology (J. E. Colligan et al. eds., Wiley & Sons.). Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, and ClonTech.

In vitro screening of DNA demethylase agonists

[0076] Screening of DNA demethylases can be accomplished using an enzyme-linked immunosorbant assay (ELISA) for 5hmC. Template oligonucleotides are synthesized from a commercial source. Biotinylated 5mCpG-containing double- stranded oligonucleotides are prepared by PCR using dATP, dTTP, dGTP and 5mCTP. The 5' primers are labelled with Biotin so the amplicons became biotinylated after PCR. Lysates from HEK293T cells transfected separately with Myc-TET2, Myc-Idax or Myc-IdaxDBM are incubated for 1.5 h at 37° C with 230 ng of substrate in 100 pL total volume (100 mM HEPES pH7.0, 50 mM NaCl, 1 mM - ketoglutarate, 2 mM ascorbic acid, 0.1 mM ferrous ammonium sulfate, 1 mM ATP, 0.1 mg/ml BSA, 1 mM dithiothreitol). As a control, 10 mM N-oxalylglycine (NOG), a well-known inhibitor of Fe(II)/-Ketoglutaratedependent dioxygenases, is added to the wells before addition of cell lysate. 25 mΐ of each sample is loaded in triplicate into individual wells of a streptavidin-coated 384-well plate.

[0077] The plate is incubated at RT on a rotating platform for 3 h. Subsequently, the wells were washed three times with 100 mΐ of lx Tris-Buffered Saline with 0.1 % Tween (TBST). Next, 50 mΐ of a 1 :2,500 dilution of anti-5hmC antibody in 1 % milk/TBST was added to each well. After incubation overnight on a rotating platform at 4C, the wells are washed three times with 100 mΐ TBST. 50 mΐ of a 1 :2,500 dilution of goat anti-rabbit peroxidase in 1% milk in TBST is added to each well, and the plate was incubated for an additional hour at RT on a rotating platform. The wells are then washed three times with 100 mΐ TBST, and 30 mΐ of a mixture with a 1 : 1 ratio of Peroxidase Substrate to Peroxidase Solution from a TMB Substrate Kit (Thermo Scientific) was added to each well. The reaction proceeds in the dark for 10-12 min, and is then quenched with 20 mΐ of 25% sulfuric acid per well.

[0078] The absorbance of each well is measured at 450 nm as a readout of 5hmC levels. This screen can be used to identify DNA demethylases including TET agonists and DNMT antagonists in the form of polynucleotides, proteins, viral vectors, or small molecules. Already approved and investigational small molecule libraries can be obtained from the National Center for Advancing Translational Sciences (NCATS) or commercial sources including Selleckchem, Sigma, and

ChemBridge.

[0079] Small molecule” TET agonists according to this invention have molecular weights less than 20,000 daltons, and are often less than 10,000, 5,000, or 2,000 daltons. Small molecule agonists are not antibody molecules or oligonucleotides, and typically have no more than five hydrogen bond donors (the total number of nitrogen-hydrogen and oxygen-hydrogen bonds), and no more than 10 hydrogen bond acceptors (all nitrogen or oxygen atoms).

Tissue and cell targets suitable for epigenetic reprogramming

[0080] Targets for therapeutic intervention according to this invention include sites in the central and peripheral nervous system. The continuous generation of new neurons in the mammalian adult brain in discrete regions, called neurogenic niches, has been documented for some time . The term“neurogenic niche” is used to define a region in which the cytoarchitecture and signaling factors within its microenvironment are able to maintain a population of neural stem cells (NSCs) with self renewal capacity that give rise to new neurons and glial cells. Cross-talk between all of the components of the niche is crucial for the survival of NSCs, as well as for controlling their fate.

[0081] The most studied and accepted niches include the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampus dentate gyrus. In these classical niches, several key cell types can be recognized, including NSCs that remain mostly in a quiescent state; a transient progenitor cell derived from the NSCs, which have a higher proliferation rate; new immature neurons or glial cells, derived from the progenitor cells; and accessory cells, including parenchymal astrocytes, endothelial, microglial, and ependymal cells, which modulate the niche and are needed for its maintenance. In the SVZ niche, differentiated immature neurons migrate to a distant region, the olfactory bulb, where they finally achieve complete differentiation. This migration occurs through a restricted path called the rostral migratory stream (RMS). Alternatively, newly generated cells remain in the SGZ niche, renewing neuronal and glial populations near their site of origin in the hippocampus.

[0082] Other areas in the nervous system contain regions of cell proliferation that may constitute a neurogenic niche, including the hypothalamus, substantia nigra, cerebellum, amygdala, and spinal cord. These non-classical neurogenic niches are summarized by Oyarce et al. (Oyarce et ah, J Stem Cell Res Ther, 2014). Neural progenitor cells in the adult neurogenic niche of the hippocampus are one attractive target for the treatment of cognitive impairment resulting from Alzheimer’s Disease, Parkinson’s Disease, or Traumatic Brain Injury for example. Motor neurons in the spinal cord are another potential target for neural regeneration as a treatment of spinal cord injury. DNA demethylation has been observed in Multiple Sclerosis white matter (Mastronard et al., J. Neurosci, 2007) and methylation changes in Parkinson’s (Masliah et al., Landes Biosci, 2013), therefore the associated myelin rich fibers and substantia nigra, respectively, may pose attractive targets.

[0083] Mature neurons in the adult peripheral nervous system can effectively switch from a dormant state with little axonal growth to robust axon regeneration upon injury. The mechanisms by which injury unlocks mature neurons’ intrinsic axonal growth competence are not well understood. Peripheral sciatic nerve lesions in adult mice lead to elevated levels of TET3 and

5-hydroxylmethylcytosine in dorsal root ganglion (DRG) neurons. Functionally, TET3 is required for robust axon regeneration of DRG neurons and behavioral recovery. Mechanistically, peripheral nerve injury induces DNA demethylation and upregulation of multiple regeneration-associated genes in a TET3- and thymine DNA glycosylase dependent fashion in DRG neurons. In addition, Pten deletion- induced axon regeneration of retinal ganglion neurons in the adult CNS is attenuated upon TET1 knockdown. Therefore, an epigenetic barrier can be removed by active DNA demethylation to permit axon regeneration in the adult mammalian nervous system. Formulation of medicaments

[0084] Preparation and formulation of pharmaceutical agents for use according to this invention can incorporate standard technology, as described, for example, in the current edition of Remington: The Science and Practice of Pharmacy. The formulation will typically be optimized for administration to the target tissue, for example, by local administration, in a manner that enhances access of the active agent to the target senolytic cells and providing the optimal duration of effect, while minimizing side effects or exposure to tissues that are not involved in the condition being treated.

[0085] Pharmaceutical preparations for use in treating neurological conditions and other diseases can be prepared by mixing an effective vector or other agent according to this invention with a pharmaceutically acceptable base or carrier and as needed one or more pharmaceutically acceptable excipients. Exemplary excipients and additives that can be used include surfactants (for example, polyoxyethylene and block copolymers); buffers and pH adjusting agents (for example, hydrochloric acid, sodium hydroxide, phosphate, citrate, and sodium cyanide); tonicity agents (for example, sodium bisulfite, sodium sulfite, glycerin, and propylene glycol); and chelating agents (for example, ascorbic acid, sodium edetate, and citric acid).

[0086] This invention provides commercial products that are kits that enclose unit doses of one or more of the agents or compositions described in this disclosure. Such kits typically comprise a pharmaceutical preparation in one or more containers. The preparations may be provided as one or more unit doses (either combined or separate). The kit may contain a device such as a syringe for administration of the agent or composition in or around the target tissue of a subject in need thereof. The product may also contain or be accompanied by an informational package insert describing the use and attendant benefits of the drugs in treating or preventing the nurological condition, and optionally an appliance or device for delivery of the composition.

Routes of administration

[0087] Depending on the pharmaceutical agent, it may be appropriate to administer a medicament of this invention either locally or systemically. If appropriate, small molecule drugs and oligonucleotides can be administered orally, subcutaneously, or intravenously. Larger expression vectors are more typically administered intravenously, by intracranial administration, intrathecal administration, intranasal administration, intracerebroventricular administration, cisterna magna administration, intrathecal (bolus or infusion pump), intraparenchymal (intraspinal into the spinal cord), intraganglionic, periganglionic, intraforaminal, or intraneural administration.

[0088] Intracranial administration of polynucleotides into the target brain region using viral or non-viral systems will ensure maximum dose on target with minimum off-target activity. Stereotactic injection is typically used to deliver miRNA knockdown agents to specific location in the brain.

These agents may be designed as antagomirs against miRNAs with locked nucleotide modifications (ENA). However, brain specific delivery of antagomirs, uniformly across different regions of the brain is achievable without the associated injury and limitation of local delivery in stereotactic injections.

[0089] For example, a complex of neurotropic, cell-penetrating peptide Rabies Vims

Glycoprotein (RVG) with antagomir against miRNA-29 can be injected into intravenous circulation to specifically deliver in the brain (Hwang et al., Biomaterials, 2011). The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioral defects. The invention can be practiced as cell-penetrating peptide Rabies Vims Glycoprotein (RVG) with antagomir against miR-125b, miR-29b, miR-29c, miR-101, and miR-7.

Incorporation by reference

[0090] For all purposes in the United States and in other jurisdictions where effective, each and every publication and patent document cited in this disclosure is hereby incorporated herein by reference in its entirety for all purposes to the same extent as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. A related study in the journal Cell Reports by G. Gontier, S.A. Villeda et al., Cell Rep. 2018 Feb 20;22(8):1974-1981, entitled“TET2 rescues age-related regenerative decline and enhances cognitive function in the adult mouse brain,” is hereby incorporated herein by reference in its entirety for all purposes.

EXAMPLES

Example 1 : TET2 and 5hmC levels decrease in the aged hippocampus

[0091] Changes in the levels of TET2 mRNA were characterized in the hippocampus and cortex of normal young and aged animals by quantitative PCR (qPCR). FIGS. 4 A and 4B show an age- related decrease in TET2 expression selectively within the aged hippocampus.

[0092] Changes in 5hmC and 5mC levels by slot blot and immunohistochemical analysis. There was an age-related decrease in the levels of hippocampal 5hmC (FIGS. 4C,D), but not 5mC

(FIGS. 5A,B). To gain further mechanistic insight into which genomic loci were affected by these changes, antibody-based 5hmC immunoprecipitation was combined with deep sequencing (hMeDIP- seq) in the young and aged hippocampus (FIGS. 4E,F; FIGS. 5C-H). There were 345 differentially 5 -hydroxy methylated regions (DhMRs) that were lost and none gained, increasing with age (FIG. 4E). Lost DhMRs were enriched in intragenic regions, in the aged compared to young hippocampus (FIG. 4E). DhMR- associated genes were determined to be involved in neurogenic processes by gene ontology analysis (FIG. 4F). These results demonstrate that 5hmC is lost during aging from regions of the genome associated with neurogenic processes in the adult hippocampus.

[0093] To investigate the relationship between age-related changes in TET2 expression and adult neurogenesis further, the temporal kinetics of decreased adult neurogenesis were compared with decreased TET2 expression in an aging cohort of mice (FIGS. 4G-I; FIG. 51). Adult neurogenesis and TET2 expression were quantified in contralateral hippocampi of the same animals by

immunohistochemical analysis and qPCR, respectively. A precipitous decline in adult neurogenesis was seen by six months of age in mature adults (FIGS. 4H,I; FIG. 51). This decline was paralleled by a sharp decrease in TET2 expression (FIG. 4G, FIG. 51). Thus, decreased TET2 and 5hmC levels in the aging hippocampus were associated with age-related impairments in adult neurogenesis.

Example 2: Reducing TET2 activity in the young adult hippocampus impairs neurogenesis

[0094] To resolve the role of TET2 in a temporally and regionally defined manner in the adult brain, TET2 expression was abrogated in the young adult hippocampus utilizing an in vivo viral- mediated RNA interference (RNAi) approach. Young adult animals were stereotaxically injected with high-titer lentivirus (LV) encoding TET2 or scramble control shRNA sequences into the DG of contralateral hippocampi, and TET2 abrogation was confirmed in vivo (FIGS. 6A-C). No changes in TET1 or TET3 were detected (FIG. 6D).

[0095] NPC function and maturation was determined in an independent cohort of young adult animals by immunohistochemical analysis (FIG. 7A). Neuronal maturation and survival was assessed using a long-term BrdU incorporation paradigm, in which mature differentiated neurons express both BrdU and the neuronal marker NeuN. Abrogation of TET2 in the DG resulted in a significant decrease in the number of Sox2/GFAPpositive NPCs, Doublecortin (DCX) -positive newly born neurons, Bromodeoxyuridne (BrdU)- positive cells and BrdU/NeuN-positive mature differentiated neurons compared with the control contralateral DG (FIG. 7B).

[0096] To gain further mechanistic insight at a cellular level, the in vivo RNAi observations were complemented with a cell type-specific temporally controlled genetic model. TET2flox/lox mice were generated carrying an inducible NestinCre-ERT2 gene, in which TET2 is excised specifically in adult NPCs (TET2-/-) upon tamoxifen administration (FIG. 7C, Fig 6E). Changes in neurogenesis were examined in young adult TET2-/- and littermate control (TET2flox/lox) mice by immunohistochemical analysis.

[0097] Absence of TET2 expression in adult NPCs resulted in a selective decrease in the number of Dcx-positive newly born neurons, BrdU-positive cells and BrdU/NeuNpositive mature differentiated neurons in TET2-/- mice compared to TET2flox/lox controls, without altering the number of Nestin -positive NPCs (FIG. 7D). This decrease in newly born and mature neurons is consistent with increased levels of 5hmC upon neuronal differentiation in vivo (FIG. 6F).

Collectively, our in vivo RNAi and genetic data suggest an involvement of TET2 in regulating adult neurogenesis at both the level of the neurogenic niche and adult NPCs.

Example 3: Reducing TET2 activity impairs hippocampal-dependent cognitive function

[0098] This experiment was designed to investigate whether decreased TET2 in the young adult DG, or selective loss of TET2 in adult NPCs, impaired cognitive processes associated with adult neurogenesis (FIG. 8A). Hippocampal-dependent learning and memory was assessed using radial arm water maze (RAWM) and contextual fear conditioning paradigms. To test the effect of decreased TET2 in the adult DG, young adult animals were given bilateral stereotaxic injections into the hippocampi of high-titer LV encoding TET2 shRNA or scramble control sequences (FIG. 8B). To test the effect of selective loss of TET2 in adult NPCs, we utilized young adult NPC TET2-/- and littermate control TET2flox/lox mice (FIG. 8E).

[0099] All mice showed similar learning capacity during the training phase of the RAWM (FIG. 8D,G). Abrogation of TET2 in the adult DG resulted in significantly more errors in locating the target platform during both short term (FIG. 8D, FIG. 9B) and long-term (FIG. 8D, FIG. 9C) learning and memory testing compared to control conditions. Interestingly, selective loss of TET2 in adult NPCs resulted in impairments only during long-term learning and memory testing compared to control mice (FIG. 8G, FIGS. 9E,F). During fear conditioning training mice exhibited no differences in baseline freezing time (FIGS. 8A,D). However, both abrogation of TET2 in the adult DG, or loss of TET2 in adult NPCs, resulted in decreased freezing time during contextual (FIGS. 8C,F), but not cued (FIGS. 9A,D), memory testing.

[0100] These data demonstrate that decreased TET2 in the adult neurogenic niche, or adult NPCs, impairs long-term hippocampal-dependent spatial learning and memory and associative fear memory acquisition.

Example 4: Decreased TET2 activity in the aged hippocampus can be reversed

[0101] The data presented above indicate that mimicking an age-related decline in TET2 impairs adult neurogenesis. This experiment was designed to investigate whether restoring TET2 in the adult brain can rescue age-related regenerative decline. To gain insight into the potential involvement of TET2 in neurogenic rejuvenation mRNA levels of TET2 were measured in the aged hippocampus after heterochronic parabiosis. An increase of TET2 in heterochronic parabionts was detected after exposure to young blood compared to age-matched isochronic parabionts exposed to old blood (FIG. 10A). No changes in TET1 or TET3 were observed (FIG. 9A). These data implicate TET2 in conditions of brain rejuvenation.

Example 5 : Restoring TET2 activity rescues age-related regenerative decline

[0102] This experiment was designed to investigate whether restoring TET2 in the adult hippocampal neurogenic niche could counteract age related regenerative decline by way of an in vivo viral mediated overexpression approach. Aging analysis demonstrates a concomitant age-related decrease in adult neurogenesis and TET2 expression in mature adult animals at six months of age (FIGS. 4G-I). Correspondingly, mature adult animals at this age were stereotaxically injected with high-titer lentivirus (LV) encoding TET2 or control into the DG of contralateral hippocampi, and TET2 overexpression was confirmed in vivo (FIG. 10B; FIG. 1 IB). No changes in TET1 or TET3 were detected (FIG. 11C).

[0103] hMeDIP-seq was performed in the mature adult hippocampi following viral-mediated TET2 overexpression (FIG. 10C-E; FIGS. 11D-H), and found 558 DhMRs gained and 110 lost after TET2 overexpression (FIG. 10D). DhMRsw as enriched in intragenic regions (FIG. 10D), associated with neurogenic processes as determined by Gene Ontology analysis (FIG. 10E). Genes whose DhMRs were lost during aging were compared with those gained by TET2 overexpression. Thirty nine overlapping genes were detected, of which 10 are involved in neurogenesis (FIG. 10F). Of these genes, mRNA expression of Igfrl, a gene implicated in lifespan regulation (Holzenberger et al.,

2003), bidirectionally changed during aging and TET2 overexpression (FIG. 10F). These data further substantiate the possibility that restoring TET2 in the mature adult hippocampal neurogenic niche promotes neurogenic rejuvenation.

[0104] The next experiment was designed to determine whether restoring TET2 was sufficient to rescue age-related decline in adult hippocampal neurogenesis by immunohistochemical analysis. Increasing TET2 in the mature adult DG at six months of age resulted in a significant enhancement in the number of Sox2/GFAP-positive NPCs, Dcx-positive newly born neurons, BrdU-positive proliferating cells, and BrdU/NeuN-positive mature differentiated neurons in the DG compared with the control contralateral DG (FIG. 10G). Fevels of adult neurogenesis achieved in the mature adult hippocampus by increased TET2 mirrored levels normally observed in the young adult hippocampus (FIG. 10G). TET2 was overexpressed in the DG of young adult animals at 3 months of age, but there were no changes in adult neurogenesis (FIG. 11 J). Together, the data indicate an age-dependent role for TET2 in regulating regenerative decline in the aging brain. Example 6: Restoring TET2 activity enhances hippocampal-dependent cognitive function.

[0105] This experiment was designed to investigate the functional consequence of increasing TET2 in the mature adult DG on hippocampal-dependent learning and memory using a contextual fear conditioning paradigm. Mature adult animals were given bilateral stereotaxic injections of high- titer LV encoding TET2 or control into the DG of the hippocampus (FIG. 10H). During fear conditioning training mice exhibited no differences in baseline freezing time (FIGS. 1 IK). However, increased TET2 expression in the mature adult DG resulted in increased freezing time during contextual (FIGS. 101), but not cued (FIGS. 11K), memory testing. These data indicate that restoring TET2 in the mature adult neurogenic niche is sufficient to enhance associative fear memory acquisition.

Example 7 : TET2 overexpression delays aged-related regenerative decline in the hippocampus

[0106] This experiment was designed to investigate the ability of increased TET2 expression to attenuate age-related decline in hippocampal neurogenesis.

[0107] Mice that were three months of age were injected with either a high-titer LV encoding TET2, or with LV control into the hippocampus of contralateral brain hemispheres of the same animal. The mice were then allowed to age to 16 months, at which point their brains were removed and analyzed for neurogenesis. Doublecortin (DCX) immunohistochemical staining in the dentate gyrus of the hippocampus was used as a marker of neurogenesis, selectively labeling newly born neurons (arrowheads). DAPI was used to label all cell nuclei (background).

[0108] FIGS. 12 A to 12C show the data from this experiment. When compared with the LV control, administration of LV encoding TET2 resulted in a significantly increased number of newly born DCX positive cells that were detected per dentate gyrus.

Figure legends

[0109] FIG. 1 : Loss of TET2 and 5hmC in the aged hippocampus associates with regenerative decline. Decreasing TET2 in the young mouse hippocampus impairs neurogenesis and cognition. Heterochronic parabiosis restores TET2 in the aged hippocampus. Restoring TET2 in the mature hippocampus rescues neurogenesis and enhances cognition.

[0110] FIG. 2A: The epigenetic clock correlates strongly with the chronological age of a person and the level of DNA methylation of his or her genome. This points to a set of possible interventions to slow, stop, and possibly reverse aging. FIG. 2B: Induced pluripotent stem cell (iPSC) reprogramming (circled dots) suggests that the epigenetic clock can be reset to a pre-birth state regardless of the age of the cell’s donor. [0111] FIG. 3: The gain, loss and maintenance of the methyl marks on cytosine in a CpG context is the result of the relative contribution of three interconnected pathways: 1) acquisition of new methylation marks by de novo methylation (de novo DNA methyltransferases DNMT3A and DNMT3B in combination with DNMT3L establish a pattern of methylation) ; (2) maintenance of existing methylation patterns across DNA replication by the maintenance methyltransferase DNMT1; and (3) active replication-independent removal of DNA methylation by the Ten-eleven translocation (TET) proteins. DNA methylation is highly regulated.

[0112] FIG. 4: TET2 expression and 5hmC levels decline in the aged hippocampus and are associated with neurogenic processes. A, Schematic of young adult (3 months) versus aged (18 months) mice. B, Quantitative reverse-transcription PCR of TET2 mRNA from hippocampal and cortical lysates of young and aged mice. (n=5 per group; t-test; *p<0.05, not significant). C, Representative slot-blot and quantification of isolated hippocampal DNA probed with anti-5hmC antibodies from young and aged mice. (n=4 per group; t-test; *p< 0.05). D, Representative field and quantification of 5hmC expression in the dentate gyms (DG) of the young and aged hippocampus. (n=4 per group; t-test; *p<0.05).

[0113] FIG. 4E: Association of regions of 5hmC lost during age with genomic elements (differentially 5-hydroxymethylated regions (DhMRs) lost between 3 and 18 months; 345 DhMRs at q=0.05). Pie chart depicts overall loss and gain of DhMRs during aging. F, Top Gene Ontology terms for genes overlapping with age-associated 5hmC loss (>2-fold enrichment; ordered by p- value). G, Quantitative reverse transcription PCR of TET2 mRNA from hippocampal lysates of aging mice. (n=5; ANOVA with Dunnett’s post hoc test; **p<0.01). H,I, Neurogenesis was analyzed by immunostaining and confocal microscopy. Representative field (G) and quantification (H) of Nestin positive, MCM2 -positive, and Doublecortin (DCX)-positive cells in aging dentate gyrus (DG) at 3, 6, 12, and 18 months. (n=5; ANOVA with Dunnett’s post hoc test; ***p<0.001, ****p<0.0001). Data are represented as mean ± SEM.

[0114] FIG. 5: Characterization of 5mC levels, hMeDIP-sequencing, and adult neurogenesis in the young and aged hippocampus. A, Representative slot-blot and quantification of isolated hippocampal DNA probed with anti-5mc antibodies from young (3 months) and aged (18 months) mice. (n=4 per group; t-test; not significant). B, Representative field and quantification of 5mc expression in the dentate gyrus (DG) of the young and aged hippocampus. (n=4 per group; t-test; not significant). C, IGV Browser track of a 20mB region of chromosome 6. At the top is an ideogram of chromosome 6 with highlighted region in red box. Normalized read counts from young (3 months, black) and aged (18 months, grey) hippocampi from this region show increased reads over genes (signified at the bottom).

[0115] FIG. 5D: Chromosome coverage of hMeDIP-Seq reads from young, aged, and input samples normalized to reads per million mapped. E, Scatterplot of young versus aged hippocampi using 50kb bins. Reads are normalized as FPKM. F, Metagene read coverage in FPKM for RefSeq genes. The gene body was scaled to 5kb for all genes. 2.5kb upstream of the TSS and 2.5kb downstream of the TES are shown. G, Read coverage in FPKM for RefSeq promoters. 2.5kb upstream and downstream of the TSS is shown. H, Two examples of lost aging DhMRs that are proximal to Trim30b and Igflr. Young (3 months, black) and aged (18 months, grey) samples are shown. I, Temporal relationship between levels of adult neurogenesis and TET2 expression in contralateral hippocampi with age (3, 6, 12, and 18 months). Quantification of Nestin-positive, MCM2 -positive, and Doublecortin (DCX)-positive cells by immunohistochemistry was correlated with TET2 mRNA levels assessed by quantitative reverse-transcription PCR. (n=3 per group). Data are represented as mean ± SEM.

[0116] FIG. 6: Abrogation of TET2 by lentiviral-mediated shRNA approach and 5hmC expression during neuronal differentiation. Panel A,B: Young adult (3 months) mice were given unilateral stereotaxic (STX) injections of lentivirus (LV) encoding either shRNA targeting TET2 or scramble control sequences in tandem with a green florescent protein (GFP) reporter into contralateral dentate gyrus (DG). Schematic of lentiviral vector generated to express small hairpin RNAs (shRNA) targeting TET2. Abbreviations: AMP, ampicillin, pCMV, cytomegalovirus promoter (A). Schematic illustrating unilateral STX injection paradigm into the DG (B). C,D, Quantitative reverse- transcription PCR of TET1, TET2, and TET3 mRNA from hippocampal lysates following STX injection. (n=5 per group; t-test; *p<0.05, not significant). F, Representative field and quantification of 5hmC expression in neural progenitors (MCM2+/DCX-), neuroblasts (MCM2+DCX+), immature neurons (MCM2-DCX+) and mature neurons by immunohistaining and confocal microscopy in the young adult (3 months) hippocampus. (n=4; ANOVA with Dunnett’ s post hoc test; ***p<0.001, not significant). Data are represented as mean+SEM.

[0117] FIG. 7 : Abrogation of TET2 in the adult dentate gyrus, or loss of TET2 in adult NPCs, impairs hippocampal neurogenesis. A,B, Young adult (3 months) mice were given unilateral stereotaxic injections of lentivirus (LV) encoding either shRNA targeting TET2 or scramble control sequences in tandem with a green florescent protein (GFP) reporter into contralateral dentate gyrus (DG). Mice were administered BrdU by intraperitoneal injections for six days and euthanized 30 days later. Neurogenesis was analyzed by immunostaining and confocal microscopy. Schematic illustrating stereotaxic injection paradigm and experimental timeline (A). Representative field and quantification of GFAP/Sox2 -positive, Doublecortin (Dcx)-positive, Brdu-positive, and NeuN/BrdU- positive cells (B). (n=5-9 per group, t-test; *p<0.05, **p<0.01,***p<0.001). C,D, A cell type specific temporally controlled TET2flox/flox/NestinCre-ERT2 genetic model was generated, in which TET2 was excised selectively in adult neural stem/progenitor cells (NPCs) upon tamoxifen administration (TET2-/-). Neurogenesis was analyzed in young adult (3 months) NPC TET2-/- and littermate control (TET2flox/lox) mice by immunostaining and confocal microscopy. C, Schematic illustrating tamoxifen injection paradigm and experimental timeline. Mice were administered BrdU by intraperitoneal injections for six days and euthanized 30 days later. D, Representative field and quantification of Nestin-positive, Dcx-positive, Brdu-positive, and NeuN/BrdU-positive cells. (n=7-8 per group; t-test; *p<0.05, **p<0.01,***p<0.001). Data are represented as mean+SEM.

[0118] FIG. 8: Abrogation of TET2 in the adult dentate gyrus, or loss of TET2 in adult NPCs, impairs hippocampal-dependent cognitive function. A, Schematic of experimental paradigm and cognitive testing timeline. Hippocampal-dependent learning and memory was assessed by radial arm water maze (RAWM) and contextual fear conditioning paradigms. B, Young adult (3 months) wild type mice were given bilateral stereotaxic injections of lend virus (LV) encoding either shRNA targeting TET2 (sh-TET2) or scramble control sequences (sh-Scramble) into the dentate gyrus (DG). C, Associative fear memory was assessed in sh-TET2 and sh-Scramble control injected mice using contextual fear conditioning. Quantification of percent freezing 24 hours after training. (n=14 per group; t-test; ***p<0.001)

[0119] FIG. 8D: Hippocampal-dependent spatial learning and memory was assessed in sh- TET2 and sh-Scramble control injected mice using RAWM. Quantification of the number of entry errors during RAWM training and testing. (n=14 per group; repeated measures ANOVA with Bonferroni posthoc correction; *p<0.05, **p<0.01). E, Young adult TET2flox/flox/NestinCre-ERT2 NPC-specific knockout (TET2-/-) or littermate control (TET2flox/flox) mice were administered tamoxifen. Data from 9-10 animals per group. F, Associative fear memory was assessed in TET2-/- and TET2flox/flox control mice using contextual fear conditioning. Quantification of percent freezing 24 hours after training. (n=9-10 per group; t-test; *p<0.05). G, Hippocampal-dependent spatial learning and memory was assessed in in TET2-/- and TET2flox/flox control mice using RAWM. Quantification of the number of entry errors during RAWM training and testing. (n=9-10 per group; repeated measures ANOVA with Bonferroni post-hoc correction; ***p<0.001). Data are represented as mean+SEM.

[0120] FIG. 9: Abrogation of TET2 in the adult dentate gyrus, or loss of TET2 in adult NPCs, selectively impairs hippocampal-dependent cognitive function. A, Young adult (3 months) wild type mice were given bilateral stereotaxic injections of lentivirus (LV) encoding either shRNA targeting TET2 (sh TET2) or scramble control sequences (sh-Scramble) into the dentate gyrus (DG). Cued fear memory was assessed in sh-TET2 and sh- Scramble control injected mice using contextual fear conditioning. Quantification of percent freezing 24 hours after training. (n=14 per group; t-test; not significant; t test). B,C, Hippocampal-dependent spatial learning and memory was assessed in sh- TET2 and sh- Scramble control injected mice using RAWM. Quantification of the number of entry errors during RAWM short-term (B) and long-term (C) learning and memory testing. (n=14 per group; t-test; *p<0.05, ***p<0.001, ****p<0.0001). D, Young adult TET2flox/flox/NestinCre- ERT2 NPC-specific knockout (TET2-/-) or littermate control (TET2flox/flox) mice were administered tamoxifen. Cued fear memory was assessed in TET2-/- and TET2flox/flox control mice using contextual fear conditioning. Quantification of percent freezing 24 hours after training. (n=9-10 per group; t-test; not significant). E,F, Hippocampal-dependent spatial learning and memory was assessed in in TET2-/- and TET2flox/flox control mice using RAWM. Quantification of the number of entry errors during RAWM short-term (E) and long-term (F) learning and memory testing. (n=9- 10 per group; t-test; *p<0.05, **p<0.01, ****p<0.0001, not significant). Data are represented as mean+SEM.

[0121] FIG. 10: Increasing TET2 in the mature adult dentate gyrus rescues age-related regenerative decline. A, Schematic and quantitative reverse-transcription PCR (qPCR) of TET2 mRNA from hippocampal lysates of aged (18 months) isochronic and heterochronic parabionts five weeks after parabiosis. (n=5; t-test- *p< 0.05). B, Mature adult (6 months) mice were given unilateral stereotaxic (STX) injections of lentivirus (LV) encoding TET2 or control LV into contralateral dentate gyrus (DG). Schematic and qPCR of TET2 mRNA from hippocampal lysates following STX injection. (n=5; t-test- *p<0.05). C, Schematic illustrating stereotaxic injection paradigm and experimental timeline. D, Association of regions of 5hmC gained after TET2 overexpression with genomic elements (DhMRs gained; 558 DhMRs at q=0.05). Pie chart depicts overall loss and gain of DhMRs after TET2 overexpression (OE). E, Top Gene Ontology terms (2-fold enrichment; ordered by p-value) for genes associated with gained DhMRs after TET2 overexpression (OE).

[0122] FIG. 10F: Venn diagram representing the overlap of genes paired with DhMRs from those lost during aging (3 month over 18 month) and those gained from TET2 overexpression (OE over Control). From the overlap, genes associated with neurogenesis using Gene Ontology are shown and mRNA expression in hippocampal lysates was measured by qPCR. (n=5; t-test; *p<0.05, **p<0.01, ***p<0.001). G, Neurogenesis was analyzed by immunostaining and confocal microscopy in mature adult (6 months) animals after LV administration. As a reference, young adult (3 months) mice were given unilateral STX injections of control LV. All mice were administered BrdU by intraperitoneal injections for six days and euthanized 30 days later. Representative field and quantification of GFAP/Sox2 -positive, Dcx-positive, BrdU-positive, and NeuN/BrdU-positive cells. (n=5; t-test; *p<0.05, **p<0.01, ***p<0.001). H, Schematic of experimental paradigm and cognitive testing timeline. Mature adult (6 months) wild type mice were given bilateral stereotaxic (STX) injections of lentivirus (LV) encoding TET2 or control LV into the dentate gyrus (DG). I, Associative fear memory was assessed using contextual fear conditioning. Quantification of percent freezing 24 hours after training. (n=12-15 per group; t-test; ***p<0.001). Data are represented as mean+SEM.

[0123] FIG. 11 : Characterization of hMeDIP- sequencing in the mature hippocampus, and adult neurogenesis in the young hippocampus, following TET2 overexpression. A, Schematic and quantitative reverse-transcription PCR of TET2 mRNA from hippocampal lysates of aged (18 months) isochronic and heterochronic parabionts five weeks after parabiosis. (n= 5 per group; t-test; not significant) A,B, Mature adult (6 months) mice were given unilateral stereotaxic (STX) injections of lentivims (LV) encoding TET2 or control LV into contralateral dentate gyrus (DG). Schematic of lentiviral vector generated to overexpress TET2. Abbreviations: AMP, ampicillin, pCMV, cytomegalovirus promoter (A). Quantitative reverse-transcription PCR of TET1 and TET3 mRNA from hippocampal lysates following STX injection (B). (n=5 per group; t-test; not significant). D, Chromosome coverage of hMeDIP-Seq reads from control (dark grey), overexpression (orange), and input samples (black) normalized to reads per million mapped. E, Scatterplot of control versus overexpression hippocampi using 50kb bins. Reads are normalized as FPKM. F, Metagene read coverage in FPKM for RefSeq genes of control (dark grey) and TET2 overexpression (orange). The gene body was scaled to 5kb for all genes. 2.5kb upstream of the TSS and 2.5kb downstream of the TES are shown.

[0124] FIG. 11G: Read coverage in FPKM for RefSeq promoters. 2.5kb upstream and downstream of the TSS is shown. H, Two examples of DhMRs that were gained after TET2 overexpression. I, Two examples of genes that were associated with DhMRs that were lost with aging and gained after TET2 overexpression are shown. In the example near Klf5, the DhMR is shared, while the example with Igflr has two associated DhMRs. Examples of young (3 month, black), aged (18 month, grey), control (dark grey), and TET2 overexpression (orange) samples are shown. J, Young adult (3 months) mice were given unilateral stereotaxic (STX) injections of lentivims (FV) encoding TET2 or control FV into contralateral dentate gyrus (DG). Neurogenesis was analyzed by immunostaining and confocal microscopy after FV administration. All mice were administered BrdU by intraperitoneal injections for six days and euthanized 30 days later. Quantification of GFAP/Sox2- positive, Dcx-positive, BrdU-positive, and NeuN/BrdU-positive cells. (n=4 per group; t-test; not significant). K, Mature adult (6 months) wild type mice were given bilateral stereotaxic injections of lentivims (FV) encoding TET2 or control FV into the dentate gyms (DG). Cued fear memory was assessed using contextual fear conditioning. Quantification of percent freezing 24 hours after training. (n=12-15 per group; t-test; not significant; t-test). Data are represented as mean ± SEM.

[0125] FIGS. 12 A to 12C provide data from an experiment in which lentivims (FV) encoding TET2 was administered to mice by injection into the dentate gyms. FIG. 12A shows the experimental design with injection occurring at 3 months of age and neurogenesis assessment occurring at 16 months of age. FIG. 12B shows the results from the dentate gyms injected with FV control (left panel) or FV TET2 (right panel). Cells were labeled for expression of Doublecortin (DCX)

(arrowheads) or for nuclear staining with DAPI (background). FIG. 12 C shows the quantification of DCX labeled cells per dentate gyms in FV control (left) and FV TET2 (right) injected hippocampi. TET2 overexpression resulted in a significant increase in DCX positive cells per dentate gyms, compared with controls. [0126] While the invention has been described with reference to the specific examples and illustrations, changes can be made and equivalents can be substituted to adapt to a particular context or intended use as a matter of routine development and optimization and within the purview of one of ordinary skill in the art, thereby achieving benefits of the invention without departing from the scope of what is claimed.

Claims

CLAIMS The invention claimed is:

1. A method of improving neurological function or preventing neurological decline in a subject, comprising increasing activity of a ten-eleven translocation methylcytosine dioxygenase (TET) in neurological tissue of the subject.

2. The method of claim 1, wherein the TET is Tet methylcytosine dioxygenase 2 (TET2).

3. The method of claim 1 or claim 2, wherein the neurological tissue is the brain.

4. The method of any of claims 1 to 3, which is a method for improving cognition or preventing cognitive decline.

5. The method of any of claims 1 to 4, wherein the activity of TET is increased by administering to the subject an expression vector encoding the TET.

6. The method of claim 5 wherein the expression vector is a non-integrating vector such as adeno-associated virus (AAV).

7. The method of claim 5 or claim 6, wherein the TET in the vector is expressed under control of a promoter that selectively causes expression in neural stem cells, neural progenitor cells, neurons, or glial cells.

8. The method of any of claims 5 to 7, wherein the TET in the vector is expressed under control of an inducible promoter.

9. The method of any of claims 5 to 8, where the TET is fused to a destabilizing domain such as ddDHFR or ddFKBP.

10. The method of any of claims 5 to 9, where the untranslated region (UTR) of the TET in the vector contains one or more copies of a miRNA target sequence, such as miR-124.

11. The method of any of claims 1 to 4, wherein the activity of TET is increased by administering to the subject an oligonucleotide that increases TET expression in the subject.

12. The method of claim 11, wherein the oligonucleotide is a decoy, blockmir or antagomir that is complementary to a negative miRNA regulator of TET, such as miR-125b, miR-29b, miR-29c, miR-101, or miR-7.

13. The method of claim 11 or claim 12, wherein the subject is administered a vector that encodes said oligonucleotide.

14. The method of any of claims 1 to 4, wherein the activity of TET is increased by administering to the subject a small molecule agonist of TET.

15. A pharmaceutical composition for improving neurological function or preventing

neurological decline in a subject, comprising an effective amount of an expression vector that encodes a translocation methylcytosine dioxygenase (TET).

16. The pharmaceutical composition of claim 15, wherein the expression vector is a

non-integrating vector such as adeno-associated vims (AAV).

17. The pharmaceutical composition of claim 15 or claim 16, wherein the TET in the vector is expressed under control of a promoter that selectively causes expression in neurons or neural progenitor cells.

18. The pharmaceutical composition of any of claims 15 to 17, wherein the TET in the vector is expressed under control of an inducible promoter.

19. The pharmaceutical composition of any of claims 15 to 18, where the TET is fused to a

destabilizing domain such as ddDHFR or ddFKBP.

20. The pharmaceutical composition of any of claims 15 to 19, where the untranslated region (UTR) of the TET in the vector contains one or more copies of a miRNA target sequence, such as miR-124.

21. A pharmaceutical composition for improving neurological function or preventing

neurological decline in a subject, comprising an effective amount of an oligonucleotide that increases TET expression in the subject.

22. The pharmaceutical composition of claim 21, wherein the oligonucleotide is a decoy, blockmir or antagomir that is complementary to a negative miRNA regulator of TET, such as miR-125b, miR-29b, miR-29c, miR-101, or miR-7.

23. The pharmaceutical composition of claim 21 or claim 22, comprising a vector that encodes said oligonucleotide.

24. A pharmaceutical composition for improving neurological function or preventing

neurological decline in a subject, comprising an effective amount of a small molecule agonist of TET.

25. The pharmaceutical composition of any of claims 15 to 24, wherein the TET is Tet

methylcytosine dioxygenase 2 (TET2).

26. The pharmaceutical composition of any of claims 15 to 25, for use in improving cognition or preventing cognitive decline in the subject.

27. A method for identifying a small molecule agonist of TET2 that is effective for improving neurological function or preventing neurological decline in a subject, comprising:

screening a library of small molecule compounds using an assay that measures TET2 activity;

identifying compounds in the library that increase TET2 activity; and

further screening the identified compounds for their ability to increase

5-hydroxymethylcytosine (5hmC) in genomic DNA of replication-capable neural cells or neural progenitor cells.

28. A pharmaceutical composition for improving neurological function or preventing

neurological decline in a subject, comprising an effective amount of a small molecule agonist identified according to the method of claim 27.