Methods and compositions for rejuvenating CNS glial populations by repression of transcription factors

Abstract

The present invention relates to methods of inducing rejuvenation in a population of adult glial progenitor cells and methods of treating a subject having a myelin deficiency.

Description

[Technical field]

[0001] This application claims priority from U.S. Provisional Patent Application No. 63 / 257,827, filed October 20, 2021, and U.S. Provisional Patent Application Nos. 63 / 350,039, 63 / 350,041, and 63 / 350,042, filed June 8, 2022, which are hereby incorporated by reference herein.

[0002] This invention was made with Government support under awards NS110776 and AG072298 given by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Field The present invention relates to methods and compositions for rejuvenating CNS glial populations using agents that repress transcription factors. [Background technology]

[0004] background Glial progenitor cells (GPCs, also called oligodendrocyte precursor cells and NG2 cells) colonize the human brain during development and persist in abundance throughout adulthood. During development, human GPCs (hGPCs) are highly proliferative bipotential cells that give rise to new oligodendrocytes and astrocytes (French-Constant and Raff, "Proliferating Bipotential Glial Progenitor Cells in Adult Rat Optic Nerve," Nature 319:499-502 (1986) and Raff et al., "A Glial Progenitor Cell that Develops in Vitro into an Astrocyte or an Oligodendrocyte Depending on Culture Medium," Nature 303:390-396 (1983)). In rodents, this ability declines during normal aging, with proliferation, migration, and differentiation competence all being attenuated in aged GPCs (Chari et al., "Decline in Rate of Colonization of Oligodendrocyte Progenitor Cell (OPC)-depleted Tissue by Adult OPCs With Age," J. Neuropathol. Exp. Neurol. 62:908-916 (2003); Gao and Raff, "Cell Size Control and a Cell-intrinsic Maturation Program in Proliferating Oligodendrocyte Precursor Cells," J. Cell Biol.138:1367~1377(1997); Moyon et al., "TET1-mediated DNA Hydroxymethylation Regulates Adult Remyelination in Mice", Nature Communications 12:3359~3359(2021); Segel et al., "Niche Stiffness Underlies the Ageing of Central Nervous System Progenitor Cells", Nature 573:130~134(2019); Tang et al., "Long-Term Culture of Purified Postnatal Oligodendrocyte Precursor Cells. Evidence for an Intrinsic Maturation Program that Plays Out Over Months", J. Cell Biol. 148:971~984(2000); Temple and Raff, "Clonal Analysis of Oligodendrocyte Development in Culture: Evidence for a Developmental Clock that Counts Cell Divisions", Cell 44:773~779(1986); Wolswijk and Noble, "Identification of an Adult-Specific Glial Progenitor Cell", Development 105:387~400(1989); and Wren et al., "In Vitro Analysis of the Origin and Maintenance of O-2A adult Progenitor Cells", J. Cell Biol.116,:167-176 (1992)). Similarly, adult human GPCs were previously found to be less proliferative, less migratory, and more readily differentiated than their fetal counterparts when transplanted into congenitally dysmyelinated mouse hosts (Windrem et al., "Fetal and Adult Human Oligodendrocyte Progenitor Cell Isolates Myelinate the Congenitally Dysmyelinated Brain," Nat. Med. 10:93-97 (2004)). However, there is a wealth of data on the distinct competence of fetal and adult hGPCs, as well as GPC transcription in rodent models of aging (Bouhrara et al., "Evidence of Demyelination in Mild Cognitive Impairment and Dementia Using a Direct and Specific Magnetic Resonance Imaging Measure of Myelin Content," Alzheimers Dement. 14:998-1004 (2018); de la Fuente et al., "Changes in the Oligodendrocyte Progenitor Cell Proteome with Ageing," Mol.Cell Proteomics 19:1281-1302 (2020); Neumann et al., "Metformin Restores CNS Remyelination Capacity by Rejuvenating Aged Stem Cells", Cell Stem Cell 25:473-485 e478 (2019); and Spitzer et al., "Oligodendrocyte Progenitor Cells Become Regionally Diverse and Heterogeneous with Age", Neuron 101:459-471 e455 (2019)), despite the lack of data addressing changes in GPC gene expression during human aging (Perlman et al., "Developmental Trajectory of Oligodendrocyte Progenitor Cells in the Human Brain Revealed by Single Cell RNA Sequencing", Glia 68:1291-1303 (2020) and Sim et al., "Complementary Patterns of Gene Expression by Human Oligodendrocyte Progenitors and Their Environmental Predict Determinants of Progenitor Maintenance and Differentiation," Ann. Neurol. 59:763-779 (2006)), or data providing a clear head-to-head comparison of transcription by fetal and adult human GPCs are largely unavailable. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention is directed to overcoming deficiencies in the art. [Means for solving the problem]

[0006] One aspect of the present invention relates to a method of inducing rejuvenation in a population of adult glial progenitor cells, comprising administering to the population of adult glial progenitor cells an effective amount of an agent that represses one or more transcription factors selected from the group consisting of: (i) zinc finger protein 274 (ZNF274), (ii) Myc-associated factor X (MAX), (iii) E2F transcription factor 6 (E2F6), (iv) zinc finger protein Aiolos (IKZF3), and (v) signal transduction and activator of transcription 3 (STAT3).

[0007] Another aspect of the present invention relates to a method for treating a subject having a glial cell-related disorder, comprising administering to the subject an effective amount of an agent that suppresses one or more transcription factors selected from the group consisting of ZNF274, MAX, E2F6, IKZF3 and STAT3. [Brief description of the drawings]

[0008] [Figure 1-1] Figure 1 shows bulk RNA-Seq characterization of human fetal GPC. Panel A: Bulk and scRNA sequencing workflow of CD140a+, CD140a-, and A2B5+ / PSA-NCAM--selected second trimester human fetal brain isolates. Panel B: Principal component analysis of all samples across two batches. [Figure 1-2] Figure 1 shows bulk RNA-Seq characterization of human fetal GPC. Panel C: Venn diagram of CD140a+ vs. CD140a- and CD140+ vs. A2B5+ / PSA-NCAM- differentially expressed gene sets (p<0.01 and absolute Log2 fold change>1). [Figure 1-3] Figure 1 shows bulk RNA-Seq characterization of human fetal GPC. Panel D: Significant Ingenuity Pathway Analysis terms for both gene sets. Size represents -log10 p-value and color represents activation Z-score (blue, CD140a+; red, A2B5+ or CD140a-). [Figure 1-3]Figure 1 shows bulk RNA-Seq characterization of human fetal GPC. Panel E: Log2 fold change of significant genes in both gene sets. No bar was not significant. [Figure 1-4] Bulk RNA-Seq characterization of human fetal GPC. Panel F: Heatmap of transformed transcripts per million (TPM) of 19 selected genes from Panel E. [Figure 2-1] Figure 1: Single-cell RNA-seq of CD140a and A2B5 selected human fetal GPCs. Panel A: UMAP plot of the main cell types identified during scRNA-Seq analysis of FACS-isolated hGPCs derived from the VZ / SVZ of human fetuses at 20 weeks gestation. [Figure 2-2] Single cell RNA-seq of CD140a and A2B5 selected human fetal GPCs. Panels B-C: UMAP of only PSA-NCAM- / A2B5+ (B) or CD140a+ (C) human fetal cells. [Figure 2-3] Single cell RNA-seq of CD140a and A2B5 selected human fetal GPCs. Panel D: Violin plot of cell type selection marker genes. [Figure 2-4] Single cell RNA-seq of CD140a and A2B5 selected human fetal GPCs, Panel E: Volcano plot of GPC vs. pre-GPC populations. [Figure 2-5] Figure 1 shows single cell RNA-seq of CD140a and A2B5 selected human fetal GPCs.Panel F: Signature plot of selected genes differentially expressed between GPCs and pre-GPCs. [Figure 2-6] Figure 1 shows single cell RNA-seq of CD140a and A2B5 selected human fetal GPC. Panel G: Selected significantly enriched GPC and pre-GPC IPA terms with -log10 p-values ​​and activation Z-scores shown. [Diagram 2-7]Figure 1 shows single cell RNA-seq of CD140a and A2B5 selected human fetal GPCs. Panel H: Selected feature plot of transcription factors predicted to be significantly activated in fetal hGPCs. Relative transcription factor regulon activation calculated using the SCENIC package is shown. [Figure 3-1] Figure 1 shows that adult human GPC is transcriptionally and functionally distinct from fetal GPC. Panel A: Workflow for bulk RNA-Seq analysis of human adult and fetal GPC. Panel B: Principal component analysis of all samples across three batches. [Figure 3-2] Figure 13: Adult human GPC is transcriptionally and functionally distinct from fetal GPC. Panel C: Venn diagram of both adult vs. fetal differentially expressed gene sets. [Figure 3-3] (A) Adult human GPC is transcriptionally and functionally distinct from fetal GPC. Panel D: IPA network of curated terms and genes. Node size is proportional to node degree. Label color corresponds to enrichment of either adult (red) or fetal (blue) populations. [Diagram 3-4] FIG. 1 shows that adult human GPC is transcriptionally and functionally distinct from fetal GPC. Panel E: Bar graph of significant IPA terms per module. Z-score indicates predicted activation of fetal (blue) or adult (red) hGPC. [Figure 3-5] Figure 1 shows that adult human GPC is transcriptionally and functionally distinct from fetal GPC. Panel F: Bar graph and heatmap of Log2 fold change in TPM of network genes. [Figure 4-1] Figure 1 shows that predicted transcription factor activity suggests the involvement of a set of transcriptional repressors in establishing adult hGPC identity. Panel A: Normalized enrichment score plot of significantly enriched transcription factors predicted to be active in fetal and adult GPCs. Each dot is a motif, the size of which indicates the number of genes in which the motif is predicted to be active, and the color represents the window near the promoter in which the motif is found to be enriched. [Figure 4-2](A) Estimation of transcription factor activity suggests the involvement of a set of transcriptional repressors in establishing adult hGPC identity. Panel B: Heatmap of enriched TF TPMs. [Figure 4-3] Figure 1 shows that estimation of transcription factor activity suggests the involvement of a set of transcriptional repressors in establishing adult hGPC identity.Panel C: Log fold change for both fetal hGPC isolates versus adult GPC. [Figure 4-4] Figure 1 shows that predicted transcription factor activity implicates a set of transcriptional repressors in establishing adult hGPC identity.Panel D: Fetal activators. [Figure 4-5] Figure 1 shows that predicted transcription factor activity suggests the involvement of a set of transcriptional repressors in establishing adult hGPC identity.Panel E: Fetal repressors. [Figure 4-6] Figure 1 shows that predicted transcription factor activity suggests the involvement of a set of transcriptional repressors in establishing adult hGPC identity.Panel F: Adult activators. [Diagram 4-7] Figure 2: Estimation of transcription factor activity suggests the involvement of a set of transcriptional repressors in establishing adult hGPC identity. Panel G: Direct transcription factor activity prediction of select genes partitioned into adult repressors. Color indicates differential expression in either adult (red) or fetal (blue) hGPCs. Shape indicates node type (octagon, repressor; rectangle, activator; oval, other target genes). Boxed and circled genes indicate functionally related genes that contribute to either glial progenitor / oligodendrocyte identity, senescence / proliferation targets, or upstream or downstream TFs that are also likely activated. [Figure 5-1]Figure 1 shows induction of the aged GPC transcriptome by adult hGPC-enriched repressors. Panel A: Schematic outline of the structure of four different doxycycline (Dox)-inducible EGFP lentiviral expression vectors, each encoding one of the transcriptional repressors: E2F6, IKZF3, MAX, or ZNF274. Panel B: Induced pluripotent stem cell (iPSC)-derived hGPC cultures (C27 line (Chambers et al., Nature biotechnology, 27:275-280, 2009; Wang et al., Cell Stem Cell 12:252-264, 2013)) were transduced with a single lentivirus or vehicle for 1 day and then treated with Dox for the remainder of the experiment. Three, seven, and ten days after the initiation of Dox-inducible transgene expression, hGPCs were FACS-isolated for qPCR. [Figure 5-2] Figure 1. Induction of the aged GPC transcriptome by adult hGPC-enriched repressors. Panel C: qPCR of Dox-treated cells showing expression of each transcription factor compared to matched timepoint controls. Panel D: qPCR fold change heatmap of selected aging-associated genes. Within-timepoint comparisons to controls were calculated by post-hoc least squares means tests of linear models after regression of cell batch effects. FDR adjusted p-values: *<0.05, **<0.01, ***<0.001. [Figure 6-1] Figure 1: miRNAs drive adult GPC transcriptional divergence in parallel with transcription factor activity.Panel A: Principal component analysis of miRNA microarray samples derived from human A2B5+ adult GPCs and CD140a+ fetal GPCs. [Figure 6-2] Figure 2: miRNAs drive adult GPC transcriptional divergence in parallel with transcription factor activity. Panel B: Bar graph and heat map of Log2 fold change of differentially expressed miRNAs. [Figure 6-3] Figure 1: miRNAs drive adult GPC transcriptional divergence in parallel with transcription factor activity.Panel C: Enrichment profile bubble plot of miRNAs against the average log2FC of their predicted gene targets. [Figure 6-4]Figure 1 shows that miRNAs drive adult GPC transcriptional divergence in parallel with transcription factor activity.Panel D: Curated signaling network of fetal (top) enriched miRNAs and their predicted targets. [Figure 6-5] Figure 1: miRNAs drive adult GPC transcriptional divergence in parallel with transcription factor activity.Panel E: Curated signaling network of adult (bottom) enriched miRNAs and their predicted targets. [Figure 7-1] Figure 1 shows enrichment of human fetal GPCs by CD140a+ or A2B5+ / PSA-NCAM-selection. Panel A: Principal component analysis of CD140a+ and A2B5+ fetal GPCs. Panel B: Volcano plot showing significant A2B5 (green) and CD140a (blue) enriched genes. [Figure 7-2] FIG. 1 shows enrichment of human fetal GPCs by CD140a+ or A2B5+ / PSA-NCAM-selection. Panel C: Principal component analysis of CD140a+ and CD140a- fetal cells. Panel D: Volcano plot showing significant CD140a- (crimson) and CD140a (blue) enriched genes. Panel E. Upset plot of significantly up- and down-regulated genes in both gene sets. [Figure 8-1] FIG. 2 shows single cell RNA-Seq quality filtering in relation to FIG. 2. Panel A: Violin plot of A2B5+ / PSA-NCAM- capture without filtering. Panel B: Violin plot of CD140a scRNA-seq capture. [Figure 8-2] FIG. 2 shows single-cell RNA-Seq quality filtering in relation to FIG. Panel C: A2B5+ / PSA-NCAM-capture. Panel D: Violin plot after quality filtering (mitochondrial gene expression percent <15% and >500 unique genes) of CD140a+ capture. [Figure 9-1] Related to Figure 2: Isolated cell RNA sequencing of PSA-NCAM- / A2B5+ vs. CD140a+ fetal hGPCs. Panel A: UMAP plot of A2B5+ fetal hGPCs and CD140a+ fetal hGPCs. [Figure 9-2] Related to Figure 2, isolated cell RNA sequencing of PSA-NCAM- / A2B5+ vs. CD140a+ fetal hGPC. Panel B: Frequency of cell types in each sorting paradigm isolate. Panel C: Scatter plot of differentially expressed bulk RNA-Seq Log2 fold change vs. pseudo bulk Log2 fold change between CD140a+ and A2B5+ fetal hGPC isolates. [Figure 10] In connection with Figure 4, shared motifs of active transcription factors in fetal or adult hGPCs: A matrix of all predicted active transcription factors in fetal and adult GPCs. Size and color indicate the degree of shared motifs between transcription factors. [Figure 11] Adult repressor isoform expression in relation to Figure 5. Bar graph of transcripts per million (TPM) of all proteins encoding adult repressor isoforms in each GPC group. [Figure 12] Related to Figure 5, bulk RNA-Seq of iPSC-derived hGPCs reveals concordance in abundance of aging-associated genes. iPSC-derived hGPCs (C27) were CD140a+ FACS isolated and assayed by bulk RNA sequencing. Abundance of relevant glial aging-associated genes, including those in our active transcription factor cohort, is shown aligned with fetal and adult hGPC data. [Figure 13-1] Related to Figure 6, transcription factor regulation of miRNAs provides post-transcriptional modulation of glial aging gene expression. Panel (A): Log2 FC violin plot of significant adult vs. fetal GPC transcription factors predicted to be upstream of differentially expressed adult vs. fetal GPC miRNAs. [Figure 13-2] In relation to Figure 6, transcription factor regulation of miRNAs provides post-transcriptional modulation of glial aging gene expression. Panel (B): Network of identified transcription factors in Figure 2 and their regulatory predictions for differentially expressed adult vs. fetal hGPC miRNAs. [Figure 14] Panel A: Design of CBh-BCL11 A-GFP and control CBh-GFP lentiviruses. Panel B: Generation of CRISPR-Cas9-modified C27 iPSC lines expressing tRFP-mScarlet derived from the AAVS1 locus. Panel C: Schematic illustrating the experimental paradigm for long-term chimerism and BCL11A overexpression in Rag1 mice. Panel D: BCL11A overexpression by CBhBCL11A-GFP confirmed by ddPCR in vitro and Panel E: immunohistochemistry in vivo. [Figure 15] Panel A: Coronal sections of Rag1-C271RFP mouse corpus callosum (CC) 3 weeks after injection with CBh-BCL11A-GFP (L) or CBH-GFP (R). Panel B: Enlarged coronal sections of (CC) showing differences in RFP-tagged human cells and OLIG2 between BCL11A-GFP and GFP-only hemispheres. Panel D: Enlarged coronal sections of (CC) showing differences in RFP-tagged human cells and mouse NG2 between BCL11A-GFP and GFP-only hemispheres. [Figure 16-1] Panel A: Coronal images of chimeric mouse corpus callosum (CC) 3 weeks after injection with CBh-BCL11A-GFP (L) or CBh-GFP (R). Human cells are marked with tRFP / mScarlet, human nuclear antigen (hNA) is stained red, and OLIG2 is stained green. Panel B: Coronal images of chimeric mouse CC 6 weeks after injection. Human cells are marked with tRFP / mScarlet, hNA is stained red, and OLIG2 is stained green. [Figure 16-2] Panel C: Coronal image of chimeric mouse CC 3 weeks after injection. Human cells are marked with tRFP / mScarlet, hNA in red, and PDGFRa in green. Panel D: Coronal image of chimeric mouse CC 3 weeks after injection. Human cells are marked with tRFP / mScarlet, hNA in red, and NG2 in green. [Figure 17]FIG. 1 shows an exemplary design of a BCL11A expression vector. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Reference shall now be made in detail to certain aspects and exemplary embodiments of the present invention, showing examples of structures and drawings in the accompanying drawings. Aspects of the present invention will be described in conjunction with exemplary embodiments, including methods, materials, and examples. Such description is not limiting, and the scope of the present invention is intended to encompass all equivalents, alternatives, and modifications, either generally known or incorporated herein. The described aspects, features, advantages, and characteristics of the present invention may be combined in any suitable manner in one or more further embodiments. Those skilled in the art will recognize that the present invention may be practiced without one or more of the specific aspects or advantages of a particular embodiment. In other instances, additional aspects, features, and advantages may be recognized and claimed in a particular embodiment, which may not be present in all embodiments of the present invention. Moreover, those skilled in the art will recognize numerous techniques and materials similar or equivalent to those described herein. They may be used in the practice of the aspects and embodiments of the present invention. The described aspects and embodiments of the present invention are not limited to the described methods and materials.

[0010] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0011] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "peptide" includes "one or more" peptides or "a plurality" of such peptides.

[0012] I. Definition As used herein, the following terms or phrases (in brackets) shall have the following meanings:

[0013] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "peptide" includes "one or more" peptides or "a plurality" of such peptides.

[0014] The term "about" or "approximately" includes being within a statistically meaningful range of values. Such a range may be within the same order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The acceptable variation encompassed by the term "about" or "approximately" depends on the particular system under study and can be readily appreciated by one of ordinary skill in the art.

[0015] The term "and / or" as used herein means that the listed items are present or used either individually or in combination. Thus, this term means that "at least one" or "one or more" of the listed items are used or present.

[0016] As will be understood by those skilled in the art, for any and all purposes, including providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of those subranges. It can be easily recognized that any recited range is fully descriptive and allows for the same range to be divided into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be easily divided into a lower third, middle third, upper third, etc. As will also be understood by those skilled in the art, all terms such as "up to" and "at least" refer to ranges that include the recited numerical values ​​and can then be divided into subranges as discussed above. Finally, as will be understood by those skilled in the art, a range includes each individual member.

[0017] In understanding the scope of the present invention, the term "comprising" and its derivatives, as used herein, are intended to be open-ended terms specifying the presence of stated features, elements, components, groups, integers, and / or steps, but not excluding the presence of other unstated features, elements, components, integers, and / or steps. The above also applies to words of similar meaning, such as the terms "including," "involving," and "having," and their derivatives. The term "consisting" and its derivatives, as used herein, are intended to be closed terms specifying the presence of stated features, elements, components, groups, integers, and / or steps, but not excluding the presence of other unstated features, elements, components, integers, and / or steps. The term "consisting essentially of," as used herein, is intended to specify the presence of the stated features, elements, groups, integers, and / or steps, as well as things that do not materially affect the basic and novel characteristic(s) of the features, elements, groups, integers, and / or steps. In embodiments or claims in which the term "comprising" (and the like) is used as a transitional phrase, embodiments in which the term "comprising" is replaced with the term "consisting of" or "consisting essentially of" can also be envisioned. The disclosed methods, kits, systems, and / or compositions may comprise, consist essentially of, or consist of the disclosed components.

[0018] In embodiments that include an "additional" or "second" component, the second component, as that term is used herein, is different from the other component or the first component. A "third" component is different from the other, first, and second components, and further recited or "additional" components are similarly different.

[0019] The term "complementary" when used with respect to nucleic acids refers to base pairing of A with T or U, and G with C. The term "complementary" refers to nucleic acid molecules that are fully complementary, i.e., A with T or U pairings and G with C pairings are formed throughout the reference sequence, as well as molecules that are partially complementary (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%).

[0020] The terms "nucleic acid," "nucleotide," and "polynucleotide" include both DNA and RNA, unless otherwise specified.

[0021] The terms "polypeptide," "peptide," or "protein" are used interchangeably and refer to a polymer of amino acid residues. These terms encompass all types of naturally occurring and synthetic proteins, including protein fragments of any length, fusion proteins, and modified proteins, including, but not limited to, glycoproteins and any other type of modified protein (e.g., phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, pegylation, biotinylation, etc.).

[0022] The terms "abrogate," "abrogation," "eliminate," or "elimination," with respect to expression of a gene or gene product (e.g., RNA or protein), refer to the complete loss of transcription and / or translation of a gene or the complete loss of a gene product (e.g., RNA or protein). Expression of a gene or gene product (e.g., RNA or protein) can be detected by methods known in the art, such as those described herein, relative to a control, e.g., an unmodified cell.

[0023] The terms "express" and "expression" mean to enable or cause the information in a gene or DNA sequence to be embodied, e.g., an RNA or protein is produced, by activating cellular functions involved in the transcription and / or translation of the corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an "expression product," such as an RNA or protein. The expression product itself, e.g., the resulting protein, can also be said to be "expressed" by the cell. Expression products can be characterized as being intracellular, extracellular, or transmembrane.

[0024] As used herein, the term "glial cells" refers to a population of non-neuronal cells that provide support and nutrients, maintain homeostasis, either form or promote myelination, and participate in signal transmission in the nervous system. "Glial cells," as used herein, encompass fully differentiated cells of the glial lineage, such as oligodendrocytes or astrocytes, as well as glial precursor cells, each of which may be referred to as astroglial cells.

[0025] As used herein, the term "adult glial progenitor cells" refers to glial progenitor cells present in a mammal at any stage of postnatal development. In some embodiments, "adult glial progenitor cells" refers to glial progenitor cells present in a human subject that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 years of age or older. In some embodiments, the term "adult glial progenitor cells" refers to glial progenitor cells present in a human subject that is 20 years of age or older, 25 years of age or older, 30 years of age or older, 35 years of age or older, 40 years of age or older, 45 years of age or older, or 50 years of age or older. In some embodiments, the term "adult glial progenitor cells" refers to glial progenitor cells present in an older human subject, such as an adult that is 55 years of age or older, 60 years of age or older, 65 years of age or older, 70 years of age or older, 75 years of age or older, or 80 years of age or older.

[0026] The term "functional variant" of a gene product (e.g., a transcription factor) refers to a modified transcription factor (e.g., modified by deletion, substitution, insertion, glycosylation, etc.) that retains at least 50% of the biological activity of the unmodified (wild-type) transcription factor in a competitive assay.

[0027] The term "effective amount" refers to that amount of an active compound or pharmaceutical agent that elicits the biological or medical response in a tissue, system, animal, individual, or human that is desired by a researcher, veterinarian, physician, or other clinician.

[0028] The term "regulatory sequence" or "regulatory element" refers to a nucleic acid sequence or element that controls, regulates, causes, or enables the expression of a gene regulated by such regulatory sequence or element. Regulatory elements / sequences can be found 5' or 3' to or within the coding region of the regulated gene, or within an intron. Examples of regulatory sequences / elements include, but are not limited to, promoters, enhancers, RNA polymerase initiation sites, ribosome binding sites, and other sequences that facilitate expression of an encoded polypeptide in a given expression system.

[0029] The term "promoter" as used herein refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, a polynucleotide of interest is located 3' of the promoter sequence. In some embodiments, the promoter is derived entirely from a native gene. In some embodiments, the promoter is composed of different elements derived from different naturally occurring promoters. In some embodiments, the promoter comprises a synthetic nucleotide sequence. Those skilled in the art will understand that different promoters will direct the expression of a gene in different tissues or cell types, or at different developmental stages, or in response to different environmental conditions, or in response to the presence or absence of a drug or transcriptional cofactor. Ubiquitous, cell type-specific, tissue-specific, developmental stage-specific, and conditional promoters, such as drug-responsive promoters (e.g., tetracycline-responsive promoters), are well known to those skilled in the art. Examples of promoters include, but are not limited to, phophoglycerate kinase (PKG) promoter, CAG, NSE (neuron specific enolase), synapsin or NeuN promoter, SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter, such as CMV immediate early promoter region (CMVIE), SFFV promoter, Rous sarcoma virus (RSV) promoter, synthetic promoters, and hybrid promoters. The promoter may be of human origin or may be derived from other species, including mouse origin. In addition, sequences derived from non-viral genes, such as mouse metallothionein gene promoter, could also be used herein. In some embodiments, the promoter is a heterologous promoter. In some embodiments, the promoter sequence consists of proximal and more distal upstream elements and may include enhancer elements.

[0030] The term "heterologous promoter," as used herein, refers to a promoter that is not found operably linked to a given coding sequence in nature.

[0031] The term "enhancer" refers to a nucleotide sequence that can stimulate promoter activity and may be a native element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of the promoter.

[0032] The terms "operably linked" or "operably linked" refer to the association of two or more nucleic acid fragments on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence if it is capable of affecting the expression of that coding sequence (e.g., the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation.

[0033] The term "transcription factor" refers to a DNA-binding protein that regulates the expression of specific genes.

[0034] Transcription factors can have a positive effect on gene transcription and therefore can be referred to as "transcription activators," "activators," or "transcriptional activation factors." An exemplary activator (or transcriptional activation factor) is signal transduction and activator of transcription 3 (STAT3). As shown in FIG. 4F of the present disclosure, in the context of adult glial progenitor cells, STAT3 is predicted to activate a set of senescence-related genes (e.g., BIN1, DMTF1, CD47, CTNNA1, RUNX2, RUNX1, MAP3K7, and OGT), glial cell-related genes (e.g., PLP1, CNP, PMP22, SEMA4D, CLDN11, GPR37, MYRF, MAG, BCAS1, ST18, ERBB4, CERS2, LPAR1, and GJB1), and downstream transcription factors (e.g., MAX, E2F6, and IKZF3).

[0035] Transcription factors can also negatively affect gene expression and therefore may be referred to as "transcriptional repressors," "repressors," or "transcriptional repressors." Exemplary repressors (or transcriptional repressors) involved in glial progenitor cell senescence include, without limitation, ZNF274, MAX, E2F6, and IKZF3. As shown in FIG. 4G, in the context of adult glial progenitor cells, ZNF274, MAX, E2F6, and IKZF3 are predicted to repress a set of proliferation-related gene targets (e.g., YAP1, LMNB1, PATZ1, TEAD1, FN1, TP53, CDK1, CCND2, CDKN2D, CENPH, MKI67, CDK4, CENPF, CDK5, CDKN3, and CHEK1), glial cell-related genes (e.g., CHRDL1, ST8SIA1, PTPRZ1, CA10, PDGFRA, BCAN, NXPH1, CSPG4, and PCDH15), and downstream transcription factors (e.g., BLC11A, EZH2, HDAC2, NF1B, MYC, HMGA2, and TEAD).

[0036] The term "transcription factor inhibitor" or "transcription factor repressor" refers to an agent that inhibits the activity or expression of a transcription factor. A "transcription factor inhibitor" or a "transcription factor repressor" can be a small molecule, a polypeptide, a polynucleotide, such as an antisense oligonucleotide (ASO), shRNA, or miRNA.

[0037] Certain terms used in the specification, examples, and claims are summarized here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0038] Preferences and options for any given aspect, feature, embodiment or parameter herein should be considered as disclosed in combination with any and all preferences and options for all other aspects, features, embodiments and parameters of the invention, unless the context indicates otherwise.

[0039] II. Methods Involving Activation of Transcription Factors One aspect of the present invention relates to a method for inducing rejuvenation in adult glial progenitor cells by activating certain transcription factors. In some embodiments, the method comprises expressing an effective amount of one or more transcription factors selected from the group consisting of B-cell lymphoma / leukemia 11A (BCL11A), histone deacetylase 2 (HDAC2), histone-lysine N-methyltransferase EZH2 (EZH2), myc proto-oncogene protein (MYC), high mobility group 15 protein HMGI-C (HMGA2), nuclear factor type 1B (NFIB) and transcriptional enhancer factor TEF-4 (TEAD2) in adult glial progenitor cells.

[0040] As described herein, the term "rejuvenation" or "rejuvenate" refers to the reversal of the aging process in cells and the return to a youthful cellular state without loss of cellular identity, particularly with respect to proliferation and / or differentiation potential.

[0041] Suitable adult glial progenitor cells for use in the methods disclosed herein include mammalian glial progenitor cells, such as human glial progenitor cells, rodent glial progenitor cells, non-human primate glial progenitor cells, ovine glial progenitor cells, bovine glial progenitor cells, butaglial progenitor cells, canine glial progenitor cells, and feline glial progenitor cells. In some embodiments, the adult glial progenitor cells are adult human glial progenitor cells.

[0042] In some embodiments, the glial progenitor cells are adult human glial progenitor cells. In some embodiments, the expressing step is performed ex vivo. In some embodiments, the expressing step is performed in vivo.

[0043] Another aspect of the present invention relates to a method for treating myelin deficiency in a subject by activating certain transcription factors in glial progenitor cells of the subject. In some embodiments, the method comprises expressing an effective amount of one or more transcription factors selected from the group consisting of BCL11A, HDAC2, EZH2, MYC, HMGA2, NFIB and TEAD2 in glial progenitor cells of the subject.

[0044] In some embodiments, the expressing step is performed ex vivo. In some embodiments, the expressing step is performed in vivo.

[0045] According to this aspect of the invention, the myelin deficiency is selected from the group consisting of multiple sclerosis, neuromyelitis optica, transverse myelitis, optic neuritis, subcortical stroke, diabetic leukoencephalopathy, hypertensive leukoencephalopathy, age-related white matter disease, spinal cord injury, radiation or chemotherapy induced demyelination, post-infectious and post-vaccination leukoencephalitis, periventricular leukomalacia, childhood leukodystrophies (e.g., Pelizaeus-Merzbach disease, Tay-Sachs disease, etc.), and the like. The present invention may be associated with a condition selected from the group consisting of: Polypeptide-binding protein 1 (P1 ...

[0046] In some embodiments, the myelin deficiency is associated with a neurodegenerative disease, e.g., Huntington's disease. As used herein, "Huntington's disease" refers to an autosomal dominant inherited brain disorder that typically manifests in adulthood. Huntington's disease pathology is characterized by myelination hypoplasia, as well as neuronal and white matter loss (see, e.g., Osipovitch et al., "Human ESC-Derived Chimeric Mouse Models of Huntington's Disease Reveal Cell-Intrinsic Defects in Glial Progenitor Cell Differentiation," Cell Stem Cell 24(1):107-122 (2019), which is hereby incorporated by reference in its entirety).

[0047] In other embodiments, the myelin deficiency is associated with a neuropsychiatric disorder, e.g., schizophrenia. As used herein, "schizophrenia" refers to a condition typically characterized by a relatively small amount of white matter and often evident hypomyelination (see, e.g., Windrem et al., "Human iPSC Glial Mouse Chimeras Reveal Glial Contributions to Schizophrenia," Cell Stem Cell 21(2):195-208 (2017), which is hereby incorporated by reference in its entirety).

[0048] As used hereinafter, the term "treating" a subject having a myelin deficiency includes: (1) preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at least one clinical or subclinical symptom of myelin deficiency developing in a subject who may be suffering from or predisposed to myelin deficiency, but who has not yet experienced or shown a clinical or subclinical symptom of myelin deficiency; or (2) inhibiting myelin deficiency, i.e., arresting, reducing, or delaying the onset of myelin deficiency or its recurrence or at least one clinical or subclinical symptom thereof; or (3) alleviating myelin deficiency, i.e., causing regression of myelin deficiency, or at least one of its clinical or subclinical symptoms. The benefit to the subject being treated is either statistically significant or at least perceptible to the patient or physician.

[0049] As used hereinafter, the term "subject" refers to an individual organism, e.g., an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, goat, cat, or dog. In some embodiments, the subject is a research animal. In some embodiments, the subject has been genetically engineered, e.g., is a genetically engineered non-human subject. The subject may be an adult subject. In some embodiments, the subject is at least 1 year old, at least 2 years old, at least 4 years old, at least 6 years old, at least 8 years old, at least 10 years old, at least 12 years old, at least 15 years old, at least 18 years old, at least 20 years old, at least 25 years old, at least 30 years old, at least 35 years old, at least 40 years old, at least 45 years old, at least 50 years old, at least 55 years old, at least 60 years old, at least 65 years old, at least 70 years old, at least 75 years old, at least 80 years old, at least 85 years old, at least 90 years old, at least 95 years old, at least 100 years old, or older. In some embodiments, the subject is an adult subject between 18-100 years old, between 20-100 years old, between 30-100 years old, between 40-100 years old, between 50-100 years old, between 50-100 years old, between 60-100 years old, between 70-100 years old, between 80-100 years old, or between 90-100 years old.

[0050] In some embodiments, the expressing step in the method of inducing rejuvenation in adult GPCs or treating myelin deficiency in a subject comprises administering to the adult GPCs or GPCs in the subject, respectively, one or more nucleic acid molecules encoding one or more transcription factors selected from the group consisting of BCL11A, HDAC2, EZH2, MYC, HMGA2, NFIB, and TEAD2.

[0051] In some embodiments, the expressing step in the method of inducing rejuvenation in adult GPCs or treating myelin deficiency in a subject comprises administering to the adult GPCs or GPCs in the subject, respectively, one or more expression vectors expressing one or more transcription factors selected from the group consisting of BCL11A, HDAC2, EZH2, MYC, HMGA2, NFIB, and TEAD2, each of the expression vectors comprising (1) a nucleotide sequence encoding one or more transcription factors selected from the group consisting of BCL11A, HDAC2, EZH2, MYC, HMGA2, NFIB, and TEAD2, and (2) a regulatory sequence operably linked to the nucleotide sequence.

[0052] As used herein, the transcription factors referred to in this application, such as BCL11A, HDAC2, EZH2, MYC, HMGA2, NFIB and TEAD2, include all their transcript variants and functional variants. The nucleotide sequences encoding one or more of the transcription factors identified herein are well known and accessible in the art. Table 1 below identifies the transcription factors and their transcript variants by their gene name, gene ID number and NCBI reference transcript accession number.

[0053] [Table 1] TIFF2024539081000003.tif255154TIFF2024539081000004.tif255156TIFF2024539081000005.tif76170

[0054] In some embodiments, the one or more expression vectors comprise an expression vector comprising a nucleotide sequence encoding BCL11A, a transcript variant or a functional variant thereof. In some embodiments, the one or more expression vectors comprise an expression vector comprising a nucleotide sequence encoding a BCL11A variant of SEQ ID NO: 21. In some embodiments, the one or more expression vectors comprise an expression vector comprising the nucleotide sequence of SEQ ID NO: 20.

[0055] In some embodiments, the one or more expression vectors include an expression vector that includes a nucleotide sequence encoding HDAC2, a transcript variant, or a functional variant thereof.

[0056] In some embodiments, the one or more expression vectors include an expression vector comprising a nucleotide sequence encoding EZH2, a transcript variant, or a functional variant thereof.

[0057] In some embodiments, the one or more expression vectors include an expression vector comprising a nucleotide sequence encoding MYC, a transcript variant or a functional variant thereof.

[0058] In some embodiments, the one or more expression vectors include an expression vector comprising a nucleotide sequence encoding HMGA2, a transcript variant or a functional variant thereof.

[0059] In some embodiments, the one or more expression vectors include an expression vector that includes a nucleotide sequence encoding HFIB, a transcript variant, or a functional variant thereof.

[0060] In some embodiments, the one or more expression vectors include an expression vector comprising a nucleotide sequence encoding TEAD2, a transcript variant, or a functional variant thereof.

[0061] In some embodiments, the expressing step in the method of inducing rejuvenation in adult GPCs or treating myelin deficiency in a subject comprises administering to the adult GPCs or GPCs in the subject, respectively, an effective amount of an inhibitor of a transcription factor repressor.

[0062] As used herein, the term "transcription factor repressor" refers to an agent that inhibits the activity or expression of a transcription factor. A "transcription factor repressor" can be a polypeptide or a polynucleotide.

[0063] In some embodiments, the inhibitor of a transcription factor repressor is a small molecule.

[0064] In some embodiments, the inhibitor of the transcription factor repressor is a polypeptide or a polynucleotide. In some embodiments, the expressing step in the method of inducing rejuvenation in an adult GPC or the method of treating myelin deficiency in a subject comprises administering to the adult GPC or the GPC in the subject, respectively, an effective amount of an expression vector comprising (1) a nucleotide sequence encoding an inhibitor of the transcription factor repressor, and (2) a regulatory sequence operably linked to the nucleotide sequence.

[0065] In some embodiments, the transcription factor repressor is aryl 20 hydrocarbon receptor (AHR). According to such embodiments, the inhibitor of the transcription factor repressor is an AHR inhibitor. In some embodiments, the AHR inhibitor is a small molecule inhibitor.

[0066] Suitable AHR inhibitors are known in the art and include, without limitation, small molecules, such as BAY-218 (see, e.g., Abstract 1288:Blocking Tumor Associated Immune Suppression with BAY-218, a Novel, Selective Aryl Hydrocarbon Receptor (AhR) Inhibitor," Proceedings of the American Association for Cancer 25 Research Annual Meeting 2019; March 29-April 3, 2019; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res. 79(Suppl 13):Abstract nr 1288 (2019), which is hereby incorporated by reference in its entirety); perillaldehyde (see, e.g., Fuyuno et al., "Perillaldehyde Inhibits AHR Signaling and Activates NRF2 Antioxidant Pathway in Human Keratinocytes," Oxid.Med.Cell Longev.2018:9524657 (2018); StemRegenin1 (SR1) (see, e.g., Boitano et al., "Aryl Hydrocarbon Receptor Antagonists Promote the Expansion of Human Hematopoietic Stem Cells," Science 329(5997):1345-1348 (2010), which is hereby incorporated by reference in its entirety); KYN-101 (see, e.g., Campesato et al., "Blockade of the AHR Restricts a Treg-Macrophage Suppressive Axis Induced by L-5 Kynurenine," NatureComm.11:4011 (2020); CH-223191 (see, e.g., Kim et al., "Novel Compound 2-Methyl-2H-Pyrazole-3-Carboxylic acid (2-Methyl-4-o-Tolylazo-Phenyl)-Amide (CH-223191) Prevents 2,3,7,8-TCDD-Induced Toxicity by Antagonizing the Aryl Hydrocarbon Receptor," Mol. Pharmaocl. 69(6):1871-1878 (2006), which is hereby incorporated by reference in its entirety); BAY 2416964 (see, e.g., International Patent Publication WO / 2018 / 146010 to Bayer Aktiengesellschaft et al., which is hereby incorporated by reference in its entirety); PDM2 (see, e.g., de Medina et al., "Synthesis and Biological Properties of New Stilbene Derivatives of Resveratrol as New Selective Aryl Hydrocarbon Modulators," J. Med. Chem. 48:287-291 (2005)) and GNF351 (see, e.g., Smith et al., "Identification of a High-Affinity Ligand that Exhibits Complete Aryl Hydrocarbon Receptor Antagonism," J. Pharmacol. Exp. Ther. 338(1):318-327 (2011), which is hereby incorporated by reference in its entirety). Exemplary AHR small molecule inhibitors are set forth below in Table 2.

[0067] [Table 2]

[0068] In some embodiments, the transcription factor repressor is Myc box-dependent interacting protein (BIN1). According to such embodiments, the inhibitor of the transcription factor repressor is a BIN1 inhibitor.

[0069] In some embodiments, the transcription factor repressor is a miRNA molecule. According to such an embodiment, the inhibitor of the transcription factor repressor is an inhibitor of miRNA. As described herein, miRNAs that function as transcription factor repressors include, without limitation, those identified in Table 3 below.

[0070] [Table 3]

[0071] In some embodiments, the inhibitor of the transcription factor repressor is an inhibitor of any one or more of the miRNAs (target miRNAs) identified above. Inhibitors of miRNAs are known in the art as "antagomirs." Antagomirs are RNA oligonucleotides or RNA oligonucleotide mimics that have complementarity to a particular miRNA and inhibit the activity of that miRNA.

[0072] In some embodiments, the inhibitor of the transcription factor repressor is an antagomir that targets one or more of the miRNAs listed in Table 3. The antagomir may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences from the complementary sequence of the miRNA it inhibits. Furthermore, the antagomir may have the same length, a longer length, or a shorter length than the miRNA it inhibits. In certain embodiments, the antagomir hybridizes to 6-8 nucleotides at the 5' end of the miRNA it inhibits. In other embodiments, the antagomir is complementary to a miRNA and is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In other embodiments, the antagomir is 5-10, 6-8, 10-20, 10-15, or 5-500 nucleotides in length.

[0073] In some embodiments, antagomir is a synthetic reverse complement of a target RNA that tightly binds to and inactivates the target miRNA. Thus, in some embodiments, antagomir specifically inhibits or blocks the expression or activity of a target miRNA.

[0074] In some embodiments, the antagomir is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to any one of the miRNA sequences provided in Table 3 above.

[0075] In some embodiments, antagomir is capable of inhibiting the expression of one or more miRNAs disclosed in Table 3.

[0076] In particular, the antagomir of the present invention can be substantially complementary to a nucleic acid sequence specific to a miRNA selected from miR-193a-5p, miR-23b-3p, miR-4687-3p, miR-4651, miR-4270, or miR-24-3p, or a portion thereof. Thus, nucleic acids or analogs thereof that exhibit substantially equivalent or altered activity are also contemplated.

[0077] In some embodiments, the antagomir contains chemical modifications that improve nuclease resistance and binding affinity. Suitable modifications include, without limitation, 2' sugar modifications, such as 2'-O-Me, 2'-O-methoxyethyl (2'-MOE) or 2'-fluoro (2'-F).

[0078] In some embodiments, the expressing step in the method of inducing rejuvenation in adult GPCs or treating myelin deficiency in a subject comprises administering to the adult GPCs or GPCs in the subject, respectively, an effective amount of (1) an inhibitor of a transcription factor repressor, and / or (2) an effective amount of one or more transcription factors selected from the group consisting of B-cell lymphoma / leukemia 11A (BCL11A), histone deacetylase 2 (HDAC2), histone-lysine N-methyltransferase EZH2 (EZH2), myc proto-oncogene protein (MYC), high mobility group protein HMGI-C (HMGA2), nuclear factor type 1B (NFIB), and transcriptional enhancer factor TEF-4 (TEAD2).

[0079] In some embodiments, the expressing step in the method of inducing rejuvenation in adult GPCs or treating myelin deficiency in a subject comprises administering to the adult GPCs or GPCs in the subject, respectively, an effective amount of (1) an expression vector encoding an inhibitor of the transcription factor repressor, and / or (2) an effective amount of an expression vector encoding one or more transcription factors selected from the group consisting of B-cell lymphoma / leukemia 11A (BCL11A), histone deacetylase 2 (HDAC2), histone-lysine N-methyltransferase EZH2 (EZH2), myc proto-oncogene protein (MYC), high mobility group protein 15-C (HMGA2), nuclear factor type 1B (NFIB), and transcriptional enhancer factor TEF-4 (TEAD2).

[0080] In some embodiments, the regulatory sequence of the expression vector in the method of inducing rejuvenation in adult GPCs or treating myelin deficiency in a subject comprises a promoter and / or enhancer for a gene selectively or specifically expressed by glial progenitor cells.

[0081] Genes selectively expressed by glial progenitor cells include platelet-derived growth factor alpha (PDGFRA), zinc finger protein 488 (ZNF488), G protein-coupled receptor (GPR17), oligodendrocyte transcription factor 2 (OLIG2), chondroitin sulfate proteoglycan 4 (CSPG4), and SRY-box transcription factor 10 (SOX10).

[0082] According to such embodiments, promoter sequences suitable for controlling expression of the nucleic acid inhibitors disclosed herein include, without limitation, the platelet-derived growth factor alpha (PDGFRA) promoter, zinc finger protein 488 (ZNF488) promoter, G protein-coupled receptor (GPR17) promoter, oligodendrocyte transcription factor 2 (OLIG2) promoter, chondroitin sulfate proteoglycan 4 (CSPG4) promoter, and SRY-box transcription factor 10 (SOX10) promoter, which are identified in Table 4 below.

[0083] [Table 4]

[0084] In some embodiments, the regulatory sequence of the expression vector in the method of inducing rejuvenation in adult GPCs or treating myelin deficiency in a subject comprises an inducible promoter or promoter system, such as a tetracycline-controlled inducible system, a cumate-controlled inducible system, and a rapamycin-controlled inducible system, which are described in more detail below.

[0085] In some embodiments, the expression vector in the method of inducing rejuvenation in adult GPCs or treating myelin deficiency in a subject is a plasmid vector, a viral vector, or a bacterial vector.

[0086] In some embodiments, the expression vector in the method of inducing rejuvenation in adult GPCs or treating myelin deficiency in a subject is a viral vector selected from the group consisting of adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, vaccinia virus, and herpes virus. In some embodiments, the expression vector is a lentiviral vector. In some embodiments, the expression vector is a retroviral vector. In other embodiments, the expression vector is an AAV vector. Methods for generating and isolating viral vectors suitable for use as expression vectors are described in more detail below.

[0087] III. Methods Involving Repression of Transcription Factors Another aspect of the present invention relates to a method for inducing rejuvenation in a population of adult glial progenitor cells by repressing certain transcription factors, comprising administering to the population of adult glial progenitor cells an effective amount of an agent that represses one or more transcription factors selected from the group consisting of zinc finger protein 274 (ZNF274), Myc-associated factor X (MAX), E2F transcription factor 6 (E2F6), zinc finger protein Aiolos (IKZF3) and signal transducer and activator of transcription 3 (STAT3).

[0088] Another aspect of the present invention relates to a method of inducing rejuvenation in a population of adult glial progenitor cells by suppressing certain senescence genes, comprising administering to the population of adult glial progenitor cells an effective amount of an agent that inhibits expression of one or more senescence genes selected from the group consisting of RUNX1, BIN1, VAMP3, DMTF1, CTNNA1, SERPINE1, CDK19, CDKN1C, RUNX2, EFEMP1, MAP3K7, AHR, OGT, PAK1, CBX7 and CYLD.

[0089] Another aspect of the present invention relates to a method of inducing rejuvenation in a population of adult glial progenitor cells by suppressing certain senescence genes, comprising administering to the population of adult glial progenitor cells an effective amount of an agent that inhibits expression of one or more glial target genes selected from the group consisting of MBP, CD9, ENPP2, PLP1, ERBIN, ZNF365, UGT8, GDNF, DUSP10, PMP22, ERBB4 and MYRF.

[0090] Suitable adult glial progenitor cells for use in the methods disclosed herein include mammalian glial progenitor cells, such as human glial progenitor cells, rodent glial progenitor cells, non-human primate glial progenitor cells, ovine glial progenitor cells, bovine glial progenitor cells, butaglial progenitor cells, canine glial progenitor cells, and feline glial progenitor cells. In some embodiments, the adult glial progenitor cells are adult human glial progenitor cells.

[0091] In some embodiments, the administering is performed ex vivo. In other embodiments of the methods according to the present disclosure, the administering is performed in vivo.

[0092] Another aspect of the present invention relates to a method of treating myelin deficiency in a subject by suppressing certain transcription factors, comprising administering to a subject having myelin deficiency an agent that suppresses one or more transcription factors selected from the group consisting of ZNF274, MAX, E2F6, IKZF3 and STAT3, wherein the agent is administered in an effective amount to suppress the one or more transcription factors in adult glial progenitor cells of the subject.

[0093] Another aspect of the present invention relates to a method of treating myelin deficiency in a subject by suppressing certain senescence genes, comprising administering to the subject an agent that inhibits expression of one or more senescence genes selected from the group consisting of RUNX1, BIN1, VAMP3, DMTF1, CTNNA1, SERPINE1, CDK19, CDKN1C, RUNX2, EFEMP1, MAP3K7, AHR, OGT, PAK1, CBX7 and CYLD, wherein the agent is administered in an amount effective to suppress the one or more senescence genes in adult glial progenitor cells of the subject.

[0094] Another aspect of the present invention relates to a method of treating myelin deficiency in a subject by suppressing certain glial target genes, comprising administering to the subject an agent that inhibits expression of one or more senescence genes selected from the group consisting of MBP, CD9, ENPP2, PLP1, ERBIN, ZNF365, UGT8, GDNF, DUSP10, PMP22, ERBB4 and MYRF, wherein the agent is administered in an amount effective to suppress the one or more glial target genes in adult glial progenitor cells of the subject.

[0095] According to this aspect of the invention, the myelin deficiency may be associated with a condition selected from the group consisting of multiple sclerosis, neuromyelitis optica, transverse myelitis, optic neuritis, subcortical stroke, diabetic leukoencephalopathy, hypertensive leukoencephalopathy, age-related white matter disease, spinal cord injury, radiation or chemotherapy induced demyelination, post-infectious and post-vaccination leukoencephalitis, periventricular leukomalacia, childhood leukodystrophies (e.g., Pelizaeus-Merzbach disease, Tay-Sachs disease, Sandhoff gangliosidosis, Krabbe disease, metachromatic leukodystrophy, mucopolysaccharidoses, Niemann-Pick disease type A, adrenoleukodystrophy, Canavan disease, vanishing white matter disease, and Alexander disease), lysosomal storage diseases, congenital dysmyelination, inflammatory demyelination, vascular demyelination, and cerebral palsy.

[0096] In some embodiments, the myelin deficiency is associated with a neurodegenerative disease, such as Huntington's disease. As used herein, "Huntington's disease" refers to an autosomal dominant inherited brain disorder that typically manifests in adulthood. Huntington's disease pathology is characterized by myelination hypoplasia, as well as neuronal and white matter loss.

[0097] In another embodiment, the myelin deficiency is associated with a neuropsychiatric disorder, for example, schizophrenia.

[0098] In some embodiments, the agent used in the method of inducing rejuvenation or treating myelin deficiency is an agent that suppresses STAT3. As shown in FIG. 4F of the present disclosure, in the context of adult glial progenitor cells, STAT3 is predicted to activate a set of senescence-related genes (e.g., BIN1, DMTF1, CD47, CTNNA1, RUNX2, RUNX1, MAP3K7 and OGT), glial cell-related genes (e.g., PLP1, CNP, PMP22, SEMA4D, CLDN11, GPR37, MYRF, MAG, BCAS1, ST18, ERBB4, CERS2, LPAR1 and GJB1) and downstream transcription factors (e.g., MAX, E2F6 and IKZF3). In some embodiments, the agent is an agent that suppresses or silences the activity of STAT3 and / or any of the above-indicated senescence-related genes that are activated by STAT3.

[0099] In some embodiments, the agent used in the methods of inducing rejuvenation and treating myelin deficiency is an agent that suppresses one or more of ZNF274, MAX, E2F6, and IKZF3. As shown in FIG. 4G of the present application, in the context of adult glial progenitor cells, ZNF274, MAX, E2F6 and IKZF3 are predicted to repress a set of proliferation-related gene targets (e.g., YAP1, LMNB1, PATZ1, TEAD1, FN1, TP53, CDK1, CCND2, CDKN2D, CENPH, MKI67, CDK4, CENPF, CDK5, CDKN3 and CHEK1), glial cell-related genes (e.g., CHRDL1, ST8SIA1, PTPRZ1, CA10, PDGFRA, BCAN, NXPH1, CSPG4 and PCDH15) and downstream transcription factors (e.g., BLC11A, EZH2, HDAC2, NF1B, MYC, HMGA2 and TEAD).

[0100] In some embodiments, the agent is one that represses or silences the activity of ZNF274, MAX, E2F6, IKZF3, or any combination thereof, to allow expression of a downstream proliferation-related gene target.

[0101] In some embodiments, the agent is an agent that represses ZNF274, MAX, E2F6, IKZF3 and / or STAT3. Agents suitable for use in the methods described herein include, without limitation, ZNF274 inhibitors, MAX inhibitors, E2F6 inhibitors, IKZF3 inhibitors and STAT3 inhibitors.

[0102] The ZNF274 inhibitor, MAX inhibitor, E2F6 inhibitor, IKZF3 inhibitor and / or STAT3 inhibitor can be (1) a small molecule inhibitor, (2) a nucleic acid molecule inhibitor (e.g., miRNA, shRNAi, siRNA and antisense oligonucleotides), (3) a nuclease-based gene editing system (e.g., CRISPR / Cas system targeted to silence ZNF274, MAX, E2F6, IKZF3 and / or STAT expression), or (4) a nucleic acid molecule encoding (2) or (3).

[0103] Small molecule inhibitors In some embodiments, the ZNF274 inhibitor, MAX inhibitor, E2F6 inhibitor, IKZF3 inhibitor and / or STAT3 inhibitor is a small molecule inhibitor. Exemplary small molecule inhibitors of these transcription factors known in the art and suitable for use according to the methods described herein are provided in Table 5 below. Analogs and derivatives of the small molecule inhibitors in Table 5 are also contemplated for use in the methods described herein.

[0104] [Table 5] JPEG2024539081000010.jpg228170JPEG2024539081000011.jpg216170JPEG2024539081000012.jpg194170JPEG2024539081000013.jpg168170

[0105] In some embodiments, the small molecule inhibitors include 10058-F4 (also known as "10058-F4(1RH)") (see, e.g., Huang et al., "A Small-Molecule c-Myc Inhibitor, 10058-F4, Induces Cell-Cycle Arrest, Apoptosis, and Myeloid Differentiation of Human Acute Myeloid Leukemia," Exp. Hematol. 34(11):1480-1489 (2006), which is hereby incorporated by reference in its entirety); MYCMI-6, MYCMI-11, and MYCMI-14 (see, e.g., Castell et al., "A Selective High Affinity MYC-Binding Compound Inhibits MYC:MAX Interaction and MYC-Dependent Tumor Cell Linear Tumors," Exp. Hematol. 34(11):1480-1489 (2006), which is hereby incorporated by reference in its entirety). Proliferation," Sci. Rep. 8:10064 (2018); 12RH, 22RH, 27RH, 28RH, 1RH-S-Me, 1RH-NCN-1, #015, #474, ​​#764, 12RH-NCN-1 and 28RH-NCN-1 (see, e.g., Wang et al., "Improved Low Molecular Weight Myc-Max inhibitors," Mol. Cancer Ther. 6(9):2399-2408 (2007), which is hereby incorporated by reference in its entirety).

[0106] In some embodiments, the small molecule inhibitor is cryptotanshinone (see, for example, Shin et al., "Cryptotanshinone Inhibits Constitutive Signal Transducer and Activator of Transcription 3 Function through Blocking the Dimerization in DU145 Prostate Cancer Cells," Cancer Res.69(1):193-202 (2009); STA-21 (see, e.g., Song et al., "A Low-Molecular-Weight Compound Discovered through Virtual Database Screening Inhibits Stat3 Function in Breast Cancer Cells," PNAS 102(13):4700-4705 (2005), which is hereby incorporated by reference in its entirety); NSC 7459 (S3I-201) (see, e.g., Siddiquee et al., "Selective Chemical Probe Inhibitor of Stat3, Identified through Structure-Based Virtual Screening, Induces Antitumor Activity," PNAS 104(18):7391-7396 (2007), which is hereby incorporated by reference in its entirety); napabucasin (BBI608) (see, e.g., Li et al., "Suppression of Cancer Relapse and Metastasis by , “Inhibiting Cancer Stemness,” PNAS 112(6):1839-1844 (2015); and Hubbard et al., “Napabucasin: An Update on the First-in-Class Cancer Stemness Inhibitor,” Drugs 77(10):1091-1103 (2017); Stattic, cucurbitacin I, cucurbitacin Q, Phpr-pTyr-Leu-cis-3,4-methanoPro-Gln-NHBn (see, e.g., McMurray, J., “A New Small-Molecule Stat3 Inhibitor,” Chem. Biol.13(11):1123-1124 (2006); LLL-3 (see, e.g., Fuh et al., "LLL-3 Inhibits STAT3 Activity, Suppresses Glioblastoma Cell Growth and Prolongs Survival in a Mouse Glioblastoma Model," Br. J. Cancer 100(1):106-112 (2009), which is hereby incorporated by reference in its entirety); LLL-12, S31-201, SF-1-066, S31-1757, STX-0119, Cpd30-12, LY5, Cpd9, and Cpd1 (see, e.g., Orlova et al., "Direct Targeting Options for STAT3 and STAT5 in Cancer," Cancers 100(1):106-112 (2009), which is hereby incorporated by reference in its entirety). 11(12):1930 (2019); SF-1-087, SF-1-121, S3I-M2001, S3I-201.1066, and BP-1-102 (see, e.g., Wu et al., "Negative Regulators of STAT Signaling Pathway in Cancers," Cancer Manag. Res. 11:4957-4969 (2019), which is hereby incorporated by reference in its entirety); HO-3867 (see, e.g., Tierney et al., "HO-3867, a STAT3 Inhibitor Induces Apoptosis by Inactivation of STAT3 Activity in BRCA1-Mutated Ovarian Cancer Cells," Cancer Biol. Ther. 13(9):766-775 (2012); corylifol A (see, e.g., Lee et al., "Phenolic Compounds Isolated from Psoralea corylifolia Inhibit IL-6-Induced STAT3 Activation," Planta. Med. 1999, 144:1311-1315, the entire contents of which are hereby incorporated by reference herein);78(9):903-906 (2012); and SD 1008 (see, e.g., Liu et al., "SOCS3 Promotes Inflammation and Apoptosis via Inhibiting JAK2 / STAT3 Signaling Pathway in 3T3-L1 Adipocyte," Immunobiology 220(8):947-953 (2015), which is hereby incorporated by reference in its entirety).

[0107] Nucleic Acid Inhibitors In some embodiments, the ZNF274 inhibitor, MAX inhibitor, E2F6 inhibitor, IKZF3 inhibitor and / or STAT3 inhibitor is a nucleic acid molecule inhibitor.

[0108] As used herein, the term "nucleic acid molecule inhibitor" refers to a nucleic acid molecule that reduces or eliminates the expression of a target gene. A nucleic acid molecule inhibitor typically contains a region that specifically targets a sequence in a target gene or target gene mRNA to achieve target-specific inhibition. Typically, the targeting region of a nucleic acid inhibitor molecule contains a sequence that is sufficiently complementary to a sequence on a target gene or target gene mRNA to direct the effect of the nucleic acid inhibitor molecule to a specified target gene or target gene mRNA. For example, a "nucleic acid molecule inhibitor of ZNF274" reduces or eliminates the expression of the ZNF274 gene. A nucleic acid inhibitor molecule can include natural ribonucleotides, natural deoxyribonucleotides, and / or modified nucleotides. Modified nucleotides include modifications, such as substitutions on positions on the sugar ring, modifications of the phosphate ester linkage between nucleotides, unnatural bases, and unnatural alternative carbon structures, such as locked nucleic acids ("LNA") and unlocked nucleic acids ("UNA").

[0109] As used herein, the term "reduce" or "reduces" refers to its meaning as generally accepted in the art. With respect to exemplary nucleic acid inhibitor molecules (e.g., nucleic acid molecule inhibitors of ZNF274, MAX, E2F6, IKZF3 and / or STAT3 selected from miRNA, shRNAi, siRNA and antisense oligonucleotides), "reduce" or "reduces" generally refers to suppression in the level of gene transcription and / or translation or gene product compared to the transcription and / or translation of the gene observed in the absence of the nucleic acid inhibitor molecule. In some embodiments, the reduction in gene transcription and / or translation or gene product levels is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, up to 100% (i.e., no detectable transcription and / or translation), or at most 2-fold, at most 5-fold, at most 10-fold, at most 20-fold, at most 30-fold, at most 40-fold, at most 50-fold or less reduction compared to that observed in the absence of a nucleic acid inhibitor molecule in accordance with the present disclosure.

[0110] Suitable nucleic acid inhibitor molecules include, without limitation, (i) a nucleic acid molecule inhibitor of ZNF274 selected from miRNA, shRNAi, siRNA, and antisense oligonucleotides (ASO); (ii) a nucleic acid molecule inhibitor of MAX selected from miRNA, shRNAi, siRNA, and antisense oligonucleotides; (iii) a nucleic acid molecule inhibitor of E2F6 selected from miRNA, shRNAi, siRNA, and antisense oligonucleotides; (iv) a nucleic acid molecule inhibitor of IKZF3 selected from miRNA, shRNAi, siRNA, and antisense oligonucleotides; and (v) a nucleic acid molecule inhibitor of STAT3 selected from miRNA, miRNA inhibitor, shRNAi, siRNA, and antisense oligonucleotides.

[0111] As used herein, the term "microRNA" or "miRNA" refers to a class of small RNA molecules that can negatively regulate gene expression (see, e.g., Lam et al., "siRNA Versus miRNA as Therapeutics for Gene Silencing," Mol. Ther. Nucleic Acids 4(9):e252 (2015), which is hereby incorporated by reference in its entirety). miRNA gene transcription is carried out in the nucleus by RNA polymerase II to generate primary miRNAs (pri-miRNAs), which are 5'-capped, 3'-polyadenylated RNAs with a double-stranded stem-loop structure. The pri-miRNAs are then cleaved by the microprocessor complex (comprising Drosha and the microprocessor complex subunit DCGR8) to form precursor miRNAs (pre-miRNAs), which are double-stranded, containing 70-100 nucleotides with interspersed mismatches and adopting a loop structure. The pre-miRNA is subsequently transported from the nucleus to the cytoplasm by exportin 5, where it is further processed by Dicer into an 18-25 nucleotide miRNA duplex. The miRNA duplex then associates with RISC to form a complex called miRISC. The miRNA duplex unwinds, releasing and discarding the passenger strand (sense strand). The mature single-stranded miRNA guides the miRISC to the target mRNA. The mature miRNA can bind to the target mRNA through partial complementary base pairing, resulting in target gene silencing via translational repression, degradation and / or cleavage. In some embodiments, the nucleic acid inhibitor molecule is a miRNA molecule. MiRNA inhibitor molecules suitable for use in the methods of the present disclosure include, without limitation, those identified in Table 6 below.

[0112] [Table 6]

[0113] In some embodiments, the MAX inhibitor is a nucleic acid molecule inhibitor of MAX, and a suitable MAX nucleic acid molecule inhibitor is a miRNA. According to such embodiments, an exemplary MAX inhibitor miRNA has a nucleotide sequence corresponding to miR-485-5p, pre-miR-485-5p, or mature miR-485-5p. For example, the miRNA may have the nucleotide sequence of SEQ ID NO:7.

[0114] In some embodiments, the E2F6 inhibitor is a nucleic acid molecule inhibitor of E2F6, and the suitable E2F6 nucleic acid molecule inhibitor is a miRNA. According to such an embodiment, the exemplary E2F6 inhibitor miRNA has a nucleotide sequence corresponding to miR-379-5p, pre-miR-379-5p or mature miR-379-5p. For example, the miRNA may have the nucleotide sequence of SEQ ID NO:8.

[0115] In some embodiments, the STAT3 inhibitor is a nucleic acid molecule inhibitor of STAT3, and a suitable STAT3 inhibitor is a miRNA. According to such embodiments, an exemplary STAT3 inhibitory miRNA has a nucleotide sequence corresponding to miR-125b-5p, pre-miR-125b-5p, mature miR-125b-5p, miR-106a-5p, pre-miR-106a-5p, mature miR-106a-5p, miR-17-5p, pre-miR-17-5p, mature miR-17-5p, miR-130a-3p, pre-miR-130a-3p, mature miR-130a-3p, miR-130b-3p, pre-miR-130b-3p, or mature miR-130b-3p. For example, the miRNA can have the nucleotide sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0116] In some embodiments, the agents used in the methods of inducing rejuvenation and treating myelin deficiency comprise one or more expression vectors encoding: (i) one or more microRNAs selected from the group consisting of miR-125b-5p, miR-106a-5p, miR-17-5p, miR-130a-3p, and miR-130b-3p, the administration of which suppresses the signal transduction and activator of transcription 3 (STAT3) signaling pathway; (ii) miR-379-5p, the administration of which suppresses the E2F transcription factor 6 (E2F6) signaling pathway; and / or (iii) miR-485-5p, the administration of which suppresses the Myc-associated factor X (MAX) signaling pathway.

[0117] In some embodiments, the agent used in the methods of inducing rejuvenation and treating myelin deficiency comprises an expression vector encoding a MAX inhibitor. In some embodiments, the MAX inhibitor is a nucleic acid molecule inhibitor. In some embodiments, the MAX nucleic acid molecule inhibitor is a miRNA. According to such embodiments, an exemplary MAX inhibitor miRNA has a nucleotide sequence corresponding to miR-485-5p, pre-miR-485-5p, or mature miR-485-5p. For example, the miRNA may have the nucleotide sequence of SEQ ID NO:7.

[0118] In some embodiments, the agent used in the method of inducing rejuvenation and treating myelin deficiency comprises an expression vector encoding an E2F6 inhibitor. In some embodiments, the E2F6 inhibitor is a nucleic acid molecule inhibitor. In some embodiments, the E2F6 nucleic acid molecule inhibitor is a miRNA. According to such embodiments, an exemplary E2F6 inhibitor miRNA has a nucleotide sequence corresponding to miR-379-5p, pre-miR-379-5p, or mature miR-379-5p. For example, the miRNA may have the nucleotide sequence of SEQ ID NO:8.

[0119] In some embodiments, the agent used in the method of inducing rejuvenation and treating myelin deficiency comprises an expression vector encoding a STAT3 inhibitor. In some embodiments, the STAT3 inhibitor is a nucleic acid molecule inhibitor. In some embodiments, the STAT3 nucleic acid molecule inhibitor is a miRNA. According to such embodiments, an exemplary STAT3 inhibitory miRNA has a nucleotide sequence corresponding to miR-125b-5p, pre-miR-125b-5p, mature miR-125b-5p, miR-106a-5p, pre-miR-106a-5p, mature miR-106a-5p, miR-17-5p, pre-miR-17-5p, mature miR-17-5p, miR-130a-3p, pre-miR-130a-3p, mature miR-130a-3p, miR-130b-3p, pre-miR-130b-3p, or mature miR-130b-3p. For example, the miRNA may have the nucleotide sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0120] In some embodiments, the agent used in the method of inducing rejuvenation and the method of treating myelin deficiency comprises an expression vector encoding one or more microRNAs selected from the group consisting of (1) miR-485-5p miR-379-5p, miR-125b-5p, miR-106a-5p, miR-17-5p, miR-130a-3p and miR-130b-3p, and (2) miR-93-3p, miR-1260b, miR-767-5p, miR-30b-5p, miR-9-3p and miR-9-5p, as shown in Table 7 below.

[0121] [Table 7]

[0122] In some embodiments, the nucleic acid inhibitor molecule of ZNF274, MAX, E2F6, IKZF3 and / or STAT3 is an shRNA molecule. A short hairpin RNA (shRNA) molecule comprises a sense sequence and an antisense sequence from a target gene connected by a loop. Once transcribed, the shRNA molecule is transported from the nucleus into the cytoplasm, where the enzyme Dicer processes them into small / short interfering RNA (siRNA) in the short hairpin RNA interference process. As used herein, the term "short hairpin RNA interference" or "shRNAi" is a process mediated by a class of small RNA molecules that negatively regulate gene expression.

[0123] In some embodiments, the ZNF274 inhibitor is a nucleic acid molecule inhibitor of ZNF274, and the suitable ZNF274 inhibitor is a ZNF274 shRNA. In some embodiments, the MAX inhibitor is a nucleic acid molecule inhibitor of MAX, and the suitable MAX inhibitor is a MAX shRNA. In some embodiments, the E2F6 inhibitor is a nucleic acid molecule inhibitor of E2F6, and the suitable E2F6 inhibitor is an E2F6 shRNA. In some embodiments, the IKZF3 inhibitor is a nucleic acid molecule inhibitor of IKZF3, and the suitable IKZF3 inhibitor is an IKZF3 shRNA. In some embodiments, the STAT3 inhibitor is a nucleic acid molecule inhibitor of STAT3, and the suitable STAT3 inhibitor is a STAT3 shRNA.

[0124] In some embodiments, the nucleic acid inhibitor molecule of ZNF274, MAX, E2F6, IKZF3 and / or STAT3 is an siRNA. As used herein, the term "small interfering RNA" or "siRNA" refers to a short nucleic acid molecule, typically 21-23 nucleotides in length, with a 3'-2 nucleotide overhang (see, e.g., McManus and Sharp, "Gene Silencing in Mammals by Small Interfering RNAs," Nat. Rev. Genet. 3(10):737-747 (2002), which is hereby incorporated by reference in its entirety). siRNA interacts with and activates the RNA-induced silencing complex ("RISC"). The endonuclease Argonaute 2 (AGO2) component of RISC cleaves the passenger strand (sense strand) of the siRNA, while the guide strand (antisense strand) remains associated with RISC.

[0125] The guide strand then guides the active RISC to its target mRNA for cleavage by AGO2. Because the guide strand only binds to mRNAs that are perfectly complementary to it, the siRNA causes specific gene silencing (see, e.g., Lam et al., "siRNA Versus miRNA as Therapeutics for Gene Silencing," Mol. Ther. Nucleic Acids 4(9):e252 (2015), which is hereby incorporated by reference in its entirety).

[0126] In some embodiments, the ZNF274 inhibitor is ZNF274 siRNA. In some embodiments, the MAX inhibitor is MAX siRNA. In some embodiments, the E2F6 inhibitor is E2F6 siRNA. In some embodiments, the IKZF3 inhibitor is IKZF3 siRNA. In some embodiments, the STAT3 inhibitor is STAT3 siRNA.

[0127] In some embodiments, the nucleic acid inhibitor molecules of ZNF274, MAX, E2F6, IKZF3 and / or STAT3 are antisense oligonucleotides. As used herein, the term "antisense oligonucleotide" or "ASO" refers to small (about 18-30 nucleotides), synthetic, single-stranded nucleic acid polymers of diverse chemistry that can be used to modulate gene expression through a variety of mechanisms (see, e.g., Roberts et al., "Advances in Oligonucleotide Drug Delivery," Nature Reviews Drug Discovery 19:673-694 (2020), which is hereby incorporated by reference in its entirety). ASOs can be sub-divided into two major categories: RNase H competent and steric block. The endogenous RNase H enzyme RNASEH1 recognizes the RNA-DNA heteroduplex substrates formed when DNA-based oligonucleotides bind to their cognate mRNA transcripts and catalyzes the degradation of the RNA. Cleavage at the site of ASO binding results in the destruction of the target RNA, thereby silencing target gene expression. Steric blocking oligonucleotides are ASOs that are designed to bind to target transcripts with high affinity but do not induce target transcript degradation because they lack RNase H competence. Thus, such oligonucleotides contain either nucleotides that do not form RNase H substrates when paired with RNA, or mixtures of nucleotide chemistries (i.e., "mixmers"), such that runs of consecutive DNA-like bases are avoided.

[0128] In some embodiments, the ZNF274 inhibitor is a ZNF274 ASO. In some embodiments, the MAX inhibitor is a MAX ASO. In some embodiments, the E2F6 inhibitor is an E2F6 ASO. In some embodiments, the IKZF3 inhibitor is an IKZF3 ASO. In some embodiments, the STAT3 inhibitor is a STAT3 ASO, e.g., danvatirsen (see, e.g., Xu et al., "Population Pharmacokinetic Analysis of Danvatirsen Supporting Flat Dosing Switch," J. Pharmacokinet. Pharmacodyn. 46(1):65-74 (2019), which is hereby incorporated by reference in its entirety).

[0129] Methods for designing nucleic acid inhibitors are well known in the art and are suitable for designing nucleic acid inhibitors for use in the methods described herein (see, e.g., Lam et al., "siRNA Versus miRNA as Therapeutics for Gene Silencing," Mol. Ther. Nucleic Acids 4(9):e252 (2015) and Kulkarni et al., "The Current Landscape of Nucleic Acid Therapeutics," Nature Nanotechnology 16:630-643 (2021), the entireties of which are hereby incorporated by reference).

[0130] The nucleic acid inhibitor molecule is designed to target ZNF274, MAX, E2F6, IKZF3 and / or STAT3 and their transcriptional variants in a sequence-specific manner.The sequences of ZNF274, MAX, E2F6, IKZF3 and / or STAT3 and their transcriptional variants are well known in the art and can be accessed through various curated databases, such as NCBI nucleotide or gene databases.In some embodiments, the nucleic acid inhibitor molecule is designed to target one or more of the transcription factors identified in Table 8 below using the sequences available through the provided NCBI accession numbers.

[0131] [Table 8] JPEG2024539081000017.jpg255158JPEG2024539081000018.jpg212170

[0132] In some embodiments, the ZNF274 inhibitor, MAX inhibitor, E2F6 inhibitor, IKZF3 inhibitor and / or STAT3 inhibitor is a nuclease-based gene editing system capable of silencing the expression of ZNF274, MAX, E2F6, IKZF3 and / or STAT3. As used herein, the term "nuclease-based gene editing system" refers to a system that includes a nuclease or its derivative that can be recruited to a target sequence in a genome. This system can include clustered regularly interspaced short palindromic repeats associated ("Cas") protein (e.g., Cas9, Cas12a and Cas12b), zinc finger nuclease ("ZFN") or transcription activator-like effector nuclease ("TALEN").

[0133] In some embodiments, the nuclease-based gene editing system is a CRISPR / Cas system targeted to silence ZNF274 expression, MAX expression, E2F6 expression, IKZF3 expression, and / or STAT3 expression. The CRISPR / Cas system may include a Cas protein or a nucleic acid molecule encoding a Cas protein and a guide RNA that includes a nucleotide sequence complementary to a portion of a target DNA sequence.

[0134] As described herein, Cas proteins form ribonucleoprotein complexes with guide RNAs that guide the Cas proteins to target DNA sequences. Suitable Cas proteins include Cas nucleases (i.e., Cas proteins capable of introducing double-stranded breaks in target nucleic acid sequences), Cas nickases (i.e., Cas protein derivatives capable of introducing single-stranded breaks in target nucleic acid sequences), and nuclease-dead (dCas) proteins (i.e., Cas protein derivatives that do not have any nuclease activity).

[0135] In some embodiments, the Cas protein is a Cas9 protein. As used herein, the term "Cas9 protein" or "Cas9" includes any recombinant or naturally occurring form of CRISPR associated protein 9 (Cas9) or variants or homologs thereof. In some embodiments, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 consecutive amino acids) compared to a naturally occurring Cas9 protein. In some embodiments, the Cas9 protein is substantially identical to a protein identified by UniProt reference numbers Q99ZW2, G3ECR1, J7RUA5, A0Q5Y3, or J3F2B0 (which are hereby incorporated by reference in their entirety) or a variant or homolog having substantial identity thereto. In some embodiments, the Cas9 protein is selected from the group consisting of Cas9 nuclease, Cas9 nickase, and nuclease-dead Cas9 ("dCas9").

[0136] In some embodiments, the Cas protein is a Cas12a protein. As used herein, the term "Cas12a protein" or "Cas12a" includes any recombinant or naturally occurring form of CRISPR associated protein 12 (Cas12a) or variants or homologs thereof. In some embodiments, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150 or 200 consecutive amino acids) compared to a naturally occurring Cas12a protein. In some embodiments, the Cas12a protein is substantially identical to a protein identified by UniProt reference numbers A0Q7Q2, U2UMQ6, A0A7C6JPC1, A0A7C9H0Z9, or A0A7J0AY55, which are hereby incorporated by reference in their entireties, or a variant or homolog having substantial identity thereto. In some embodiments, the Cas12a protein is selected from the group consisting of Cas12a nuclease, Cas12a nickase, and nuclease-dead Cas12a ("dCas12a").

[0137] In some embodiments, the Cas protein is a Cas12b protein. As used herein, the term "Cas12b protein" or "Cas12b" includes any recombinant or naturally occurring form of CRISPR associated protein 12 (Cas12b) or variants or homologs thereof. In some embodiments, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150 or 200 consecutive amino acids) compared to a naturally occurring Cas12b protein. In some embodiments, the Cas12b protein is substantially identical to a protein identified by UniProt reference numbers T0D7A2, A0A6I3SPI6, A0A6I7FUC4, A0A6N9TP17, A0A6M1UF64, A0A7Y8V748, A0A7X7KIS4, A0A7X8X2U5, or A0A7X8UMW7, which are hereby incorporated by reference in their entireties, or a variant or homolog having substantial identity thereto. In some embodiments, the Cas12b protein is selected from the group consisting of Cas12b nuclease, Cas12b nickase, and nuclease-dead Cas12b ("dCas12b").

[0138] As used herein, the term "guide RNA" or "gRNA" refers to a ribonucleotide sequence that can bind to a nuclear protein, thereby forming a ribonucleoprotein complex. According to the methods and systems disclosed herein, the guide RNA comprises (i) a DNA targeting sequence that is complementary to the target nucleic acid sequence of ZNF274, MAX, E2F6, IKZF3 or STAT3 sequence, and (ii) a binding sequence for a Cas protein (e.g., Cas9 nuclease, Cas9 nickase, dCas9, Cas12a nuclease, Cas12a nickase or dCas12a).

[0139] In some embodiments, the guide RNA is a single guide RNA molecule (single RNA nucleic acid), which may include "single guide RNA" or "sgRNA". In other embodiments, the nucleic acid of the present disclosure includes two RNA molecules (e.g., connected together via hybridization at a binding sequence). Thus, the term guide RNA is inclusive, referring to both bimolecular and single molecule nucleic acids (e.g., sgRNA).

[0140] In some embodiments, the gRNA is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleic acid residues long. In some embodiments, the gRNA is 10-30 nucleic acid residues long. In some embodiments, the gRNA is 20 nucleic acid residues long. In some embodiments, the gRNA is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleic acid or sugar residues in length. In some embodiments, the gRNA is 5-50, 10-50, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 5-75, 10-75, 15-75, 20-75, 25-75, 30-75, 35-75, 40-75, 45-75, 50-75, 55-75, 60-75, 65-75, 70-75, The gRNA may be 5, 5-100, 10-100, 15-100, 20-100, 25-100, 30-100, 35-100, 40-100, 45-100, 50-100, 55-100, 60-100, 65-100, 70-100, 75-100, 80-100, 85-100, 90-100, 95-100, or more residues in length. In some embodiments, the gRNA is 10-15, 10-20, 10-30, 10-40, or 10-50 residues in length.

[0141] In some embodiments, the CRISPR / Cas system is targeted to silence ZNF274 gene expression, and the guide RNA comprises a nucleotide sequence complementary to a portion of the ZNF274 gene sequence.

[0142] In some embodiments, the CRISPR / Cas system is targeted to silence MAX gene expression and the guide RNA comprises a nucleotide sequence complementary to a portion of the MAX gene sequence.

[0143] In some embodiments, the CRISPR / Cas system is targeted to silence E2F6 gene expression, and the guide RNA comprises a nucleotide sequence complementary to a portion of the E2F6 gene sequence.

[0144] In some embodiments, the CRISPR / Cas system is targeted to silence IKZF3 gene expression, and the guide RNA comprises a nucleotide sequence complementary to a portion of the IKZF3 gene sequence.

[0145] In some embodiments, the CRISPR / Cas system is targeted to silence STAT3 DNA expression, and the guide RNA comprises a nucleotide sequence complementary to a portion of the STAT3 gene sequence.

[0146] In some embodiments, the CRISPR / Cas system targeted to silence ZNF274 expression, MAX expression, E2F6 expression, IKZF3 expression, and / or STAT3 expression is a CRISPRi system. As used herein, the term "CRISPR interference" or "CRISPRi" refers to a system that allows sequence-specific suppression of gene expression. The CRISPRi system includes nuclease-dead Cas ("dCas") proteins (i.e., nuclease-inactivated Cas proteins) to block transcription of target genes without cleaving the target DNA sequence. Nuclease-inactivated Cas proteins and methods for generating nuclease-inactivated Cas proteins are well known in the art (see, for example, Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression," Cell 152(5):1173-1183 (2013), which is hereby incorporated by reference in its entirety).

[0147] A CRISPRi system suitable for use as described herein may include (i) a nuclease-dead Cas (dCas) protein (i.e., a nuclease-inactivated Cas protein) or a nucleic acid molecule encoding a dCas protein, and (ii) a guide RNA comprising a nucleotide sequence complementary to a portion of ZNF274, MAX, E2F6, IKZF3, and STAT3.

[0148] In some embodiments, the nuclease-dead Cas (dCas) protein is selected from the group consisting of dCas9, dCas12a, and dCas12b.

[0149] In some embodiments, the nuclease-dead Cas (dCas) protein is a fusion protein comprising a Cas protein and one or more epigenetic modulators that repress or silence the expression of target genes, namely ZNF274, MAX, E2F6, IKZF3 and STAT3.

[0150] Suitable epigenetic modulators include DNA methyltransferase enzymes (e.g., DNA methyltransferase 3 alpha ("DNMT3A") and DNA methyltransferase 3-like ("DNMT3L")), histone demethylases (e.g., lysine-specific histone demethylase 1 ("LSD1")), histone methyltransferase enzymes (e.g., G9A and SuV39h1), transcription factor recruitment domains (e.g., Krüppel-associated box domain ("KRAB"), KRAB-methyl-CpG binding protein 2 domain ("KRAB-MeCP2"), enhancer of Zeste 2 ("EZH2")), zinc finger transcriptional repressor domains (e.g., spalt-like transcription factor 1 ("SALL1"), and suppressor of defective silencing protein 3 ("SDS3")) (see, e.g., Brezgin et al., "Dead Caspase Genetics: A Novel Genetic Modulator for the Treatment of Alzheimer's Disease," vol. 14, no. 1, 2002, which is hereby incorporated by reference in its entirety). These include, but are not limited to, "Systems: Types, Principles, and Applications," Int. J. Mol. Sci. 20:6041 (2019)).

[0151] In some embodiments, the epigenetic modulator is selected from the group consisting of DNMT3A, DNMT2L, LSD1, KRAB, KRAB-MeCP2, EZH2, SALL1, SDS3, G9A, and Suv39h1 (see, e.g., Yeo et al., "An Enhanced CRISPR Repressor for Targeted Mammalian Gene Regulation," 15(8):611-616 (2018); Alerasool et al., "An Efficient KRAB Domain for CRISPRi Applications in Human Cells," Nature Methods 17:1093-1096 (2020); and Duke et al., "An Improved CRISPR / dCas9 Interference Tool for Neuronal Gene Suppression," Frontiers in Genome Editing 2:9 (2020), which are hereby incorporated by reference in their entireties).

[0152] In some embodiments, the ZNF274 inhibitor is a CRISPRi system targeted to silence ZNF274 DNA expression, and the guide RNA comprises a nucleotide sequence complementary to a portion of the ZNF274 gene sequence.

[0153] In some embodiments, the MAX inhibitor is a CRISPRi system targeted to silence MAX DNA expression, and the guide RNA comprises a nucleotide sequence complementary to a portion of the MAX gene sequence.

[0154] In some embodiments, the E2F6 inhibitor is a CRISPRi system targeted to silence E2F6 DNA expression, and the guide RNA comprises a nucleotide sequence complementary to a portion of the E2F6 gene sequence.

[0155] In some embodiments, the IKZF3 inhibitor is a CRISPRi system targeted to silence IKZF3 DNA expression, and the guide RNA comprises a nucleotide sequence complementary to a portion of the IKZF3 gene sequence.

[0156] In some embodiments, the STAT3 inhibitor is a CRISPRi system targeted to silence STAT3 DNA expression, and the guide RNA comprises a nucleotide sequence complementary to a portion of the STAT3 gene sequence.

[0157] In some embodiments, the agent that suppresses ZNF274, MAX, E2F6, IKZF3, and / or STAT3 comprises one or more expression vectors expressing one or more transcription factor inhibitors selected from the group consisting of a ZNF274 inhibitor, a MAX inhibitor, an E2F6 inhibitor, an IKZF3 inhibitor, and an IKZF3 inhibitor. Each expression vector comprises (1) a nucleotide sequence encoding one or more nucleic acid inhibitors for ZNF274, MAX, E2F6, IKZF3, and / or IKZF3, and (2) a regulatory sequence operably linked to the nucleotide sequence. In some embodiments, the regulatory sequence comprises a glial cell-specific promoter selected from the group consisting of a PDGFRA promoter, a ZNF488 promoter, a GPR17 promoter, an OLIG2 promoter, a CSPG4 promoter, and a SOX10 promoter. In some embodiments, the regulatory sequence comprises an inducible promoter or promoter system, such as a tetracycline-controlled inducible system, a cumate-controlled inducible system, and a rapamycin-controlled inducible system, which are described in more detail below.

[0158] In some embodiments, the one or more expression vectors comprise a plasmid vector, a viral vector, or a bacterial vector. In some embodiments, the one or more expression vectors comprise a viral vector selected from the group consisting of adenovirus, AAV, retrovirus, lentivirus, vaccinia virus, and herpes virus. In some embodiments, the one or more expression vectors comprise a lentiviral vector. In some embodiments, the one or more expression vectors comprise a retroviral vector. In other embodiments, the one or more expression vectors comprise an AAV vector. Methods for generating and isolating viral vectors suitable for use as expression vectors are described in more detail below.

[0159] IV. Expression Vectors Another aspect of the present invention relates to an expression vector as described herein. In some embodiments, the expression vector expresses one or more transcription factors selected from the group consisting of BCL11A, HDAC2, EZH2, MYC, HMGA2, NFIB and TEAD2, and the expression vector comprises (1) a nucleotide sequence encoding one or more transcription factors selected from the group consisting of BCL11A, HDAC2, EZH2, MYC, HMGA2, NFIB and TEAD2, and (2) a regulatory sequence operably linked to the nucleotide sequence.

[0160] In some embodiments, the expression vector comprises (1) a nucleotide sequence encoding one or more nucleic acid inhibitors of ZNF274, MAX, E2F6, IKZF3 and / or IKZF3, and (2) a regulatory sequence operably linked to the nucleotide sequence.

[0161] In some embodiments, the regulatory sequence comprises a glial cell-specific promoter selected from the group consisting of a PDGFRA promoter, a ZNF488 promoter, a GPR17 promoter, an OLIG2 promoter, a CSPG4 promoter, and a SOX10 promoter.

[0162] In some embodiments, the regulatory sequence comprises an inducible promoter and / or operator system sequence. Inducible promoters and / or operator systems that may be used in carrying out the disclosed methods or included in the disclosed systems include those regulated by hormones and hormone analogs, such as progesterone, ecdysone and glucocorticoids, as well as promoters regulated by tetracycline, heat shock, heavy metal ions, interferons and lactose operon activating compounds. An inducible promoter and / or operator system is capable of directly or indirectly activating transcription of a nucleic acid molecule to which it is operably coupled in response to a "regulator" (e.g., a chemical agent or a biological molecule, e.g., a metabolite, a small molecule) or stimulus. In the absence of a "regulator" or stimulus, a nucleotide sequence operably linked to an inducible promoter and / or operator system is not transcribed or is not substantially expressed.

[0163] The terms "not transcribed" or "not substantially expressed" mean that the level of transcription is at least 50-fold lower than the level of transcription observed in the presence of an appropriate stimulus or regulator; preferably at least 100-, 250-, or 500-fold lower than the level of transcription observed in the presence of an appropriate stimulus or regulator. For a review of these systems, see Gingrich and Roder, "Inducible Gene Expression in the Nervous System of Transgenic Mice," Annu. Rev. Neurosci. 21:377-405 (1998), which is hereby incorporated by reference in its entirety.

[0164] Suitable inducible promoter and / or operator systems for inclusion in the expression vectors of the present disclosure are well known in the art and include, without limitation, tetracycline-regulated operator systems, cumate-regulated operator systems, rapamycin-inducible systems, FKCsA-inducible systems, and ABA-inducible systems (see, e.g., Kallunki et al., "How to Choose the Right Inducible Gene Expression System for Mammalian Studies?" Cells 8(8):796 (2019); U.S. Patent No. 8,728,759; and U.S. Patent No. 7,745,592, which are hereby incorporated by reference in their entireties).

[0165] In some embodiments, the tetracycline-controlled operator system comprises a repression-based configuration, in which a Tet operator ("TetO") is inserted between a constitutive promoter and a gene of interest, and binding of a Tet repressor ("TetR") to the operator represses downstream transcription of the nucleic acid sequence of interest (see, e.g., Kallunki et al., "How to Choose the Right Inducible Gene Expression System for Mammalian Studies?" Cells 8(8):796 (2019), which is hereby incorporated by reference in its entirety). According to such embodiments, addition of tetracycline (or the synthetic tetracycline derivative doxycycline) results in disruption of the association between TetR and TetO, thereby inducing TetO-dependent transcription of the nucleic acid sequence of interest.

[0166] In some embodiments, the tetracycline-controlled operator system comprises a Tet-off configuration, in which tandem TetO sequences are positioned upstream of a minimal promoter followed by a nucleic acid sequence of interest (see, e.g., Kallunki et al., “How to Choose the Right Inducible Gene Expression System for Mammalian Studies?” Cells 8(8):796 (2019), hereby incorporated by reference in its entirety). According to such embodiments, a chimeric protein consisting of TetR and VP16, a eukaryotic transactivator derived from Herpes Simplex Virus Type 1 ("tTA"), is converted into a transcriptional activator, and an expression plasmid is transfected together with the operator plasmid. Thus, the presence of tetracycline (or the synthetic tetracycline derivative doxycycline) switches off expression of the system or its components, while removal of tetracycline switches it on.

[0167] In some embodiments, the tetracycline-controlled operator system comprises a Tet-On construct, and the system or its components are transcribed when tetracycline is present (see, e.g., Kallunki et al., "How to Choose the Right Inducible Gene Expression System for Mammalian Studies?" Cells 8(8):796 (2019), which is hereby incorporated by reference in its entirety). According to such embodiments, tandem TetO sequences are positioned upstream of a minimal promoter followed by a nucleic acid sequence of interest. In the presence of tetracycline (or the 5 synthetic tetracycline derivative doxycycline), mutant rTa ("rtTa") binds to the TetO sequence, thereby activating the minimal promoter.

[0168] In some embodiments, the inducible promoter and / or operator system is a cumate-regulated operator system. Similar to the tetracycline-regulated operator system, the cumate-regulated operator system, the cumate operator ("CuO") and its repressor ("CymR"), can be engineered into repressor, activator and reverse activator configurations (see, e.g., Kallunki et al., "How to Choose the Right Inducible Gene Expression System for Mammalian Studies?" Cells 8(8):796 (2019), which is hereby incorporated by reference in its entirety).

[0169] In some embodiments, a cumate-controlled operator system comprises a repression-based construct, in which a cumate operator ("CuO") is inserted between a constitutive promoter and a gene of interest, and binding of a cumate repressor ("CymR") to the operator represses downstream transcription of a system (or system component) described herein. According to such embodiments, addition of cumate releases CymR, thereby inducing CuO-dependent expression of the system or system component.

[0170] In some embodiments, the cumate-controlled operator system comprises an activator construct, in which a chimeric molecule ("cTA") is formed via the fusion of CymR with VP16. In this construct, a minimal promoter is placed downstream of a multimerized operator binding site (e.g., 6xCuO). Transcription of the nucleic acid sequence of interest is controlled by the minimal promoter, which is activated in the absence of cumate.

[0171] In some embodiments, the cumate-controlled operator system comprises a reverse activator construct, and the nucleic acid sequence is transcribed when cumate is present. According to such embodiments, tandem CuO sequences are positioned upstream of a minimal promoter followed by a nucleic acid sequence of interest. In the presence of cumate, the cTA mutant ("rcTA") binds to the CuO sequence, thereby activating the minimal promoter.

[0172] In some embodiments, the inducible promoter and / or operator system is a rapamycin-inducible system. According to such embodiments, the promoter is a rapamycin-inducible promoter (e.g., the minimal IL-2 promoter). In this system, the DNA 25 binding domain (ZFHD1) and the transcription factor activation domain (NF-ΚBp65) are expressed separately as fusion proteins with the rapamycin binding domains of FKBP12 and FRAP (mTOR), respectively (see, e.g., Koh et al., "Use of a Stringent Dimerizer-Regulated Gene Expression System for Controlled BMP2 Delivery," Mol. Ther. 14(5):P684-691 (2006), which is hereby incorporated by reference in its entirety). Upon addition of rapamycin (or the rapamycin analog FK506), the fusion proteins are reversibly crosslinked to drive transcription of the nucleic acid sequence of interest. Mutations in the rapamycin binding region of mTOR activation domain fusion proteins result in lines that are responsive to rapamycin-like compounds (rapalogs) that, unlike rapamycin, do not bind to the endogenous mTOR protein and therefore have little immunosuppressive or antiproliferative activity.

[0173] In some embodiments, the regulatory sequences include the human elongation factor 1 alpha promoter ("EF1A"), the cytomegalovirus ("CMV") promoter, the human ubiquitin C promoter ("UBC"), the chicken beta-actin promoter, the hybrid chicken beta-actin promoter (CBh) and the phosphoglycerokinase ("PGK") promoter. According to such embodiments, the promoters may be modified to include one, two, three or more Tet operator (TetO) sites, or one, two, three, four, five, six or more CuO sites.

[0174] Further suitable promoters for inclusion in the expression vector of the present invention include, but are not limited to, H1 promoter and U6 promoter. In some embodiments, when an inducible promoter and / or operator system is operably linked to a nucleotide sequence encoding one or more guide RNAs in a Cas system, the promoter is an H1 promoter or a U6 promoter. The H1 promoter can be a modified H1 promoter that contains one, two, three or more Tet operator (TetO) sites. The U6 promoter can be a modified U6 promoter that contains one, two or three Tet operator (TetO) sites or one, two, three, four, five, six or more CuO sites (see, e.g., Sun et al., "Development of Drug-20 Inducible CRISPR-Cas9 Systems for Large-Scale Functional Screening," BMC Genomics 20:225 (2019), which is hereby incorporated by reference in its entirety).

[0175] In some embodiments, the regulatory sequence may further comprise transcriptional enhancer binding sites, RNA polymerase initiation sites, ribosome binding sites, and / or other sites that facilitate expression of a nucleotide sequence operably linked to the regulatory sequence in an expression vector.

[0176] In some embodiments, the expression vector encodes a system for inducing rejuvenation of a population of adult glial progenitor cells or for treating a myelin deficiency in a subject.

[0177] In some embodiments, the system for inducing rejuvenation of a population of adult glial progenitor cells or treating myelin deficiency in a subject is a nuclease-dead Cas (dCas) system, comprising: a first nucleic acid molecule encoding a fusion protein comprising a nuclease-dead Cas (dCas) protein fused to an epigenetic modulator; a second nucleic acid molecule encoding one or more guide RNAs, each of the one or more guide RNAs comprising an RNA sequence that hybridizes to a portion of the DNA sequence of ZNF274, MAX, E2F6, IKZF3 and / or STAT3; and an inducible promoter and / or operator system operably linked to the first nucleic acid molecule, the second nucleic acid molecule, or both. In some embodiments, the inactivated Cas protein (dCas) is selected from the group consisting of dCas9, dCas12a, and dCas12b (see, e.g., Brezgin et al., "Dead Cas Systems: Types, Principles, and Applications," Int. J. Mol. Sci. 20:6041 (2019), which is hereby incorporated by reference in its entirety).

[0178] Suitable epigenetic modulators are described in detail above. In some embodiments, the epigenetic modulator is selected from the group consisting of DNMT3A, DNMT2L, LSD1, KRAB, KRAB-MeCP2, EZH2, SALL1, SDS3, G9A, Suv39h1, Cs, and WRPW (see, e.g., Yeo et al., "An Enhanced CRISPR Repressor for Targeted Mammalian Gene Regulation," 15(8):611-616 (2018); Alerasool et al., "An Efficient KRAB Domain for CRISPRi Applications in Human Cells," Nature Methods 17:1093-1096 (2020); and Duke et al., "An Improved CRISPR / dCas9 Interference Tool for Neuronal Gene Suppression," Frontiers in Genome Editing 2:9 (2020), which are hereby incorporated by reference in their entireties). In some embodiments, the epigenetic modulator is a methyltransferase, where methylation of a gene or a gene promoter is effective to repress its transcription.

[0179] In some embodiments, the epigenetic modulator is selected from the group consisting of Tet methylcytosine dioxygenase 1 ("TET1"), SunTag-TET1, MS2 / MCP-TET1, p300Core, four tandem copies of herpes simplex virus protein 16 ("VP64"), VP160, NF-KappaB p65 activation domain ("p65"), Epstein-Barr virus R transactivator ("Rta"), SunTag-VP64, VP64-p65-Rta ("VPR"), SunTag-p65-HSF1, TV, synergistic activation mediator ("SAM"), Three-Component Repurposed Technology for Enhanced Expression ("TREE"), Casilio, Scaffold, and CMV (see, e.g., Brezgin et al., "Dead Cas," which is hereby incorporated by reference in its entirety). (See, "Epigenetic Modulators: Types, Principles, and Applications," Int. J. Mol. Sci. 20(23):6041 (2019)). In some embodiments, the epigenetic modulator is a demethylase (e.g., TET1), where demethylation of a gene or gene protein is effective to repress its transcription.

[0180] In some embodiments, the system for inducing rejuvenation in a population of adult glial progenitor cells or treating a myelin deficiency in a subject comprises a dCas fusion protein in which dCas is fused to a methyltransferase. In any embodiment, the system for inducing rejuvenation in a population of adult glial progenitor cells or treating a myelin deficiency in a subject comprises a dCas fusion protein in which dCas is fused to a demethylase.

[0181] Exemplary dCas fusion proteins and dCas fusion protein systems for use in accordance with the methods of the invention are identified in Table 9, below.

[0182] [Table 9] JPEG2024539081000020.jpg32170

[0183] In some embodiments, the dCas protein is a dCas9 or dCas12 protein.

[0184] In some embodiments, the first and second nucleic acid molecules of the dCas system are contained in a single expression vector. In some embodiments, the first and second nucleic acid molecules of the system are contained in separate expression vectors.

[0185] In some embodiments, the system for inducing rejuvenation of a population of adult glial progenitor cells or treating myelin deficiency in a subject is a Cas system, comprising a first nucleic acid molecule encoding a Cas protein, a second nucleic acid molecule encoding one or more guide RNAs, each of the one or more guide RNAs comprising an RNA sequence that hybridizes to a portion of a DNA sequence of ZNF274, MAX, E2F6, IKZF3 and / or STAT3, and an inducible promoter and / or operator system operably linked to the first nucleic acid molecule, the second nucleic acid molecule, or both.

[0186] In some embodiments, the Cas nuclease system comprises a Cas9 protein or a Cas12 protein (e.g., Cas12a or Cas12b). Suitable Cas proteins and derivatives thereof for inclusion in a system according to the present disclosure are well known in the art and are described in more detail above.

[0187] In some embodiments, the first and second nucleic acid molecules of the Cas nuclease system are contained in a single expression vector. In some embodiments, the first and second nucleic acid molecules are contained in separate expression vectors.

[0188] In some embodiments, the system for inducing rejuvenation of a population of adult glial progenitor cells or treating myelin deficiency in a subject is a gene editing nuclease system, comprising: a first nucleic acid molecule encoding a first sequence-specific gene editing nuclease comprising a first DNA-binding motif, the first DNA-binding motif binding to a first DNA sequence of ZNF274, MAX, E2F6, IKZF3 and / or STAT3, respectively; a second nucleic acid molecule encoding a second sequence-specific gene editing nuclease comprising a second DNA-binding motif, the second DNA-binding motif binding to a second DNA sequence of ZNF274, MAX, E2F6, IKZF3 and / or STAT3, respectively; and an inducible promoter and / or operator system sequence operably coupled to the first nucleic acid molecule, the second nucleic acid molecule, or both the first and second nucleic acid molecules.

[0189] Suitable sequence-specific gene editing nucleases for inclusion in a system according to the invention are well known in the art and include, without limitation, zinc finger nucleases ("ZFNs") and transcription activator-like effector nucleases ("TALENs").

[0190] In one embodiment, the sequence-specific gene editing nuclease of the system described herein is a ZFN. A ZFN is an artificial endonuclease that comprises at least one zinc finger motif (e.g., at least two, three, four or five zinc finger motifs) fused to a nuclease domain (e.g., the cleavage domain of FokI restriction enzyme). Heterodimerization of two individual ZFNs in a target nucleic acid sequence can result in the cleavage of the target sequence. For example, two individual ZFNs can bind to opposite strands of a target DNA sequence to induce a double-stranded break in the target nucleic acid sequence. Methods for designing appropriate ZFNs for inclusion in the systems of the present disclosure are well known in the art (see, e.g., Urnov et al., "Genome Editing with Engineered Zinc Finger Nucleases," Nat. Rev. Genet. 11(9):636-646 (2010); Gaj et al., "Targeted Gene Knockout by Direct Delivery of Zinc-Finger Nuclease Proteins," Nat. Methods 2011, 11(9):636-646 (2011), which are hereby incorporated by reference in their entireties. 9(8):805-807 (2012); U.S. Patent No. 6,534,261; U.S. Patent No. 6,607,882; U.S. Patent No. 6,746,838; U.S. Patent No. 6,794,136; U.S. Patent No. 6,824,978; U.S. Patent No. 6,866,997; U.S. Patent No. 6,933,113; U.S. Patent No. 6,979,539; U.S. Patent No. 7,013,219; U.S. Patent No. 7,030,215; U.S. Patent No. 7,220,719; U.S. Patent No. 7,241,573; U.S. Patent No. 7,241,574; U.S. Patent No. 7,585,849; U.S. Patent No. 7,595,376; U.S. Patent No. 6,903,185; and U.S. Patent No. 6,479,626). In some embodiments, the first and second gene-editing nucleases are FokI nucleases. According to such embodiments, the first and second DNA-binding motifs are zinc finger motifs.

[0191] In some embodiments, the sequence-specific gene editing nuclease of the system described herein is a TALEN. A TALEN is an engineered transcription activator-like effector nuclease that includes a DNA binding domain and a nuclease domain (e.g., the cleavage domain of FokI restriction enzyme). The DNA binding domain includes a series of 33-35 amino acid repeat domains that each recognize a single base pair. Heterodimerization of two individual TALENs in a target nucleic acid sequence can result in cleavage of the target sequence. For example, two individual TALENs can bind to opposite strands of a target DNA sequence to induce a double-stranded break in the target nucleic acid sequence. Methods for designing appropriate ZFNs for inclusion in the systems of the present disclosure are well known in the art (see, e.g., Scharenberg et al., "Genome Engineering with TAL-Effector Nucleases and Alternative Modular Nuclease Technologies," Curr. Gene Ther. 13(4):291-303 (2013); Gaj et al., "Targeted Gene Knockout by Direct Delivery of Zinc-Finger Nuclease Proteins," Nat. Methods 9(8):805-807 (2012); Beurdeley et al., "Compact Designer TALENs for Efficient Genome Synthesis," 2013, which are hereby incorporated by reference in their entireties. (see U.S. Patent Nos. 8,440,431; 8,440,432; 8,450,471; 8,586,363; and 8,697,853). In some embodiments, the first and second gene-editing nucleases are FokI nucleases. According to such embodiments, the first and second DNA-binding motifs are TALE motifs.

[0192] In some embodiments, the first and second nucleic acid molecules of the sequence-specific gene editing nuclease system described herein are contained in a single expression vector. In some embodiments, the first and second nucleic acid molecules are contained in separate expression vectors.

[0193] In all of the systems described herein for rejuvenating glial progenitor cells or treating a myelin deficiency in a subject, the first and / or second nucleic acid molecules of the system are operably coupled to an inducible promoter and / or operator system, as described in more detail above.

[0194] In some embodiments, an expression vector of the invention is a plasmid vector, a viral vector, or a bacterial vector.

[0195] In some embodiments, an expression vector of the invention is a lentiviral vector (see, e.g., U.S. Pat. No. 748,529 to Fang et al.; Ura et al., "Developments in Viral Vector-Based Vaccines," Vaccines 2:624-641 (2014); and Hu et al., "Immunization Delivered by Lentiviral Vectors for Cancer and Infection Diseases," Immunol. Rev. 239:45-61 (2011), 15, which are hereby incorporated by reference in their entireties).

[0196] In some embodiments, expression vectors of the invention are retroviral vectors (see, e.g., U.S. Pat. No. 748,529 to Fang et al. and Ura et al., "Developments in Viral Vector-Based Vaccines," Vaccines 2:624-641 (2014), which are hereby incorporated by reference in their entireties), vaccinia virus, replication-deficient adenoviral vectors, and gutless adenoviral vectors (see, e.g., U.S. Pat. No. 5,872,005, which is hereby incorporated by reference in its entirety).

[0197] In other embodiments, an expression vector of the invention is an adeno-associated virus (AAV) vector (see, e.g., Krause et al., "Delivery of Antigens by Viral Vectors for Vaccination," Ther. Deliv. 2(1):51-70 (2011); Ura et al., "Developments in Viral Vector-Based Vaccines," Vaccines 2:624-641 (2014); Buning et al., "Recent Developments in Adeno-associated Virus Vector Technology," J. Gene Med. 10:717-733 (2008), each of which is hereby incorporated by reference in its entirety).

[0198] Methods for generating and isolating viral expression vectors suitable for use as vectors are known in the art (see, e.g., Bulcha et al., "Viral Vector Platforms within the Gene Therapy Landscape," Nature 6:53 (2021); Bouard et al., "Viral Vectors: From Virology to Transgene Expression," Br. J. Pharmacol. 157(2):153-165 (2009); Grieger and Samulski, "Adeno-associated Virus as a Gene Therapy Vector: Vector Development, Production and Clinical Applications," Adv. Biochem. Engin / Biotechnol. 99:119-145 (2005); Buning et al., "Recent Developments in Adeno-associated Virus Vector Technology," J. Gene Med. 10:717-733 (2008), each of which is hereby incorporated by reference in its entirety).

[0199] V. Genetically Modified Glial Progenitor Cells Aspects of the present disclosure also relate to glial progenitor cells genetically modified with an expression vector or nucleic acid molecule of the present application. In some embodiments, the expression vector or nucleic acid molecule is integrated into the genome of the genetically modified glial progenitor cell. In some embodiments, the expression vector or nucleic acid molecule is present in an epichromosomal form in the genetically modified glial progenitor cell.

[0200] In some embodiments, the glial precursor cells are genetically modified with a nuclease-dead Cas (dCas) system, a Cas nuclease system, or a gene-editing nuclease system according to the present invention.

[0201] In some embodiments, the genetically modified glial precursor cells are mammalian glial precursor cells. In any embodiment, the genetically modified glial precursor cells are human glial precursor cells.

[0202] Glial progenitor cells suitable for the genetic modifications described herein can be derived from multipotent cells (e.g., neural stem cells) or pluripotent cells (e.g., embryonic stem cells and induced pluripotent stem cells) using methods known in the art or described herein. In yet another embodiment, glial progenitor cells can be extracted directly from embryonic, fetal or adult brain tissue containing a mixed population of cells by using promoter-specific isolation techniques, as described in U.S. Patent Application Publication Nos. 20040029269 and 20030223972 to Goldman, which are hereby incorporated by reference in their entireties. According to this embodiment, glial progenitor cells are isolated from the ventricular or subventricular zone of the brain or from subcortical white matter.

[0203] In some embodiments, the genetically modified glial precursor cells are genetically modified bipotential glial precursor cells. In some embodiments, the genetically modified glial precursor cells are genetically modified oligodendrocyte-biased glial precursor cells. In some embodiments, the genetically modified glial precursor cells are genetically modified astrocyte-biased glial precursor cells.

[0204] In some embodiments, it may be preferred to enrich cell preparations containing glial progenitor cells before or after genetic modification to increase the concentration and / or purity of glial progenitor cells modified to contain the expression vectors or systems described herein. Thus, in one embodiment, the A2B5 monoclonal antibody (mAb), which recognizes and binds to gangliosides present on glial progenitor cells early in the development or differentiation process, is utilized to separate glial progenitor cells from a mixed population of cells (Nunes et al., "Identification and Isolation of Multipotential Neural Progenitor Cells From the Subcortical White Matter of the Adult Human Brain." Nat Med. 9(4):439-47 (2003), which is hereby incorporated by reference in its entirety). Using the A2B5 mAb, glial progenitor cells can be separated, enriched or purified from a mixed population of cell types. In another embodiment, selection of CD140α / PDGFRα positive cells is used to produce a purified or enriched preparation of bipotential glial progenitor cells. In another embodiment, CD9 positive cell selection is used to generate a purified or enriched preparation of glial precursor cells biased towards oligodendrocytes. In yet another embodiment, both CD140α / PDGFRα and CD9 positive cell selection are used to generate a purified or enriched preparation of glial precursor cells biased towards oligodendrocytes. In another embodiment, CD44 positive cell selection is used to generate a purified or enriched preparation of glial precursor cells biased towards astrocytes (Liu et al., "CD44 Expression Identifies Astrocyte-Restricted Precursor Cells," Dev. Biol. 276(1):31-46 (2004), which is hereby incorporated by reference in its entirety). In another embodiment, both CD140α / PDGFRα and CD44 positive cell selection are used to generate a purified or enriched preparation of glial precursor cells biased towards oligodendrocytes.In another embodiment, CD140α / PDGFRα, CD9 and CD44 positive cell selection is used to produce purified or enriched preparations of glial progenitor cells that are differentiation biased towards oligodendrocytes.

[0205] A further aspect of the invention relates to a preparation of glial precursor cells expressing a genetic construct according to the invention.

[0206] The following examples are intended to illustrate the practice of embodiments of the present invention, but are in no way intended to limit its scope. EXAMPLES

[0207] Materials and Methods cell line Human iPSC line C27 was used to generate hGPCs and validate the predicted transcripts of interest in hGPCs. The C27 line is male. Cells were differentiated into GPCs as detailed for human iPSC-derived GPC production (Chambers et al., "Highly Efficient Neural Conversion of Human ES and iPS Cells by Dual Inhibition of SMAD Signaling," Nat Biotechnol 27:275-280 (2009), which is hereby incorporated by reference in its entirety).

[0208] Adult and fetal brain processing for cell isolation Human brain samples were obtained from consenting patients at the University of Rochester Strong Memorial Hospital under an Institutional Review Board approved protocol. Brain tissue was obtained from normal GW18-24 cortical and / or VZ / SVZ dissections or adult white matter / cortical epilepsy resections (18F, 19M, and 27F for mRNA, 8M, 20F, 43M, and 54F for miRNA). Fetal GPC procurement, dissociation and immunomagnetic sorting of A2B5+ / PSA-NCAM- cells were as described (Windrem et al., "Fetal and Adult Human Oligodendrocyte Progenitor Cell Isolate Myelinate the Congenitally Dysmyelinated Brain," Nat. Med. 10:93-97 (2004), which is hereby incorporated by reference in its entirety). GPC were isolated from the dissociated tissue using a dual immunomagnetic sorting strategy. Microbead-tagged rat anti-mouse IgM (Miltenyi Biotech) was used to deplete mouse anti-PSA-NCAM+ (Millipore, DSHB) cells, and then A2B5+ (clone 105; ATCC, Manassas, VA) were selected from the PSA-NCAM- pool as described (Windrem et al., "Fetal and Adult Human Oligodendrocyte Progenitor Cell Isolates Myelinate the Congenitally Dysmyelinated Brain," Nat. Med. 10:93-97 (2004) and Windrem et al., "Neonatal Chimerization with Human Glial Progenitor Cells can both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse," Cell Stem Cell 2:553-565 (2008), which are hereby incorporated by reference in their entireties). After sorting, cells were maintained in DMEM-F12 / N1 with 10 ng / ml bFGF and 20 ng / ml PDGF-AA for 1-14 days.Alternatively, CD140a / PDGF R defined GPCs were isolated and sorted using MACS as previously described (Sim et al., "CD140a Identifies a Population of Highly Myelogenic, Migration-Competent and Efficiently Engrafting Human Oligodendrocyte Progenitor Cells," Nat. Biotechnol. 29:934-941 (2011b), which is hereby incorporated by reference in its entirety) to obtain an enriched population of CD140+ glial progenitor cells.

[0209] Bulk RNA sequencing RNA was purified from the isolates using the Qiagen RNeasy kit, and bulk RNA sequencing libraries were constructed. Samples were extensively sequenced on an Illumina HiSeq2500 at the University of Rochester Genomics Research Center. Raw FASTQ files were trimmed and adapters removed using fastp (Chen et al., "fastp: An Ultra-fast All-in-one FASTQ Preprocessor," Bioinformatics 34:i884-i890 (2018), which is hereby incorporated by reference in its entirety), aligned to GRCh38 using Ensembl95 gene annotations by STAR in two-pass mode across all samples (Dobin et al., "STAR: Ultrafast Universal RNA-seq Aligner," Bioinformatics 29:15-21 (2013), which is hereby incorporated by reference in its entirety), and quantified with RSEM version (Li and Dewey, "RSEM: Accurate Transcript Quantification From RNA-Seq Data With or Without a Reference Genome," BMC Bioinformatics 12:323 (2011), which is hereby incorporated by reference in its entirety).Subsequent analyses were performed in R (R Core Team R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing (2017), which is hereby incorporated by reference in its entirety), in this case importing RSEM gene-level results with tximport (Soneson et al., “Differential Analyses for RNA-seq: Transcript-Level Estimates Improve Gene-Level Inferences,” F1000Research 4:1521 (2015), which is hereby incorporated by reference in its entirety). DE analyses were performed with DESeq2 (Love et al., "Moderated Estimation of Fold Change and Dispersion for RNA-seq Data With DESeq2," Genome Biology 15:550 (2014), which is hereby incorporated by reference in its entirety), where paired analyses (fetal A2B5+ vs. CD140a+, fetal CD140a+ vs. CD140a-) added paired information to their models. For adult vs. fetal DE analyses, age was concatenated with the sorting marker (CD140a- samples were not included) to define group variables, and sequencing batch was also added to the model to account for technical variability. Genes with adjusted p-values ​​<0.01 and absolute Log2 fold change >1 were considered significant. These data were then further filtered by meaningful abundance, defined as a median TPM of 1 (calculated by RSEM) in at least one group (20,663 genes met this criterion before DE).

[0210] scRNA-Seq analysis Fetal brain samples were processed as described above for bulk rna-seq until single cells were sorted by FACS for either CD140a+ or PSA-NCAM- / A2B5+ surface expression. Single cells were then captured on a 10X genomics chrome controller using V2 chemistry and libraries were generated according to the manufacturer's instructions.

[0211] Samples were sequenced on an Illumina HISeq2500 system. The demultiplexed samples were then aligned and quantified using Cell Ranger against an index generated from GRCh38 and Ensembl95 gene annotations using only protein-coding lncRNA or miRNA biotypes. Analysis of the scRNA-Seq samples was performed with Seurat in R (Butler et al., "Integrating Single-cell Transcriptomic Data Across Different Conditions, Technologies, and Species," Nat Biotechnol 36:411-420 (2018), which is hereby incorporated by reference in its entirety). Both samples were merged and low-quality cells, defined as having >15% mitochondrial gene expression or <500 unique genes, were filtered out. Samples were then normalized using SCTransform, taking care to remove contributions from differences in total number of UMIs, percent mitochondrial gene content, or S-phase and G2M-phase scores of each cell by regression. We then computed PCA and ran UMAP using the first 30 dimensions with n.neighbors=60 and repulsion.strength=0.8. We then ran FindNeighbors followed by FindClusters with resolution set to 0.35. Based on the expression profiles of each cluster, some similar clusters were merged into broader cell type clusters.Static differential expression of clusters was calculated using the MAST test (Finak et al., "MAST: A Flexible Statistical Framework for Assessing Transcriptional Changes and Characterizing Heterogeneity in Single-cell RNA Sequencing Data," Genome Biology 16:278 (2015), which is hereby incorporated by reference in its entirety), and an adjusted p-value of <0.01 and an absolute Log2 fold change of >0.5 were considered significant. Prediction of active transcription factor regulons was performed with the SCENIC package in R (Aibar et al., "SCENIC: Single-cell Regulatory Network Inference and Clustering," Nat. Methods 14:1083-1086 (2017), which is hereby incorporated by reference in its entirety). Genes were included in the coexpression analysis if they were expressed in at least 1% of cells.

[0212] Ingenuity pathway analysis and network construction Differentially expressed genes were entered into Ingenuity Pathway Analysis (Qiagen) to determine significant canonical, functional, and upstream signaling terms. To construct the IPA network, terms were filtered with adjusted p-values ​​below 0.001. The iGraph package (Csardi, GN, Tamas, "The Igraph Software Package for Complex Network Research", InterJournal Complex Systems 1695 (2006), which is hereby incorporated by reference in its entirety) was used to remove irrelevant IPA terms, along with highly redundant functional terms, as assessed by the Jaccard similarity index. Modularity was established within Gephi (Bastian et al., Gephi: An Open Source Software for Exploring and Manipulating Networks, (2009), which is hereby incorporated by reference in its entirety) so that the final networks could be visualized using Cytoscape (Shannon P., Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks, Genome Res 13:2498-2504 (2003), which is hereby incorporated by reference in its entirety). Genes and terms of interest were retained for visualization. Modules were partitioned from each other and organized using the yFiles organic layout.

[0213] Estimation of transcription factor activity The adult enriched and fetal enriched gene lists were input separately into RcisTarget (Aibar et al., "SCENIC: Single-cell Regulatory Network Inference and Clustering," Nat. Methods 14:1083-1086 (2017), which is hereby incorporated by reference in its entirety) to identify overrepresentation of motifs in windows (500bp upstream / 100bp downstream and 10kb upstream and downstream) near the promoters of genes. Transcription factors associated with significantly enriched motifs (NES>3) were then filtered by significant differential expression of the input gene list. Within each window and gene list, only the appropriate TF-gene interactions (repressors that downregulate the gene and activators that upregulate the gene) were retained. The scan windows were then merged to generate TF-gene edge lists of predicted fetal / adult repressors / activators. TFs of interest were narrowed down to those that have been reported in the literature primarily only as activators or repressors.

[0214] miRNA microarray analysis A2B5+ adult (n=3) and CD140a+ fetal (n=4) cell suspensions were isolated by MACS as described above, and their miRNAs were isolated using the miRNeasy kit according to the manufacturer's instructions (QIAGEN). Purified miRNAs were then prepared and profiled on Affymetrix GeneChip miRNA3.0 arrays as directed by standard protocols. Raw CEL files were then loaded into R with the Oligo (Carvalho and Irizarry, "A Framework for Oligonucleotide Microarray Preprocessing," Bioinformatics 26:2363-2367 (2010), which is hereby incorporated by reference in its entirety) package, and samples were normalized with robust multiarray averaging (RMA). Probes were then filtered for only human miRNAs according to Affymetrix annotations and differential expression was performed in limma (Ritchie et al., "Limma Powers Differential Expression Analyses for RNA-Sequencing and Microarray Studies," Nucleic Acids Res 43:e47 (2015), which is hereby incorporated by reference in its entirety), with significance established at an adjusted p-value of <0.01. Finally, differentially expressed miRNAs were surveyed across five independent miRNA prediction databases using miRNAtap (Pajak M., "miRNAtap: microRNA Targets-Aggregated Predictions," R Package Version 1.22.0 (2020), which is hereby incorporated by reference in its entirety) with min_src set to 2 and method set to "geom".Transcription factor regulation of miRNAs was performed by querying the TransmiR V2.0 database (Tong et al., "TransmiR v2.0: An Updated Transcription Factor-microRNA Regulation Database," Nucleic Acids Res 47:D253-D258 (2019), which is hereby incorporated by reference in its entirety).

[0215] Exploratory Analysis and Visualization PCA of bulk RNA-Seq or microarray samples was calculated with prcomp using the default variance stabilization values ​​of the DESeq2 object. PCA was plotted with autoplot from the ggfortify package. Volcano plots were generated using EnhancedVolcano. Graphs were further edited or newly generated using ggplot2 and aligned using patchwork.

[0216] Human iPSC-derived GPC production Human induced pluripotent stem cells (C27 (Chambers et al., "Highly Efficient Neural Conversion of Human ES and iPS Cells by Dual Inhibition of SMAD Signaling," Nat Biotechnol 27:275-280 (2009), which is hereby incorporated by reference in its entirety)) were differentiated into GPCs using our previously described protocol (Osipovitch et al., "Human ESC-Derived Chimeric Mouse Models of Huntington's Disease Reveal Cell-Intrinsic Defects in Glial Progenitor Cell Differentiation," Cell Stem Cell 24:107-122e107 (2019); Wang et al., "Human iPSC-Derived Oligodendrocyte Progenitor Cells Can Myelinate and Rescue a Mouse Model of Congenital Hypomyelination," Cell Stem Cell 24:107-122e107 (2019)). 12:252-264 (2013); and Windrem et al., "Human iPSC Glial Mouse Chimeras Reveal Glial Contributions to Schizophrenia," Cell Stem Cell 21:195-208.e196 (2017), which are hereby incorporated by reference in their entireties. Briefly, cells were differentiated first into neuroepithelial cells, then into pre-GPCs, and finally into GPCs. GPCs were maintained in glial medium supplemented with T3, NT3, IGF1, and PDGF-AA.

[0217] Lentiviral overexpression For overexpression of E2F6, ZNF274, IKZF3, or MAX, we first identified the most abundant protein-coding transcript of each of these genes from the adult hGPC dataset. The cDNA of each transcript was cloned downstream of the tetracycline response element promoter in the pTANK-TRE-EGFP-CAG-rtTA3G-WPRE vector. Viral particles pseudotyped with vesicular stomatitis virus G glycoprotein were produced by transient transfection of HEK293FT cells, concentrated by ultracentrifugation, and titrated by QPCR (qPCR Lentivirus Titer Kit, ABM-Applied Biological Materials Inc). iPSC(C27)-derived GPC cultures (160-180 days in vitro) were infected in glial medium at 1.0 MOI for 24 h. Cells were washed with HBSS and maintained in glial medium supplemented with 1 μg / ml doxycycline (Millipore-Sigma, St. Louis, MO) for the remaining experiments. Transduced hGPCs were isolated by FACS for DAPI- / EGFP+ expression 3, 7, and 10 days after the initial addition of doxycycline. Doxycycline control cells were sorted with DAPI only.

[0218] quantitative PCR RNA for overexpression experiments was extracted using RNeasy micro kit (Qiagen, Germany). First-strand cDNA was synthesized using TaqMan reverse transcription reagents (Applied Biosystems, USA). qPCR reactions were performed in triplicate by loading 1 ng of RNA mixed with FastStart Universal SybrGreen Mastermix (Roche Diagnostics, Germany) per reaction and analyzed on a real-time PCR instrument (CFX Connect Real-Time System Thermocycler; Bio-Rad). Results were normalized to the expression of 18S in each sample.

[0219] Quantification and statistical analysis For qPCR experiments, significant differences in delta CT for each gene were analyzed with a linear model built by the interaction of overexpression condition and time point with the addition of cell batch covariate. Post-hoc pairwise comparisons were performed with least-squares means tests against Dox control within time points using the lsmeans package (Lenth, RV, "Least-Squares Means: The R Package lsmeans", Journal of Statistical Software, Foundation for Open Access Statistics Vol. 69(i01) (2016), which is hereby incorporated by reference in its entirety). P values ​​were adjusted for multiple comparisons using the false discovery rate method, where a p value <0.05 was considered significant.

[0220] Example 1 CD140a selection enriches human fetal glial progenitor cells more efficiently than A2B5 To identify transcriptional associates with GPC aging, we first used bulk and single-cell RNA-Seq to identify the CD140a epitope of PDGFRα (Sim et al., "CD140a Identifies a Population of Highly Myelinogenic, Migration-Competent and Efficiently Engrafting Human Oligodendrocyte Progenitor Cells," Nature Biotechnology 29:934-941 (2011a), which is hereby incorporated by reference in its entirety) or the glial ganglioside recognized by the monoclonal antibody A2B5 (Dietrich et al., "Characterization of A2B5+ Glial Precursor Cells From Cryopreserved Human Fetal Brain Progenitor Cells," Glia 40:65-77 (2002); Sim et al., "CD140a Identifies a Population of Highly Myelinogenic, Migration-Competent and Efficiently Engrafting Human Oligodendrocyte Progenitor Cells," Nature Biotechnology 29:934-941 (2011a), which is hereby incorporated by reference in its entirety) In this study, we characterized hGPCs derived from second trimester fetal human tissue, whether isolated by targeting human fibroblasts (e.g., Fischer et al., "Engraftment and Efficiently Engrafting Human Oligodendrocyte Progenitor Cells," Nature Biotechnology 29:934-941 (2011a); and Windrem et al., "Fetal and Adult Human Oligodendrocyte Progenitor Cell Isolates Myelinate the Congenitally Dysmyelinated Brain," Nat. Med. 10:93-97 (2004), which are hereby incorporated by reference in their entireties).To this end, we performed two sample-matched experiments in which the ventricular / subventricular zone (VZ / SVZ) of 18-22 gestational age (ga) fetal brains were dissociated and sorted by fluorescence-activated cell sorting (FACS) for either CD140a+ and A2B5+ / PSA-NCAM- (A2B5+) GPCs or CD140a+ GPCs isolated from the same fetal brains (n=3), as well as CD140a-depleted remnants (n=5; Figure 1, panel A). Bulk RNA-Seq libraries were then generated and sequenced in depth for both experiments. Principal component analysis (PCA) demonstrated the separation of CD140a+ and A2B5+ cells, with further separation of both cells from the CD140a-depleted sample (Figure 1, panel B). Differential expression (p<0.01, absolute Log2 fold change>1) in both paired cohorts identified 723 genes as differentially expressed between CD140a+ and A2B5+ GPCs (435 for CD140a and 288 for A2B5, Table S1). In contrast, 2,629 genes distinguished CD140a+ GPCs from CD140a- cells (Figure 1, Panel C). When comparing CD140+ cells with either A2B5+ or CD140- cells, the differential gene expression direction was highly consistent, with all but four genes matching.

[0221] Pathway enrichment analysis using Ingenuity Pathway Analysis (IPA) of both of these gene sets identified similar pathways that were relatively active in CD140+ GPCs. These pathways included cell motility, oligodendrocyte differentiation, lipid synthesis, and downstream PDGF, SOX10, and TCF7L2 signaling (Figure 1, Panel D). As expected, stronger activation Z-scores were typically observed when comparing CD140a+ GPCs to CD140a- cells but not to A2B5+ GPCs. Interestingly, CD140a+ cells also differentially expressed a number of pathways related to the immune system, possibly due to a small amount of microglial contamination as a result of re-expression of the PDGF R epitope on the microglial surface. A2B5+ samples further showed upregulation of ST8SIA1, the enzyme responsible for A2B5 synthesis (Sim et al., "Fate Determination of Adult Human Glial Progenitor Cells," Neuron Glia Biol 5:45-55 (2009), which is hereby incorporated by reference in its entirety), as well as the pre-neuronal pathway.

[0222] Among the genes differentially upregulated in CD140a+ isolates were PDGFRA itself, as well as a number of early oligodendroglial genes, including OLIG1, OLIG2, NKX2-2, SOX10, and GPR17 (Figure 1, Panel E-1, Panel F). In addition, the CD140a+ fraction also exhibited increased expression of late myelination-related genes, including MBP, GAL3ST1, and UGT8. In addition to enrichment for the oligodendroglial lineage, CD140a isolates were also enriched for a number of genes typically associated with microglia, including CD68, C2, C3, C4, and TREM2. In contrast, A2B5+ isolates showed enrichment of astroglial (AQ4, CLU) and early neuronal (NEUROD1, NEUROD2, GABRG1, GABRA4, EOMES, HTR2A) genes, suggesting expression of A2B5 by immature astrocytes and neurons as well as by GPCs and oligodendroglial lineage cells. Overall, oligodendroglial enrichment was significantly higher in CD140a+ GPCs than A2B5-defined GPCs when compared to each depleted fraction, and CD140a isolates were more enriched in hGPCs, thus suggesting that CD140a is a more appropriate phenotype for direct comparison with adult hGPCs.

[0223] Example 2 Single-cell RNA sequencing revealed cellular heterogeneity within human fetal GPC isolates To further delineate the composition of fetal hGPC isolates at single-cell resolution, both CD140a+ and A2B5+ hGPCs were isolated from 20-week ga fetal VZ / SVZ by FACS and the transcriptome of each was assayed by single-cell RNA-Seq (Figure 1, Panel A, 10X Genomics V2). We sought to capture >1,000 cells of each. After filtering low-quality cells (<500 unique genes, mitochondrial gene percentage >15%), 1,053 PSA-NCAM- / A2B5+ and 957 CD140a+ high-quality cells remained (median 6,845 unique molecular identifiers and 2,336 unique genes per cell). Dimensionality reduction by uniform manifold approximation and projection (UMAP), followed by shared nearest neighbor modularity-based clustering of all cells using Seurat (Butler et al., "Integrating Single-cell Transcriptomic Data Across Different Conditions, Technologies, and Species," Nat Biotechnol 36:411-420 (2018), which is hereby incorporated by reference in its entirety), revealed 11 clusters with eight major cell types defined by differential enrichment of marker genes. These major cell types included GPCs, pre-GPCs, neural progenitor cells (NPCs), immature neurons, neurons, microglia, and a cluster of endothelial cells and pericytes. We found that CD140a+ FACS isolates were more enriched for GPC and pre-GPC populations than fetal A2B5+ / PSA-NCAM- cells (Figure 2, Panel A-2, Panel D). Furthermore, CD140a-sorted cells were primarily restricted to GPC and pre-GPC with only scattered microglial contamination, whereas A2B5+ / PSA-NCAM- isolates also contained astrocytes and neural lineage cells.The latter were included despite prior depletion of neural PSA-NCAM. These data support the more selective and phenotypically restricted nature of CD140a-based, rather than A2B5-based, GPC isolation.

[0224] Based on this, we next explored the gene expression profiles of the predominant cell populations in CD140a+ fetal isolates, GPCs, and pre-GPCs. Differential expression between these two pools yielded 269 genes (143 upregulated and 126 downregulated, p<0.01, Log2 fold change>0.5, Figure 2, Panel E). During the transition from pre-GPCs to GPCs, early oligodendroglial lineage genes were rapidly upregulated (OLIG2, SOX10, NKX2-2, PLLP, APOD), whereas genes expressed in pre-GPCs were effectively lost (VIM, HOPX, TAGLN2, TNC). Interestingly, genes involved in the human leukocyte antigen system, including HLA-A, HLA-B, HLA-C, and B2M, were all downregulated as cells transitioned to the GPC stage (Figure 2, Panel F). IPA analysis showed that pre-GPCs were relatively enriched for terms associated with migration, proliferation, and predictors of astroglial identity (BMP4, AGT, and VEGF signaling), whereas GPCs showed enrichment for terms associated with acquisition of oligodendroglial identity (PDGF-AA, FGFR2, CCND1) in addition to activation of the MYC and MYCN pathways (Figure 2, Panel G). Next, using single-cell co-expression data along with promoter motif enrichment using the SCENIC package (Aibar et al., "SCENIC: Single-cell Regulatory Network Inference and Clustering," Nat Methods 14:1083-1086 (2017), which is hereby incorporated by reference in its entirety), we identified 262 transcription factors that were predicted to be relatively more activated in GPCs compared to pre-GPCs (Wilcoxon rank sum test, p<0.01). These included SATB1 and the early GPC specificity factors OLIG2, SOX10, and NKX2-2 (Figure 2, panel H).

[0225] Example 3 Human adult and fetal GPC are transcriptionally distinct In this study, we next investigated how adult hGPCs may transcriptionally differ from fetal hGPCs. To this end, A2B5+ hGPCs were isolated from surgically resected adult human temporal neocortex (ages 19–21, n=3) and paired with four additional fetal CD140a+ samples to assess bulk RNA expression. A2B5 selection has previously been noted to be sufficient to isolate GPCs from adult human brain, and in that respect is more sensitive than CD140a, given the maturation-associated downregulation of PDGFRA expression in adult hGPCs (Sim et al., "Complementary Patterns of Gene Expression by Human Oligodendrocyte Progenitors and their Environment Predict Determinants of Progenitor Maintenance and Differentiation," Ann Neurol 59:763-779 (2006) and Windrem et al., "Fetal and Adult Human Oligodendrocyte Progenitor Cell Isolates Myelinate the Congenitally Dysmyelinated Brain," Nat. Med. 10:93-97 (2004), which are hereby incorporated by reference in their entireties).

[0226] PDGFRA in A2B5+ adult GPCs was found to be expressed at a median TPM of 0.55 compared to a median TPM of 47.56 in fetal A2B5+ cells, confirming previous observations. Pairing sequencing with analysis of fetal CD140a-selected cells allowed regression of sequencing batch effects while improving power (Figure 3, Panel A). Depletion of PSA-NCAM+ cells was not necessary in adult hGPC samples because expression of PSA-NCAM is silenced in adult cortex and white matter (Seki et al., "Distribution and Possible Roles of the Highly Polysialylated Neural Cell Adhesion Molecule (NCAM-H) in the Developing and Adult Central Nervous System," Neurosci. Res. 1:265-290 (1993), which is hereby incorporated by reference in its entirety). As a result, PCA of human adult and fetal GPCs showed tight clustering of adult GPCs, clearly separating them from the sorted fetal hGPC pool (Figure 3, Panel B). Differential expression of adult GPCs compared to either the A2B5+ or CD140a+ fetal GPC populations yielded 3,142 and 5,282 significant genes, respectively (p<0.01; absolute Log2 fold change>1) (Figure 3, Panel C). To increase the accuracy in defining differential expression, downstream analysis was performed on the 2,720 common genes (Figure 3, Panel D; 1,060 upregulated and 1,660 downregulated in adult GPCs compared to fetal hGPCs). Strikingly, within these two sets of differentially expressed genes, 100% of the genes were directionally concordant.

[0227] To better understand the differences between adult and fetal GPCs, we constructed a gene ontology network of non-redundant significant IPA terms and their contributing differentially expressed genes (Figure 3, Panels D-3, Panel E). Spin glass community detection of this network (Reichardt et al., "Statistical Mechanics of Community Detection," Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 74:016110 (2006), which is hereby incorporated by reference in its entirety) revealed three modules (modules M1-M3) of highly connected functional terms (Figure 3E) and genes (Figure 3F, Table S3). M1 contained terms and genes related to glial development, proliferation, and motility. Notably, a number of genes associated with GPC ontogeny were downregulated in adult GPCs.These genes included CSPG4 / NG2, PCDH15, CHRDL1, LMNB1, PTPRZ1, and ST8SIA1 (Huang et al., "Origins and Proliferative States of Human Oligodendrocyte Precursor Cells," Cell 182:594-608e511 (2020); McClain et al., "Pleiotrophin Suppression of Receptor Protein Tyrosine Phosphatase-β / ζ Maintains the Self-renewal Competence of Fetal Human Oligodendrocyte Progenitor Cells," J Neurosci 32:15066-15075 (2012); Nishiyama et al., "Co-Localization of NG2 Proteoglycan and PDGF α-Receptor on O2A Progenitor Cells in the Developing Rat Brain," Journal of Neuroscience Research 43:299-314 (1996); Sim et al., "Fate Determination of Adult Human Glial Progenitor Cells," Neuron Glia Biol 5:45-55 (2009); and Yattah et al., "Dynamic Lamin B1-Gene Association During Oligodendrocyte Progenitor Differentiation," Neurochem Res 45:606-619 (2020), which are hereby incorporated by reference in their entireties.) In contrast, numerous genes whose appearance precedes and continues throughout oligodendrocyte differentiation and myelination were upregulated in adult GPCs, including MAG, MOG, MYRF, PLP1, CD9, CLDN11, CNP, ERBB4, GJB1, PMP22, and SEMA4D.

[0228] Module 2 contained numerous terms associated with cellular aging and modulation of proliferation and senescence. Growth factors including MKI67, TOP2A, CENPF, CENPH, CHEK1, and EZH2, as well as numerous cyclins including CDK1 and CDK4, were strongly enriched, predicting that cell cycle progression and mitosis are activated in fetal GPCs. Furthermore, proliferation-inducing pathways were also predicted to be activated. These pathways included MYC, CCND1, and YAP1 signaling, of which both YAP1 and MYC transcripts were similarly upregulated (Bretones et al., "Myc and Cell Cycle Control," Biochim. Biophys Acta 1849:506-516 (2015); Bunt et al., "Regulation of Cell Cycle Genes and Induction of Senescence by Overexpression of OTX2 in Medulloblastoma Cell Lines," Mol. Cancer Res. 8:1344-1357 (2010);and Xie et al., "YAP / TEAD-Mediated Transcription Controls Cellular Senescence," Cancer Res 73:3615-3624 (2013), which are hereby incorporated by reference in their entireties.) In this regard, it has recently been shown that transient overexpression of MYC in aging rodent GPCs restores both proliferation and differentiation capacity (Neumann et al., "Myc Determines the Functional Age State of Oligodendrocyte Progenitor Cells," Nature Aging 1:826-837 (2021), which are hereby incorporated by reference in their entireties). Conversely, adult GPCs exhibited upregulation of senescence-associated transcripts, including E2F6, MAP3K7, DMTF1 / DMP1, OGT, AHR, RUNX1, and RUNX2 (Ferrand et al., "Screening of a Kinase Library Reveals Novel Pro-senescence Kinases and Their Common NF-κB-dependent Transcriptional Program," Aging (Albany NY) 7:986-1003 (2015); Inoue et al., "Disruption of the ARF Transcriptional Activator DMP1 Facilitates Cell Immortalization, Ras Transformation, and Tumorigenesis," Genes Dev 14:1797-1809 (2000);Lee and Zhang, "O-Linked N-Acetylglucosamine Transferase (OGT) Interacts With the Histone Chaperone HIRA Complex and Regulates Nucleosome Assembly and Cellular Senescence," Proceedings of the National Academy of Sciences 113:E3213-E3220 (2016); Wotton et al., "RUNX1 Transformation of Primary Embryonic Fibroblasts is Revealed in the Absence of p53," Oncogene 23:5476-5486 (2004); and Kilbey et al., "Runx2 Disruption Promotes Immortalization and Confers Resistance to Oncogene-induced Senescence in Primary Murine Fibroblasts," Cancer Res 67:11263-11271 (2007), which are hereby incorporated by reference in their entireties. Concomitantly, adult hGPCs exhibited downregulation of fetal transcripts, including LMNB1, PATZ1, BCL11A, HDAC2, FN1, EZH2, and YAP1, as well as its cofactor TEAD1 (Cho et al., "POZ / BTB and AT-hook-containing Zinc Finger Protein 1 (PATZ1) Inhibits Endothelial Cell Senescence Through a p53 Dependent Pathway," Cell Death Differ 19:703-712 (2012); Fan et al., "EZH2-dependent Suppression of a Cellular Senescence Phenotype in Melanoma Cells by Inhibition of p21 / CDKN1A Expression," Mol. Cancer Res. 9:418-429 (2011);Freund et al., "Lamin B1 Loss is a Senescence-associated Biomarker," Mol. Biol. Cell 23:2066-2075 (2012); Luc et al., "Bcl11a Deficiency Leads to Hematopoietic Stem Cell Defects with an Aging-like Phenotype," Cell Rep. 16:3181-3194 (2016); Lukjanenko et al., "Loss of Fibronectin From the Aged Stem Cell Niche Affects the Regenerative Capacity of Skeletal Muscle in Mice," Nat Med 22:897-905 (2016); Sundar et al., "Genetic Ablation of Histone Deacetylase 2 Leads to Lung Cellular Senescence and Lymphoid Follicle Formation in COPD / Emphysema," FASEB Journal:Official Publication of the Federation of American Societies for Experimental Biology 32:4955-4971 (2018);(See, e.g., Xie et al., “YAP / TEAD-Mediated Transcription Controls Cellular Senescence,” Cancer Res 73:3615-3624 (2013), which are hereby incorporated by reference in their entireties.) As a result, functional terms predicted to be active in adult hGPCs included senescence, the onset of the rapid aging observed in Hutchinson-Gilford progeria, and cyclin-dependent kinase inhibitory pathways downstream of CDKN1A / p21 and CDKN2A / p16. Furthermore, AHR and its signaling pathway have been implicated in driving senescence through inhibition of MYC (Yang et al., "The Aryl Hydrocarbon Receptor Constitutively Represses C-Myc Transcription in Human Mammary Tumor Cells," Oncogene 24:7869-7881 (2005), which is hereby incorporated by reference in its entirety), and were similarly upregulated in adult GPCs;

[0229] Module 3 consisted primarily of developmental and disease-related signaling pathways that are also associated with aging. This included predicting activation of ASCL1 signaling and BDNF signaling in fetal hGPCs, and MAPT / Tau signaling, APP signaling, and REST signaling in adult GPCs (Ahlin et al., "High Expression of Cyclin D1 is Associated to High Proliferation Rate and Increased Risk of Mortality in Women With ER-positive But Not in ER-negative Breast Cancers," Breast Cancer Res. Treat 164:667-678 (2017); Erickson et al., "Brain-derived Neurotrophic Factor is Associated With Age-related Decline in Hippocampal Volume," J. Neurosci. 30:5368-5375 (2010); and Harris et al., "Coordinated Changes in Cellular Behavior Ensure the Lifelong Maintenance of the Hippocampal Stem Cell Population," Cell Stem Cell (2021), which are hereby incorporated by reference in their entireties). Overall, transcriptional and functional profiling of adult GPCs revealed a reduction in transcripts associated with proliferative capacity, as well as a shift towards a senescent and more mature phenotype.

[0230] Example 4 Estimated transcription factor activity suggests involvement of an adult GPC transcriptional repressor Given the significant transcriptional disparity between adult and fetal GPCs, this study next investigated whether it was possible to predict which transcription factors direct their identity. To achieve this, two promoter windows (500bp upstream / 100bp downstream, 10kb upstream / 10kb downstream) of adult or fetal enriched GPC gene sets were first scanned to predict significantly enriched TF motifs (Aibar et al., "SCENIC: Single-cell Regulatory Network Inference and Clustering," Nat. Methods 14:1083-1086 (2017), which is hereby incorporated by reference in its entirety). This led to the identification of 48 TFs that were also differentially expressed in the common dataset scanned. Among these, we first investigated TFs whose primary means of DNA interaction was either exclusively inhibitory or stimulatory, while also considering the enrichment of known cofactors. This analysis yielded 12 potential upstream regulators to explore (Figure 4, Panels A-4, Panel C): four adult repressors E2F6, ZNF274, MAX, and IKZF3; one adult activator STAT3; three fetal repressors BCL11 AHDAC2, and EZH2; and four fetal activators MYC, HMGA2, NFIB, and TEAD2. Interestingly, among these predicted TFs, three groups shared high concordance motif similarities within their target promoters: 1) E2F6, ZNF274, MAX, and MYC; 2) STAT3 and BCL11A; and 3) EZH2 and HDAC2, suggesting that they may cooperate or compete for DNA binding at shared loci (Figure 4, Panel A).

[0231] Next, to predict genes targeted by the set of identified TFs, we constructed four potential signaling pathways based on the curated transcriptional interactions ( Figure 4 , Panels D-4 and G). Among the activators enriched in fetal GPCs (Figure 4, panel D), the growth factor MYC (Dang, CV, "c-Myc Target Genes Involved in Cell Growth, Apoptosis, and Metabolism", Molecular and Cellular Biology 19:1 (1999), which is hereby incorporated by reference in its entirety), NFIB, a key determinant of gliogenesis (Deneen et al., "The Transcription Factor NFIA Controls the Onset of Gliogenesis in the Developing Spinal Cord", Neuron 52:953-968 (2006), which is hereby incorporated by reference in its entirety), the YAP / TAZ effector TEAD2, and another growth factor, HMGA2, were each predicted to activate a cohort of progenitor stage genes, including both mitosis-associated transcripts and those that have been shown to inhibit the onset of senescence ... Growth, Apoptosis, and Metabolism," Molecular and Cellular Biology 19:1 (1999); Deneen et al., "The Transcription Factor NFIA Controls the Onset of Gliogenesis in the Developing Spinal Cord," Neuron 52:953-968 (2006);Diepenbruck et al., "Tead2 Expression Levels Control the Subcellular Distribution of Yap and Taz, Zyxin Expression and Epithelial- mesenchymal Transition," Journal of Cell Science 127:1523-1536 (2014); and Yu et al., "HMGA2 Regulates the in Vitro Aging and Proliferation of Human Umbilical Cord Blood-Derived Stromal Cells Through the mTOR / p70S6K Signaling Pathway," Stem Cell Res 10:156-165 (2013), which are hereby incorporated by reference in their entireties. Direct positive regulation between these four fetal activators was also predicted, with NFIB driven by HMGA2 and TEAD2, MYC driven by TEAD2 and NFIB, HMGA2 driven by MYC and TEAD2, and TEAD2 reciprocally driven by MYC (Figure 4, Panel D). In contrast to these fetal activators, fetal repressors including the C2H2-type zinc finger BCL11A, the polycomb repressive complex subunit EZH2, and the histone deacetylase HDAC2 were each predicted to repress more mature oligodendrocyte gene expression at this stage (Figure 4, Panel E) (Laherty et al., "Histone Deacetylases Associated With the mSin3 Corepressor Mediate Mad Transcriptional Repression," Cell 89:349-356 (1997);Laible et al., "Mammalian Homologues of the Polycomb-group Gene Enhancer of Zeste Mediate Gene Silencing in Drosophila Heterochromatin and at S.cerevisiae Telomeres," EMBO J 16:3219-3232 (1997); and Nakamura et al., "Evi9 Encodes a Novel Zinc Finger Protein that Physically Interacts with BCL6, a known Human B-Cell Proto-Oncogene Product," Mol Cell Biol 20:3178-3186 (2000), which are hereby incorporated by reference in their entireties. Moreover, all three of these TFs were predicted to inhibit targets implicated in senescence. Thus, these factors are believed to directly regulate downstream transcriptional events leading to the maintenance of a cycling progenitor state.;

[0232] These predicted adult GPC signaling networks were then evaluated for potential mechanisms responsible for their aging-associated gene expression changes. STAT3 was predicted to shift GPC identity toward glial maturation by upregulating a large cohort of early differentiation- and myelination-associated oligodendrocyte genes (Figure 4, Panel F). In addition, STAT3 was predicted to activate a set of aging-associated genes, including BIN1, RUNX1, RUNX2, DMTF1, CD47, MAP3K7, CTNNA1, and OGT. Concurrently, repression in adult GPCs was predicted to be achieved through the Ikaros family zinc finger IKZF3 / Aiolos, the KRAB (kruppel-associated box) zinc finger ZNF274, the MYC-associated factor MAX, and the cell cycle regulator E2F6 (Figure 4, panel G) (Blackwood and Eisenman, "Max: A Helix-loop-helix Zipper Protein That Forms a Sequence-specific DNA-binding Complex With Myc," Science 251:1211-1217 (1991); Frietze et al., "ZNF274 Recruits the Histone Methyltransferase SETDB1 to the 3'Ends of ZNF Genes," PLoS One 5:e15082 (2010); Ma et al., "Ikaros and Aiolos Inhibit Pre-B-cell Proliferation by Directly Suppressing c-Myc Expression," Mol Cell 2014, 14:111-112 (2014)). Biol 30:4149-4158 (2010b); and Ogawa et al., "A Complex with Chromatin Modifiers that Occupies E2F- and Myc-Responsive Genes in G0 Cells," Science 296:1132-1136 (2002), which are hereby incorporated by reference in their entireties.Targeting by this set of transcription factors predicted repression of a set of genes that contribute to the fetal GPC signature. This was indeed observed in the downregulation of the early progenitor genes PDGFRA and CSPG4, and the cell cycle genes CDK1, CDK4, and MKI67. It also predicted repression of YAP1, LMNB1, and TEAD1, whose expression slows or prevents the onset of senescence. Interestingly, this set of four adult repressors predicted downregulated expression of each of the fetal-enriched activators NFIB, MYC, TEAD2, and HMGA2, in addition to the fetal-enriched repressors BCL11A, EZH2, and HDAC2.

[0233] Example 5 Expression of adult-enriched repressors induces age-associated transcriptional changes in GPCs We next investigated whether the four adult-enriched transcriptional repressors E2F6, IKZF3, MAX, and ZNF274 identified in panel G of Figure 4 were individually sufficient to induce aspects of age-associated changes in gene expression by otherwise young GPCs. To accomplish this, doxycycline (Dox)-inducible overexpression lentiviruses were designed for each transcription factor (Figure 5, panel A). Briefly, to best mimic endogenous age-associated upregulation, we first identified for each repressor which protein-coding isoform was most abundant in adult GPCs. These candidates were E2F6-202, IKZF3-217, MAX-201, and ZNF274-201. These cDNAs were cloned downstream of a tetracycline-responsive element promoter and upstream of a T2A self-cleaving EGFP reporter (Figure 5, panel A). Human induced pluripotent stem cell (iPSC)-derived hGPC cultures prepared from the C27 line were then infected for 24 hours as previously described (Wang et al., "Human iPSC-Derived Oligodendrocyte Progenitor Cells Can Myelinate and Rescue a Mouse Model of Congenital Hypomyelination," Cell Stem Cell 12:252-264 (2013), which is hereby incorporated by reference in its entirety) and then treated with Dox to induce transgene overexpression.C27 iPSC-derived GPCs were chosen because their transcriptome resembles that of fetuses, they engraft similarly upon transplantation, and they are able to myelinate hypomyelinated mice (Wang et al., “Human iPSC-Derived Oligodendrocyte Progenitor Cells Can Myelinate and Rescue a Mouse Model of Congenital Hypomyelination,” Cell Stem Cell 12:252-264 (2013); and Windrem et al., “Human iPSC Glial Mouse Chimeras Reveal Glial Contributions to Schizophrenia,” Cell Stem Cell 21:195-208.e196 (2017), which are hereby incorporated by reference in their entireties). At 3, 7, and 10 days after Dox addition, overexpressing cells were selected by FACS for EGFP expression (FIG. 5, Panel B, n=3-5). Uninfected cultures receiving Dox were used as controls.

[0234] RNA was extracted and aging-associated genes of interest were analyzed by qPCR. Significant induction of each adult-enriched repressor was observed at each time point after Dox complementation (Figure 5, panel C). MKI67 and CDK1, genes whose upregulation is associated with active cell division, were significantly repressed at two or more time points in each overexpression paradigm (Figure 5, panel D). This was consistent with their decreased expression in adult GPCs (Figure 3, panel F), suggesting their direct repression by E2F6, MAX, and ZNF274 (MKI67) or by all four (CDK1). The GPC stage marker PDGFRA, the cognate receptor for PDGF-AA, was also significantly repressed at two time points in day 3 IKZF3-transduced GPCs as well as E2F6-transduced GPCs, consistent with repression in normal adult GPCs. Interestingly, the senescence-associated cyclin-dependent kinase inhibitor CDKN1A / p21 was upregulated at all time points in response to each of the repressors tested, whereas CDKN2A / p16 was similarly upregulated at all time points in ZNF274-transduced hGPCs as well as in E2F6-overexpressing GPCs at day 7 (Figure 5, panel D). In addition, both MBP and IL1A, both of which are strongly upregulated in adult hGPCs compared to fetuses, showed clear trends towards upregulated expression in response to repressor transduction, although time point-related variability prevented their increases from achieving statistical significance. Taken together, these data supported our prediction that forced premature expression of the adult-enriched GPC repressors E2F6, IKZF3, MAX, and ZNF274 are individually sufficient to induce multiple features of the aged GPC transcriptome in young iPSC-derived GPCs.

[0235] Example 6 miRNA expression patterns in fetal hGPCs predict suppression of aging To identify potential post-transcriptional regulators of gene expression, we assessed the differences in miRNA expression between adult and fetal GPCs (n=4) using Affymetrix GeneChip miRNA 3.0 arrays. PCA showed that both GPC populations were separated as defined by their miRNA expression profiles (Figure 6, Panel A). Differential expression (adjusted p-value <0.01) between both ages resulted in 56 genes (23 enriched in adult GPCs and 33 enriched in fetal GPCs; Figure 6, Panels B-C). Notably, among these differentially expressed miRNAs are the fetal progenitor miRNAs miR-9-3p, miR-9-5p (Lau, P., Verrier, JD, Nielsen, JA, Johnson, KR, Notterpek, L., and Hudson, LD (2008). Identification of dynamically regulated microRNA and mRNA networks in developing oligodendrocytes. J Neurosci 28, 11720-11730), and miR-17-5p (Budde, H., Schmitt, S., Fitzner, D., Opitz, L., Salinas-Riester, G., and Simons, M. (2010). Control of oligodendroglial cell number by the miR-17-92 cluster. Development 137, 2127), as well as the adult oligodendrocyte regulators miR-219a-3p and miR-338-5p (Dugas, JC, Cuellar, TL, Scholze, A., Ason, B., Ibrahim, A., Emery, B., Zamanian, JL, Foo, LC, McManus, MT, and Barres, BA (2010). Dicer1 and miR-219 Are Required for Normal Oligodendrocyte Differentiation and Myelination. Neuron 65, 597-611).

[0236] In this study, we then used this cohort of miRNAs to predict genes whose expression might be expected to be suppressed by miRNA upregulation, analyzing both adult and fetal GPC pools separately.To achieve this, miRNAtap was used to query five miRNA gene target databases: DIANA (Maragkakis, M., Vergoulis, T., Alexiou, P., Reczko, M., Plomaritou, K., Gousis, M., Kourtis, K., Koziris, N., Dalamagas, T., and Hatzigeorgiou, A. G. (2011). DIANA-microT Web server upgrade supports Fly and Worm miRNA target prediction and bibliographic miRNA to disease association. Nucleic Acids Res 39, W145-148), Miranda (Enright, A. J., John, B., Gaul, U., Tuschl, T., Sander, C., and Marks, D. S. (2003). MicroRNA targets in Drosophila. Genome biology 5, R1), PicTar (Lall, S., Grun, D., Krek, A., Chen, K., Wang, YL, Dewey, CN, Sood, P., Colombo, T., Bray, N., Macmenamin, P., et al. (2006). A genome-wide map of conserved microRNA targets in C. elegans. Current biology:CB16, 460-471), TargetScan (Friedman, RC, Farh, KK, Burge, CB, and Bartel, DP (2009). Most mammalian mRNAs are conserved targets of microRNAs. Genome Res 19, 92-105), and miRDB (Wong, N. and Wang, X. (2015). miRDB: an online resource for microRNA target prediction and functional annotation. Nucleic Acids Res Volume 43, pages D146-152).

[0237] To maximize accuracy, genes were considered targets only if they appeared in at least two databases. This approach predicted that fetal-enriched miRNAs would repress an average of 36.3 (SD=24.5) genes per miRNA. In contrast, adult hGPC-enriched miRNAs predicted an average of 46.4 (SD=37.8) genes per miRNA (Figure 6, Panel C). Collectively, this identified that 48.8% of adult GPC-enriched genes could be repressed by fetal miRNAs and 39.9% of fetal GPC-enriched genes could be repressed by adult miRNAs.

[0238] To assess the functional significance of these miRNA-dependent post-transcriptional regulatory mechanisms, in this study we curated fetal and adult networks according to miRNA targeting of functionally related and differentially expressed genes (Figure 6, Panels D-E). The proposed upstream adult transcriptional regulators STAT3, E2F6, and MAX were predicted to be inhibited by seven miRNAs in fetal GPCs (Figure 24, Panel D). These include the previously validated repression of STAT3 in other cell types by miR-126b-5p, miR-106a-5p, miR-17-5p, miR-130a-3p, and miR-130b-3p (Du, W., Pan, Z., Chen, X., Wang, L., Zhang, Y., Li, S., Liang, H., Xu, C., Zhang, Y., Wu, Y. et al. (2014a). By Targeting Stat3 microRNA-17-5p Promotes Cardiomyocyte Apoptosis in Response to Ischemia Followed by Reperfusion. Cellular Physiology and Biochemistry 34, 955-965). In parallel, a number of early and mature oligodendrocyte genes were simultaneously targeted for inhibition, all consistent with the maintenance of a progenitor state. These included MBP, UGT8, CD9, PLP1, MYRF, and PMP22 (Goldman, SA and Kuypers, NJ (2015). How to make an oligodendrocyte. Development 142:3983-3995). Importantly, a cohort of genes implicated in either induction of senescence or inhibition of proliferation, or both, were also predicted to be actively repressed in fetal GPCs.These included RUNX1, RUNX2, BIN1, DMTF1 / DMP1, CTNNA1, SERPINE1, CDKN1C, PAK1, IFI16, EFEMP1, MAP3K7, AHR, OGT, CBX7, and CYLD (Eckers, A., Jakob, S., Heiss, C., Haarmann-Stemmann, T., Goy, C., Brinkmann, V., Cortese-Krott, MM, Sansone, R., Esser, C., Ale-Agha, N. et al. (2016). The aryl hydrocarbon receptor promotes aging phenotypes across species. Sci Rep 6, 19618). Additionally, several of the miRNAs identified herein, including miR-17-5p, miR-93-3p, miR-1260b, miR-106a-5p, miR-767-5p, miR-130a-3p, miR-9-3p, miR-9-5p, and miR-130b-3p, inhibited senescence or activated proliferation. Collectively, these data provide a complementary mechanism by which fetal hGPCs can maintain their characteristic progenitor transcriptional state and signature.

[0239] Example 7 Adult miRNA signaling may suppress the proliferative progenitor state and portend senescence Next, this study investigated potential miRNA regulatory networks within adult hGPCs (Figure 6, Panel E). This suggested the involvement of five miRNAs that control five identified active fetal transcriptional regulators, including HDAC2, NFIB, BCLL1A, TEAD2, and HMGA2, whose silencing by miR-4651 has previously been shown to inhibit proliferation (Han, X., Yang, R., Yang, H., Cao, Y., Han, N., Zhang, C., Shi, R., Zhang, Z, Fan, Z. (2020). miR-4651 inhibits proliferation of gingival mesenchymal stem cells by inhibiting HMGA2 under nifedipine treatment. Int J Oral Sci vol. 12, p. 10). This cohort of miRNAs was predicted to act in parallel with adult transcriptional repressors to inhibit the expression of genes involved in maintaining the GPC progenitor state, including PDGFRA, PTPRZ1, ZBTB18, SOX6, EGFR, and NRXN1. Furthermore, the adult miRNA milieu was predicted to repress numerous genes known to induce a proliferative state or delay senescence. These genes include LMNB1 (Freund, A., Laberge, RM, Demaria, M., and Campisi, J. (2012). Lamin B1 loss is a senescence-associated biomarker. Mol Biol Cell 23, 2066-2075), PATZ1 (Cho, JH, Kim, MJ, Kim, KJ, and Kim, JR (2012). POZ / BTB and AT-hook-containing zinc finger protein 1 (PATZ1) inhibits endothelial cell senescence through a p53 dependent pathway. Cell Death Differ 19, 703-712), and GADD45A (Hollander, MC, Sheikh, MS, Bulavin, DV, Lundgren, K., Augeri-Henmueller, L., Shehee, R., Molinaro, T. A., Kim, K. E., Tolosa, E., Ashwell, J. D. et al. (1999). Genomic instability in Gadd45a-deficient mice. Nat Genet 23, 176-184), YAP1 and TEAD1 (Xie, Q., Chen, J., Feng, H., Peng, S., Adams, U., Bai, Y., Huang, L., Li, J., Huang, J., Meng, S. et al. (2013). YAP / TEAD-mediated transcription controls cellular senescence. Cancer Res 73, 3615-3624), CDK1 (Diril, M. K., Ratnacaram, C. K., Padmakumar, V. C., Du, T., Wasser, M., Coppola, V., Tessarollo, L., and Kaldis, P. (2012). Cyclin-dependent kinase 1 (Cdk1) is essential for cell division and suppression of DNA re-replication but not for liver regeneration. Proc Natl Acad Sci USA 109, 3826-3831), TPX2 (Rohrberg, J., Van de Mark, D., Amouzgar, M., Lee, J. V., Taileb, M., Corella, A., Kilinc, S., Williams, J., Jokisch, M. L., Camarda, R. et al. (2020). MYC Dysregulates Mitosis, Revealing Cancer Vulnerabilities. Cell Rep 30, 3368-3382), S1PR1 (Liu, Y., Zhi, Y., Song, H., Zong, M., Yi, J., Mao, G., Chen, L., and Huang, G. (2019).S1PR1 promotes proliferation and inhibits apoptosis of esophageal squamous cell carcinoma through activating STAT3 pathway. Journal of Experimental & Clinical Cancer Research 38, 369), RRM2 (Aird, K.M., Zhang, G., Li, H., Tu, Z., Bitler, B.G., Garipov, A., Wu, H., Wei, Z., Wagner, S.N., Herlyn, M. et al. (2013). Suppression of nucleotide metabolism underlies the establishment and maintenance of oncogene-induced senescence. Cell Rep 3, 1252 - 1265), CCND2 (Bunt, J., de Haas, T.G., Hasselt, N.E., Zwijnenburg, D.A., Koster, J., Versteeg, R., and Kool, M. (2010). Regulation of cell cycle genes and induction of senescence by overexpression of OTX2 in medulloblastoma cell lines. Mol Cancer Res 8, 1344 - 1357), SGO1 (Murakami-Tonami, Y., Ikeda, H., Yamagishi, R., Inayoshi, M., Inagaki, S., Kishida, S., Komata, Y., Jan, K., Takeuchi, I., Kondo, Y. et al. (2016). SGO1 is involved in the DNA damage response in MYCN-amplified neuroblastoma cells. Scientific Reports 6, 31615), MCM4 and MCM6 (Mason, D.X., Jackson, T.J., and Lin, A.W. (2004).Molecular signature of oncogenic ras-induced senescence. Oncogene vol. 23, pp. 9238-9246), ZNF423 (Hernandez-Segura, A., de Jong, TV, Melov, S., Guryev, V., Campisi, J., and Demaria, M. (2017). Unmasking Transcriptional Heterogeneity in Senescent Cells. Current biology: CB 27, pp. 2652-2660), PHB (Piper, PW, Jones, GW, Bringloe, D., Harris, N., MacLean, M., & Mollapour, M. (2002). The shortened replicative life span of prohibitin mutants of yeast appears to be due to defective mitochondrial segregation in old mother cells. Aging cells 1, pp. 149-157), WLS (Poudel, SB, So, HS, Sim, HJ, Cho, JS, Cho, ES, Jeon, YM, Kook, SH, and Lee, JC (2020). Osteoblastic Wntless deletion differentially regulates the fate and functions of bone marrow-derived stem cells in relation to age. Stem Cells.), and ZMAT3 (Kim, BC, Lee, HC, Lee, JJ, Choi, CM, Kim, DK, Lee, JC, Ko, YG, and Lee, JS (2012). Wig1 prevents cellular senescence by regulating p21 mRNA decay through control of RISC recruitment. EMBO J 31, pp. 4289-4303). More directly, induction of senescence or inhibition of proliferation was associated with miR-584-5p (Li, Q., Li, Z., Wei, S., Wang, W., Chen, Z., Zhang, L., Chen, L., Li, B., Sun, G., Xu, J., et al. (2017). Overexpression of miR-584-5p inhibits proliferation and induces apoptosis by targeting WW domain-containing E3 ubiquitin protein ligase 1 in gastric cancer. J Exp Clin Cancer Res 36, 59), miR-193a-5p (Chen, J., Gao, S., Wang, C., Wang, Z., Zhang, H., Huang, K., Zhou, B., Li, H., Yu, Z., Wu, J., et al. (2016). Pathologically decreased expression of miR-193a contributes to metastasis by targeting WT1-E-cadherin axis in non-small cell lung cancers. J Exp Clin Cancer Res 35, 173), miR-548ac (Song, F., Yang, Y., and Liu, J. (2020). MicroRNA-548ac induces apoptosis in laryngeal squamous cell carcinoma cells by targeting transmembrane protein 158. Oncol Lett 20, 69), miR-23b-3p (Campos-Viguri, G. E., Peralta-Zaragoza, O., Jimenez-Wences, H., Longinos-Gonzalez, A. E., Castanon-Sanchez, C. A., Ramirez-Carrillo, M., Camarillo, C. L., Castaneda-Saucedo, E., Jimenez-Lopez, M. A., Martinez-Carrillo, D. N., et al. (2020).MiR-23b-3p reduces the proliferation, migration and invasion of cervical cancer cell lines via the reduction of c-Met expression.Sci Rep vol. 10, p. 3256), miR-140-3p(Wang, M., Wang, X., & Liu, W. (2020a).MicroRNA- 130a-3p promotes the proliferation and inhibits the apoptosis of cervical cancer cells via negative regulation of RUNX3. Mol Med Rep Volume 22. , pp. 2990-3000), and miR-330-3p (Wang, Y., Chen, J., Wang, X., and Wang, K. (2020b). miR-140-3p inhibits bladder cancer cell proliferation and invasion by targeting FOXQ1. Aging 12, pp. 20366-20379). Taken together, these data suggest that these miRs are active participants in maintaining the progenitor state of fetal hGPCs and that their modulation is a possible mechanism by which adult hGPCs exhibit their signature gene expression profile.

[0240] Example 8 Transcription factor regulation of miRNAs establishes and reinforces GPC identity Next, this study sought to predict the upstream regulation of miRNAs differentially expressed in fetal and adult GPCs by querying the TransmiR transcription factor miRNA regulation database (Tong et al. (2011). TransmiR v2.0: an updated transcription factor-microRNA regulation database. Nucleic Acids Res 47: D253-D258). This approach predicted that 54 of the 56 age-specific GPC miRNAs were regulated by 66 transcription factors that were similarly determined to be significantly differentially expressed between fetal and adult GPCs. Interestingly, the top four predicted miRNA-regulating TFs were all MYC-associated factors, including MAX, MYC itself, E2F6, and the fetal-enriched MYC-associated zinc finger protein MAZ, which targeted 36, 33, 30, and 28 unique differentially expressed miRNAs, respectively.

[0241] Examination of the proposed relationships in the context of 12 candidate TFs showed that a number of fetal hGPC-enriched miRNAs were predicted to be targeted by both fetal activators and adult repressors, whereas miRNAs enriched in adult GPCs were more uniquely targeted. MYC was predicted to drive expression of numerous miRNAs in fetal GPCs, many of which were predicted to be repressed by E2F6, MAX, or both in adulthood. Notably, miR-130a-3p was predicted to be targeted by MYC, MAX, and E2F6 in addition to being activated by TEAD2. Of note, among the TF-miRNA interactions validated in other cell types, upregulation of the rejuvenating miR-17-5p by MYC and its repression by MAX (Du et al. (2014b). miR-17 extends mouse lifespan by inhibiting senescence signaling mediated by MKP7. Cell Death Dis 5, e1355) has been reported. Similarly, parallel activation of proliferative miR-130-3p by MYC or TEAD2 and YAP1 has been reported (Shen et al. (2015). A miR-130a- YAP positive feedback loop promotes organ size and tumorigenesis. Cell Res 25, 997-1012). It has also been reported that it decreases with oligodendrocyte maturity (Lau, P. et al. (2008). Identification of dynamically regulated microRNA and mRNA networks in developing oligodendrocytes. J Neurosci 28, 11720-11730), and activation of both arms of miR-9 by MYC (Ma, L. et al. (2010a). miR-9, a MYC / MYCN-activated microRNA, regulates E-cadherin and cancer metastasis. Nat Cell Biol 12, 247-256).

[0242] In adult GPCs, enriched miRNAs predicted to be regulated by our significantly enriched TF cohort were targeted exclusively by adult activators of fetal repressors, and only miR-151a-5p and miR-4687-3p, predicted inhibitors of HMGA2, were more likely to be inversely targeted by STAT3 versus BCL11A and EZH2, respectively. Besides this, miR-1268b was predicted to be inhibited in parallel by both EZH2 and HDAC2. Of note, miR-219a-2-3p, a key oligodendrocyte microRNA, was predicted to remain inhibited by EZH2 in fetal GPCs, while STAT3 likely drives the expression of the seven other miRs independently. Interestingly, STAT3, whose increased activity has been linked to senescence (Kojima et al. 2013. IL-6-STAT3 signaling and premature senescence. JAKSTAT 2:e25763), was also predicted to drive the expression of a cohort of miRNAs that have been independently associated with senescence induction, including miR-584-5p, miR-330-3p, miR-23b-3p, and miR-140-3p.

[0243] Integrating transcriptional and miRNA profiling, pathway enrichment analysis, and target prediction, we propose a model of human GPC aging in which fetal hGPCs maintain progenitor gene expression, activate proliferation programs, and prevent senescence, while repressing oligodendrocyte and senescence gene programs both transcriptionally and post-transcriptionally via microRNAs. With adult maturation and time and population doubling, hGPCs begin to upregulate repressors of these fetal progenitor-associated networks, while also activating programs to promote a progressively more differentiated and ultimately senescent phenotype.

[0244] Example 9 Expression of BCL11A in the brain of a chimeric animal model leads to the expansion of BCL11A-expressing GPCs in the host In this study, by analyzing RNA sequencing data of fetal and adult human glial progenitor cells (GPCs) sorted from fresh tissue samples, we identified several transcription factors as central to a gene regulatory network that distinguishes "young" from "old" GPCs. Among them, the transcriptional repressor BCL11A (B-cell CLL / lymphoma 11A) was one of the most prominent differentially expressed genes, high in fetal hGPCs and low in adult cells, suggesting that it plays a role in preserving the fetal hGPC phenotype. BCL11A was never known to play such a role in the central nervous system or in regulating the expansion, fate, and aging of glial progenitor cells.

[0245] To investigate whether BCL11A can then resume or accelerate self-renewal in aging GPCs, this study generated lentiviruses expressing BCL11A and GFP driven by the CBh promoter (herein referred to as CBh-BCL11A), as well as a green fluorescent protein (GFP) only control virus (CBh-GFP) (Figure 14a). GPCs were generated from the human C27 iPSC line, tagged with a red fluorescent protein (RFP) transgene (Figure 14b), and then transplanted into the corpus callosum (CC) of postnatal day 1 Rag1 immunodeficient mice as previously described (Windrem et al., J. Neurosci 34:16153-16161 (2014); Windrem et al., Cell Stem Cell 21:195-208.e6 (2017)). Chimerized mice were allowed to age for 2 years after engraftment, at which point they received stereotactic injections of CBh-BCL11A in the left hemisphere and CBh-GFP control virus in the right hemisphere. Virus was deposited in the striatum, corpus callosum, and cortex. At either 3 or 6 weeks post-injection, mice were either dissected for single-cell analysis or perfused with 4% PFA for sectioning and immunohistochemistry (Figure 14c).

[0246] The study confirmed overexpression of BCL11A by both RNA expression in vitro (Figure 14d) and protein staining in vivo (Figure 14e). The study observed an expanded proliferation of GPC populations in the CBh-BCL11A-treated hemisphere compared to the control side of the brain 3 weeks after infection (Figure 15a). The study found a robust increase in the presence of RFP-tagged human and OLIG2+ cells of the GPC lineage (quantified in Figures 15b, 15c), as well as an increase in resident mouse GPCs identified by mouse NG2 antigen (quantified in Figures 15d, 15e). This effect was still present 6 weeks after infection (Figure 16a), with more RFP+ and OLIG2+ cells detected in the CBh-BCL11A-infected hemisphere than in the CBh-GFP-treated control (Figure 16b). At that 6-week time point, donor cell distribution was widespread and relatively uniform, with no tumors or ectopic areas found. These observations indicated that BCL11A transduction similarly activates both aged resident human and mouse GPCs to resume mitotic expansion and migratory colonization of the host brain. Furthermore, (red) membrane tagging of BCL11A-mobilized human donor cells allowed us to define the morphological characteristics of the majority of donor cells in the white matter as myelinating oligodendrocytes, suggesting that the typical age-associated myelin loss in these aged mice was at least partially reversed.

[0247] While various embodiments have been described above, it should be understood that such disclosure has been presented by way of example only, and not limitation. Thus, the breadth and scope of the compositions and methods of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0248] The above description is intended to teach those skilled in the art how to carry out the present invention, and is not intended to detail all obvious modifications and variations of the present invention that would become apparent to those skilled in the art upon reading the description. However, all such obvious modifications and variations are intended to be included within the scope of the present invention as defined by the following claims. The claims are intended to cover any sequence of components and steps that are effective to achieve the intended purpose, unless the circumstances specifically indicate otherwise.

Claims

1. A drug that represses one or more transcription factors selected from the group consisting of: (i) zinc finger protein 274 (ZNF274), (ii) Myc-related factor X (MAX), (iii) E2F transcription factor 6 (E2F6), (iv) zinc finger protein Aiolos (IKZF3), and (v) signal transduction and activator of transcription 3 (STAT3), for use in a method of inducing rejuvenation in a population of adult glial progenitor cells by introducing an effective amount thereof into the population of adult glial progenitor cells in vivo.

2. 1. An agent that represses one or more transcription factors selected from the group consisting of: (i) zinc finger protein 274 (ZNF274), (ii) Myc-related factor X (MAX), (iii) E2F transcription factor 6 (E2F6), (iv) zinc finger protein Aiolos (IKZF3), and (v) signal transducer and activator of transcription 3 (STAT3), for administration in an effective amount to treat a subject having a glial cell-related disorder.

3. The method of claim 2, wherein the glial cell-related disorder is a myelin deficiency.

4. 4. The method of claim 3, wherein the myelin deficiency is associated with a condition selected from the group consisting of multiple sclerosis, neuromyelitis optica, transverse myelitis, optic neuritis, subcortical stroke, diabetic leukoencephalopathy, hypertensive leukoencephalopathy, age-related white matter disease, spinal cord injury, radiation- or chemotherapy-induced demyelination, post-infectious and post-vaccination leukoencephalitis, periventricular leukomalacia, childhood leukodystrophies, lysosomal storage diseases, congenital dysmyelination, inflammatory demyelination, vascular demyelination, and cerebral palsy.

5. The method of claim 2, wherein the glial cell-related disorder is a neurodegenerative disease selected from the group consisting of Huntington's disease, frontotemporal dementia, Parkinson's disease, multiple system atrophy, and amyotrophic lateral sclerosis.

6. The method of claim 5, wherein the glial cell-related disorder is Huntington's disease.

7. The method of claim 2, wherein the glial cell-related disorder is a neuropsychiatric disorder selected from the group consisting of schizophrenia, autism spectrum disorder, and bipolar disorder.

8. The agent of any one of claims 2 to 7, wherein the agent comprises an antisense oligonucleotide.

9. The agent according to claim 2 , wherein the agent comprises a nucleic acid molecule comprising a nucleic acid sequence encoding a miRNA, shRNA, or siRNA.

10. 8. The agent of any one of claims 2 to 7, wherein the agent comprises one or more nucleic acid molecules comprising a first nucleic acid sequence encoding a Cas protein and a second nucleic acid sequence encoding a guide RNA.

11. The therapeutic agent of claim 10 , wherein the first nucleic acid sequence and the second nucleic acid sequence are located on the same nucleic acid molecule.

12. The agent of claim 10, wherein the Cas protein is a nuclease-dead Cas protein.

13. The agent of claim 9 , wherein the agent is a non-viral expression vector.

14. 10. The agent of claim 9, wherein the agent is a viral expression vector.

15. The therapeutic agent of claim 14 , wherein the viral expression vector is a lentiviral vector or an AAV vector.

16. 10. The agent of claim 9, wherein the nucleic acid sequence encoding a miRNA, shRNA or siRNA, the first nucleic acid sequence encoding a Cas protein, and the second nucleic acid sequence encoding a guide RNA are operably linked to regulatory elements.

17. The method of claim 16, wherein the regulatory element is a glial cell-specific promoter or an inducible promoter.

18. The therapeutic agent of claim 17, wherein the inducible promoter is a tet-on or tet-off promoter.

19. A drug that represses one or more transcription factors selected from the group consisting of: (i) zinc finger protein 274 (ZNF274), (ii) Myc-related factor X (MAX), (iii) E2F transcription factor 6 (E2F6), (iv) zinc finger protein Aiolos (IKZF3), and (v) signal transduction and activator of transcription 3 (STAT3), for use in a method of inducing rejuvenation in a population of adult glial progenitor cells by introducing an effective amount thereof ex vivo into the population of adult glial progenitor cells.

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