Competitive replacement of glial cells

Abstract

The present application relates to reducing the adverse effects of oligodendrocyte loss, astrocyte loss, or white matter loss, including age-related oligodendrocyte loss, astrocyte loss, or white matter loss, in the brain of a subject. The present application also relates to rejuvenating glial progenitor cells or their progeny, or enhancing the developmental potential of glial progenitor cells or their progeny.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 482,862, filed February 2, 2023, and U.S. Provisional Patent Application No. 63 / 493,451, filed March 31, 2023, the contents of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing Reference This application is filed with an electronic Sequence Listing, which is provided as a file entitled SeqList-161118-04702.xml, created on January 29, 2024, and is 18,319 bytes in size. The information in the electronic format of this Sequence Listing is incorporated herein by reference in its entirety.

[0003] The present application relates to competitive replacement of glial cells and uses thereof in the treatment of oligodendrocyte loss, astrocyte loss, or white matter loss, including age-related oligodendrocyte loss, age-related astrocyte loss, or age-related white matter loss. [Background technology]

[0004] The central nervous system (CNS) is generally divided into gray matter, which contains neuronal cell bodies and dendritic networks, and white matter, which consists of axon bundles wrapped in myelin produced by oligodendrocytes. Loss of white matter, oligodendrocytes, or astrocytes can lead to poor outcomes, including cognitive impairment, dementia, urinary incontinence, gait disturbances, depression, and an increased risk of stroke and death. This loss is accompanied by partial loss of myelin, axons, and oligodendrocytes, mild reactive astrogliosis, sparsely distributed macrophages, and stenosis due to hyaline fibrosis of arterioles and smaller blood vessels. There is a need for therapeutic agents and methods for treating disorders and conditions mediated by or characterized by loss of white matter, oligodendrocytes, or astrocytes. The present disclosure is directed to overcoming these and other deficiencies in the art. Summary of the Invention

[0005] The present disclosure addresses the above-mentioned needs in several aspects.

[0006] In one aspect, the present disclosure provides a method for rejuvenating or enhancing the developmental potential of glial progenitor cells or their progeny. The method comprises increasing the level or activity of (i) a transcription factor selected from the group consisting of CEBPZ, CTCF, E2F1, MYC, NFYB, and ETV4, or (ii) a target of the transcription factor, in the glial progenitor cells or their progeny. In some embodiments, the transcription factor is selected from the group consisting of CTCF, E2F1, and ETV4.

[0007] The targets are RPL6, RPS27, RPS16, RPS21, DOHH, PCCB, UTP11, RPS8, RPL27A, EIF2A, UBLCP1, RPL32, GIN1, PATZ1, TNFRSF1A, MRPL10, RFXANK, BORCS8, ENOPH1, RPS16, SNHG11, SLC35A5, RAB1B, RPL23A, YBX1, TMEM129, DOHH, CCND1, MRPL24, RPL14, HMGA1, DCTPP1, ENOPH, ZNF436, RPLP2, CCND1, TNFRSF1A, FBXL12, NTMT1, IMPDH2, MRPL18, LIMS1, CD82, POLR2H, LRRC8A, EXOSC5, RAN, DYNLT1, FDPS, ACTL6A, RPS5, DOLPP1, GGCT, RPS2, SYCE1L, MRPL17, ZDHHC16, YBX1, RPLP0, ELK1, RPSA, NME2, RPS15, TSPAN33, B3GNT9, DCLRE1B, CMC1, RPLP1, ACAT2, RPL6, RPL28, RPL18A, CCDC51, RACK1, RPS14, RPS19, RPL12, RPS5, RPL19, RPL27A, RPS19, RPS21, POLR2H, RPS14, CCDC51, EIF2A, UBLCP1, SNHG19, RPL32, ZNF579, RPS27, RPL6, GIN1, PATZ1, RACK1, LRRC8A, TNFRSF1A, RPL28, RPS18, MRPL10, RFXANK, BORCS8, FBXL12, PEF1, ENOPH1, PEX7, RPS16, SNHG11, RPL10, ZDHHC16, HMGA1, ZNF436, UTP11, DCLRE1B, RPS5, EXOSC, MRPL17, RPL19, PRMT1, NME2, CCND1, NRN1, YBX1, LIMS1, RPL23A, CD82, NTMT1, RPL18A, DOLPP1, GGCT, ELK1, ACTL6A, FDPS, MRPL18, RPLP0, ACAT2, SLC35A5, RAB1B, RAN, DYNLT1, TMEM129, RPSA, RPS15, RPL13A, PCCB, DCTPP1, RPLP2, B3GNT9, IMPDH2, RPL14, MRPL24, RPS8, RPS2, SYCE1L, RPL12, CMC1, DOHH, RPLP1, BTBD17The gene may be selected from the group consisting of TSPAN33, ACAT2, GGCT, FDPS, SNHG19, PRMT1, RPL13A, FBXL12, RPS19, RFXANK, NRN1, DCTPP1, ZNF579, YBX1, MRPL18, NTMT1, RPL14, PEF1, RPS21, PEX7, BTBD17, RPL10, and RPS18 (listed in Table 1 and Figure 5).

[0008] In some embodiments, the glial progenitor cells are senescent glial progenitor cells. In some embodiments, the progeny are oligodendrocytes or astrocytes. In some embodiments, the increasing step comprises expressing or introducing a transcription factor or target in the glial progenitor cells or progeny. In some embodiments, the increasing step comprises contacting the glial progenitor cells or progeny with an agent that increases the level or activity of the transcription factor or target.

[0009] In some embodiments, the method further comprises suppressing in the glial precursor cells or progeny a transcriptional repressor selected from the group consisting of E2F6, ZNF274, MAX, and IKZF3.

[0010] In another aspect, the present disclosure provides a cell or progeny thereof prepared according to the methods described herein.

[0011] In a further aspect, the present disclosure provides a method for treating a condition mediated by white matter loss, oligodendrocyte loss, or astrocyte loss. The method comprises administering to a subject in need thereof (a) a therapeutically effective amount of (i) a transcription factor selected from the group consisting of CEBPZ, CTCF, E2F1, MYC, NFYB, and ETV4, or (ii) an agent that increases the level or activity of a target of the transcription factor, or (b) a therapeutically effective amount of a cell or progeny thereof prepared according to the methods described herein. In some embodiments, the transcription factor is selected from the group consisting of CTCF, E2F1, and ETV4. The target is selected from the above group and is listed in Table 1 and Figure 5.

[0012] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a suppressor of a transcriptional repressor selected from the group consisting of E2F6, ZNF274, MAX, and IKZF3.

[0013] In some embodiments, the subject is a human.

[0014] In some embodiments, the agent comprises a small molecule compound, an oligonucleotide, a nucleic acid, a peptide, a polypeptide, a CRISPR / Cas system, or an antibody or antigen-binding portion thereof. In some embodiments, the nucleic acid encodes a transcription factor or target as described above.

[0015] In some embodiments, the suppressor comprises a small molecule compound, an oligonucleotide, a nucleic acid, a peptide, a polypeptide, a CRISPR / Cas system, or an antibody or antigen-binding portion thereof.

[0016] In some embodiments, the agent, suppressor, or cell is administered intraparenchymally, intracallosally, intracerebroventricularly, intrathecally, intracerebrally, intracisternally, or intravenously. In some embodiments, the cell or progeny is administered to the forebrain, striatum, and / or cerebellum.

[0017] In some embodiments, the condition is a lysosomal storage disease, an autoimmune demyelinating condition (e.g., multiple sclerosis, neuromyelitis optica, transverse myelitis, and optic neuritis), a vascular leukoencephalopathy (e.g., subcortical stroke, diabetic leukoencephalopathy, hypertensive leukoencephalopathy, age-related white matter disease, and spinal cord injury), a radiation-induced demyelinating condition, a leukodystrophy (e.g., Pelizaeus-Merzbacher disease, Tay-Sachs disease, Sandhoff gangliosidosis, Krabbe disease, metachromatic leukodystrophy, mucopolysaccharidosis, Niemann-Pick disease type A, adrenoleukodystrophy, Canavan disease, vanishing white matter disease, and Alexander disease), or periventricular leukomalacia or cerebral palsy. In some embodiments, the condition is Huntington's disease or subcortical dementia. In some embodiments, the condition is Parkinson's disease.

[0018] In some embodiments, the glial progenitor cells are derived from pluripotent stem cells. In some embodiments, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. In some embodiments, the cells or progeny are heterologous, xenogenic, allogeneic, syngeneic, or autologous to the subject.

[0019] In some embodiments, the white matter loss, oligodendrocyte loss, or astrocyte loss is age-related. [Brief explanation of the drawings]

[0020] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0021] [Figure 1A-J] We show that adult-transplanted WT human GPCs outcompeted and replaced neonatally resident HD hGPCs. [Figure 1A] Experimental design and analytical endpoints are shown. STR, striatum (caudate-putamen); LV, lateral ventricle; CTX, cortex. Dashed rectangles (orange) represent inserts (B'). Scale: B, 500 μm; C', 100 μm; D, 50 μm; E, 10 μm; I, 100 μm; I', 10 μm. [Figure 1B] Engraftment of WT glia (mCherry+, red) into the striatum of HD chimeras resulted in the gradual replacement of HD glia (EGFP+, green), creating extensive exclusion domains as they advanced. The dashed outline (white) demarcates the outline of the striatum where human cells were mapped and quantified. [Figure 1C-D] Figure 1 shows that the boundary between advancing WT GPCs and retreating HD hGPCs was typically sufficiently linear that an exclusion domain formed as WT GPCs (Olig2+, white) displaced their HD counterparts. [Figure 1E]Within areas colonized by WT hGPCs, stray HD astrocytes (hGFAP+, white) can still be found, indicating that GPC replacement precedes astrocyte replacement. [Figure 1F] Figure 1 shows the mapped distribution of human glia in the host striatum. Human glia were mapped at 15 equidistant sections (five are shown as examples) and reconstructed in three dimensions. Their distribution was measured radially as a function of distance to the injection site. [Figure 1G] A rendering example of a mapped striatum is shown. [Figure 1H] Volumetric quantification shows that WT cells gradually replaced their HD counterparts as they expanded from their transplantation sites. H1: WT vs. HD (allograft; n = 8, 54 weeks; n = 7, 72 weeks). The advance of WT cells was accompanied by the gradual elimination of HD glia from the tissue compared with untransplanted HD chimeras (HD control). H2: HD (allograft; n = 8, 54 weeks; n = 7, 72 weeks) vs. HD control (n = 4 for both time points; two-way ANOVA with Sidak's multiple comparison test. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05; data presented as mean ± SEM). [Figure 1I] At the boundary between WT and HD glia, we can see that the incidence of high Ki67+ (white) cells can only be seen within the WT glial population. I'. High magnification of two WT daughter cells at the edge of the competing boundary. [Figure 1J] Quantification of Ki67+ glia within each population as a function of time shows a significant proliferative advantage of WT glia that persists throughout the experiment. HD control: 54 weeks (n=4), 72 weeks (n=4); WT control: 54 weeks (n=5), 72 weeks: n=3; WT vs. HD allograft: 54 weeks (n=5), 72 weeks (n=3). Comparisons by two-way ANOVA with Sidak's multiple comparison test; mean ± SEM. [Figure 2A-I] We show that WT glia acquired a dominant competitor transcriptional profile when confronted with resident HD glia. [Figure 2A] The experimental design is shown. [Figure 2B-C] Uniform manifold approximation projection (UMAP) visualization of the combined (B) and group-partitioned (C) scRNA-seq data identifies six major cell populations. [Figure 2D] Tacked bar plot percentages of cell types in each group are shown. [Figure 2E] Figure 1 shows cell cycle analysis notched box plots of circulating GPCs and GPCs in G2 / M phase. Boxes indicate interquartile ranges, notches indicate 95% confidence intervals with the median at the center of the notches, and error bars represent the minimum and maximum non-outlier values. [Figure 2F] Venn diagram of pairwise differentially expressed GPC genes is shown (Log2 fold change >0.15, adjusted p-value <0.05). [Figure 2G] Curated ingenuity pathway analysis of differentially expressed genes between GPC groups is shown. Circle size represents p-value, and shaded areas indicate activation Z-scores, with red being more active in the top group and green being more active in the bottom group. [Figure 2H] Heatmap of curated pairwise differentially expressed GPC genes is shown, adjusted p-values ​​*≦0.05, **<0.01, ***≦0.001, ****≦0.0001. [Figure 2I] Violin plots of pairwise differentially expressed GPC ribosomal gene log2 fold changes are shown. Comparisons between groups in (E) were performed using a Kruskal-Wallis test followed by a Dunn's test with Benjamini-Hochberg adjustment for multiple comparisons. [Figure 3A-I] This shows that the difference in cell age was sufficient to drive competitive regrowth in the humanized striatum. B-C: STR, striatum (caudate-putamen); LV, lateral ventricle; CTX, cortex. Scale: B, 500 μm; C, 100 μm; E, 100 μm; G, 50 μm. [Figure 3A] The experimental design and analytical evaluation items are shown. [Figure 3B]Figure 1 shows that engraftment of younger WT glia (EGFP+, green) into the striatum of WT chimeras resulted in the selective replacement of their aged counterparts (mCherry+, red). The dashed outline demarcates the region of the striatum where human cells were mapped and quantified. [Figure 3C] WT chimeric controls engrafted only at birth are shown. [Figure 3D] A rendered example of the mapped striatum is shown. Volumetric quantification shows that younger WT glia replace their older allogeneic counterparts as they expand from the injection site. [Figure 3E] Aged vs. young (syngeneic grafts). n=3. Their progression tracked the gradual elimination of aged WT glia from the tissue compared to control WT chimeras (aged controls). [Figure 3F] Aged (syngraft) vs. aged (control), n=3 each; two-way ANOVA with Sidak's multiple comparison test; interactions or main effects are shown as numerical P values, and post-hoc comparisons are indicated as follows: ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05; data are shown as mean±SEM. [Figure 3G] We show that at the boundary between young and senescent WT glia, a higher incidence of Ki67+ (white) cells can be seen within the younger population. [Figure 3H] The intercalated color division is shown in dashed square in FIG. 3G. [Figure 3I] Quantification of Ki67+ cells shows that younger WT glia are significantly more proliferative than their aged counterparts. n = 3 for all experimental groups; one-way ANOVA with Sidak's multiple comparison test; data are presented as mean ± SEM with individual data points. [Figure 4A-I] We show that WT glia acquired a dominant transcriptional profile when confronted with their aged counterparts. Adjusted p-values ​​*≤0.05, **<0.01, ***≤0.001, ****≤0.0001. [Figure 4A] The experimental design is shown. [Figure 4B-C] Uniform manifold approximation projection (UMAP) visualization of the combined (B) and group-partitioned (C) scRNA-seq data identifies six major cell populations. [Figure 4D] Stacked bar plot percentages of cell types in each group are shown. [Figure 4E] Figure 1 shows cell cycle analysis notched box plots of circulating GPCs and GPCs in G2 / M phase. Boxes indicate interquartile ranges, notches indicate 95% confidence intervals with the median at the center of the notches, and error bars represent the minimum and maximum non-outlier values. [Figure 4F] Venn diagram of pairwise differentially expressed GPC genes is shown (Log2 fold change >0.15, adjusted p-value <0.05). [Figure 4G] Curated Ingenuity Pathway analysis of differentially expressed genes between GPC groups is shown. Circle size represents p-value, and shaded area indicates activation Z-score, with red being more active in the top group and green being more active in the bottom group. [Figure 4H] A heatmap of curated pairwise differentially expressed GPC genes is shown. [Figure 4I] Violin plots of pairwise differentially expressed GPC ribosomal gene log2 fold changes are shown. Comparisons between groups in E utilized Kruskal-Wallis tests followed by Dunn's test with Benjamini-Hochberg adjusted multiple comparisons. [Figure 5A-F] Figure 1 shows the transcriptional signature of competitive advantage. [Figure 5A] A schematic diagram of the protocol for identifying transcription factors (TFs) specifically associated with competitive advantage is shown. [Figure 5B] Box plots of identified WGCNA module eigengenes of interest (blue) in competitor and non-competitor cells are shown. [Figure 5C] We show that GSEA highlighted prioritized transcription factors whose regulons were enriched with genes upregulated in dominant young WT cells. [Figure 5D] An analysis of the relative contribution of each biological factor (age vs. genotype) to the composition of each module eigengene is shown. [Figure 5E] The key transcription factors predicted by SCENIC for establishing competitive advantage and their relative activities among the groups are shown. [Figure 5F] The regulatory network including downstream targets and their functional signaling pathways is shown. Target expression is controlled by at least one other key transcription factor in (E). NES: Network enrichment score. [Figure 6A-J] Generation of human HD glial chimeric striatum. C-D; G-H: Data presented as mean ± sem with individual data points (n=4). C-D: One-way ANOVA with Tukey's multiple comparison test; 12 weeks (n=3), 24 weeks (n=3), 36 weeks (n=4). Scale: B, 500 μm; H, 10 μm. [Figure 6A] The experimental design and analytical evaluation items are shown. [Figure 6B] Neonatal engraftment of HD glia (EGFP+, green) expanded within the mouse striatum, resulting in substantial tissue humanization over time. The dashed line defines the boundary of the striatum where human cells were mapped and quantified. [Figure 6C-D] Their expansion was at the expense of their Ki67+ proliferating cell pool (D), concomitant with a time-dependent increase in the number of HD glia harbored in the mouse striatum (C). [Figure 6E] Figure 1 shows the strategy used to assess the degree of striatal humanization 36 weeks after neonatal transplantation of HD GPCs. HD cell distribution was mapped in 15 equidistant sagittal sections (five shown as examples) and reconstructed in three dimensions for analysis. [Figure 6F] Rendered example of a mapped and reconstructed striatum for volumetric analysis. [Figure 6G] Volumetric quantification is shown demonstrating that by week 36, HD glia had expanded throughout the entire striatum, assuming a uniform distribution. Data are shown as mean (line) and individual data points (n=4). [Figure 6H-J] As they colonized the mouse striatum, HD glia either expanded and persisted as Olig2+ GPCs (arrows point to Olig2+ / EGFP+ (red / green) cells) or differentiated into hGFAP+ (red) astrocytes. Proliferating (Ki67+, red) HD glia are still visible after 36 weeks of expansion (D), albeit in reduced numbers. STR, neostriatum; LV, lateral ventricle; CTX, cortex. [Figure 7A-H] Replacement of HDs with WT glial progenitors resulted in proportional phenotypic substitution. Scale: D-E, G-H, 50 μm. [Figure 7A] The experimental design and analytical endpoints for the WT control group are shown. [Figure 7B] Stereological estimates show that as WT glia expanded within the humanized striatum, the total number of HD glia progressively decreased compared to HD chimeric controls. Two-way ANOVA with Sidak's multiple comparison test. HD controls: n = 4 at both time points; WT controls: n = 5 at 54 weeks, n = 3 at 72 weeks; allografts: n = 5 at 54 weeks, n = 3 at 72 weeks. Data are shown as mean ± SEM with individual data points. [Figure 7C-E] WT glia expanded as Olig2+ (white) GPCs replaced their HD counterparts. [Figure 7F-H] Within the region where they became dominant, they further differentiated into hGFAP+ (white) astrocytes. The proportion of GPCs and astrocytes in both populations was maintained as they competed for dominance in the striatum. Orange arrows point to co-labeled cells. [Figure 8A-D] We show that adult engrafted hGPCs outpopulated already resident mice more rapidly than human hGPCs. [Figure 8A] The experimental design and analytical endpoints for the WT control group are shown. [Figure 8B] Engraftment of WT glia (mCherry+, red) into the adult striatum of Rag1(- / -) mice resulted in significant humanization of the mouse striatum over time. Scale: B, 500 μm [Figure 8C-D] Figure 1 shows that adult transplanted WT glia infiltrated (C) and dispersed (D) throughout the mouse striatum over time, and volumetric quantification demonstrates that they were significantly more extensively infiltrated and dispersed than those already transplanted into HD chimeric mice. WT allografts (to HD chimeras; compare data with Figure 2): n = 8, 54 weeks; n = 7, 72 weeks; WT control: n = 8, 54 weeks; n = 5, 72 weeks. Two-way ANOVA with Sidak's multiple comparison test; main effects are shown as P values. [Figure 9A-F] This shows that expression of the fluorescent transgene did not affect competitive advantage. Co-engrafted isogenic clones of tagged and untagged WT hGPCs intermingled while displacing resident HD glia. Scale: B, 500 μm; C-D, 100 μm; E, 10 μm. [Figure 9A] The experimental design is shown for mice receiving a 1:1 mixture of mCherry-tagged (WT-mCherry) and untagged (WT-untagged) WT glia. [Figure 9B] Immunolabeling for human nuclear antigen (hN) shows that both WT-mCherry (mCherry+ hN+, red, white) and WT-untagged (mCherry- EGFP- hN+, white) glia expanded within the pre-humanized striatum and were progressively replaced by HD glia (EGFP+ hN+, green, white). [Figure 9C] We show that as admixed WT glia expanded and replaced resident HD glia, vast homotypic domains formed. [Figure 9D] In contrast, a mixture of homologous WT-mCherry and WT-untagged was observed. [Figure 9E] As mentioned above, we show that within domains dominated by WT glia, only more complex astrocyte-like HD glia could be seen, typically within white matter tracts. [Figure 9F]Quantification of the percentage of WT-mCherry glia and WT-untagged glia within the striatum shows that there was no significant difference between the two populations at any quantified time point (n = 6 per time point; samples were pooled from both experimental groups). Two-way ANOVA with Sidak's multiple comparison test; data are presented as mean ± SEM with individual data points. [Figure 10A-D] Figure 10 shows that human GPC chimeric mice were established by neonatal striatal injection of either HD EGFP-tagged glia (Figure 10A; G20 only), G19 mCherry-tagged wild-type glia transplanted into naive 36-week-old mice (Figure 10B; G19 only), or 36-week-old mice previously treated with G20-EGFP chimerism (Figure 10C). All mice were sacrificed at 72 weeks, and human cells were mapped on equidistant serial brain sections. In all groups, cells dispersed beyond the cell-injected striatum and colonized most of the forebrain. Figure 10D shows the stereological counts of human cells. Mean ± SEM. ns: not significant; *p<0.05 (one-way ANOVA with Tukey's post-hoc test). [Figure 11A-C] Aged human glia were eliminated by their younger counterparts through induced apoptosis. Scale: A-100 μm, B-50 μm. [Figure 11A] At the boundary between young WT glia (EGFP+, green) and senescent WT glia (mCherry+, red), a higher incidence of apoptotic TUNEL+ (white) cells was evident in the senescent population. [Figure 11B] Higher magnification shows the competition boundary between these different populations indicating that resident glia selectively undergo apoptosis. [Figure 11C] Quantification of TUNEL+ cells is shown, demonstrating a significantly higher incidence of TUNEL+ cells among aged resident WT glia compared to both their younger allogeneic counterparts and aged WT chimeric controls not challenged with younger cells.

[0022] Quantification was performed on pooled samples at 60 and 80 weeks (n=5 for all experimental groups). One-way ANOVA with Sidak's multiple comparison test; data are presented as mean±SEM with individual data points. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present disclosure is based at least in part on the unexpected discovery that aged and diseased human glia can be widely replaced by younger healthy glial progenitor cells in the adult brain.Therefore, the present disclosure relates to compositions and methods for treating conditions mediated by oligodendrocyte loss, astrocyte loss, or white matter loss, including age-related oligodendrocyte loss, astrocyte loss, or white matter loss.The present disclosure also relates to (a) rejuvenating glial progenitor cells or their progeny, or (b) enhancing the developmental potential of glial progenitor cells or their progeny.

[0024] As used herein, "glial progenitor cells" refer to cells that have the potential to differentiate into cells of the glial lineage, such as oligodendrocytes and astrocytes. Glial progenitor cells may be astrocyte-biased. Glial progenitor cells may be oligodendrocyte-biased. Examples of glial progenitor cells include astrocyte progenitor cells and oligodendrocyte progenitor cells. As used herein, the term "glial cells" refers to a population of non-neuronal cells that provide support and nutrition, maintain homeostasis, either form myelin or promote myelination, and participate in signal transduction in the nervous system. As used herein, "glial cells" encompass fully differentiated cells of the glial lineage, e.g., oligodendrocytes or astrocytes, as well as glial progenitor cells, each of which may be referred to as astroglia. In some embodiments, glial progenitor cells are also known as oligodendrocyte progenitor cells or NG2 cells.

[0025] Conditions and associated disorders mediated by white matter / oligodendrocyte / astrocytic loss Certain aspects of the present disclosure relate to compositions and methods for treating conditions or disorders mediated by oligodendrocyte loss, astrocyte loss, or white matter loss. Such conditions are often accompanied by a lack of myelin in the central nervous system ("CNS"). Examples of such conditions or disorders include any disease or condition associated with demyelination, insufficient myelination and remyelination, or hypomyelination in a subject. Such conditions or disorders may be hereditary, acquired, or may result from the aging process, i.e., age-related. In some embodiments, the condition is an age-related white matter disease condition defined or characterized by oligodendrocyte loss, astrocyte loss, or white matter atrophy in the setting of normal, otherwise healthy aging.

[0026] In humans, aging represents the accumulation of changes in a person over time and can encompass physical, psychological, and social changes. Aging increases the risk of human diseases such as cancer, diabetes, cardiovascular disease, and stroke, including demyelination in the CNS, which is often seen in various neurodegenerative diseases. Thus, in some embodiments of the present disclosure, the condition or disorder is mediated by age-related oligodendrocyte loss, age-related astrocyte loss, or age-related white matter loss.

[0027] Demyelination in the CNS can occur in response to genetic mutations (leukodystrophies), autoimmune diseases (e.g., multiple sclerosis), or trauma (e.g., traumatic brain injury, spinal cord injury, or ischemic stroke). Disruption of myelin function can play an important role in neurological and psychiatric disorders such as autism spectrum disorder (ASD), Alzheimer's disease, Huntington's disease, multiple system atrophy, Parkinson's disease, fragile X syndrome, schizophrenia, and various leukodystrophies.

[0028] Leukodystrophies are a group of rare, primarily inherited neurological disorders resulting from abnormal production, processing, or development of myelin and are the result of gene defects (mutations). Some forms are present at birth, while others may not cause symptoms until the child is older. Some primarily affect adults. Leukodystrophies include Canavan disease, Pelizaeus-Merzbacher disease, hypomyelination with atrophy of the basal ganglia and cerebellum, Krabbe disease (globoid cell leukodystrophy), X-linked adrenoleukodystrophy, metachromatic leukodystrophy, Pelizaeus-Merzbacher-like disease (or hypomyelinating leukodystrophy 2), Niemann-Pick disease type C (NPC), autosomal dominant leukodystrophy with autonomic neuropathy (ADLD), and 4H leukodystrophy (Pol These include: cerebrospinal fluid-associated leukodystrophy (HPE-III-related leukodystrophy), Zellweger spectrum disorder (ZSD), childhood ataxia with central nervous system hypomyelination or CACH (also called vanishing white matter disease or VWMD), celebretendinous xanthomatosis (CTX), Alexander disease (AXD), SOX10-associated peripheral demyelinating neuropathy, central demyelinating leukodystrophy, Waardenburg syndrome, Hirschung disease (PCWH), adult polyglucosan body disease (APBD), hereditary diffuse leukoencephalopathy with axonal spheroid formation (HDLS), Aicardi-Goutières syndrome (AGS), and adult Refsum disease.

[0029] Suitable subjects for treatment with the methods described herein include any human subject with a condition mediated by a deficiency in myelin, which may be manifested by age-related oligodendrocyte loss, age-related astrocyte loss, or age-related white matter loss.

[0030] In another embodiment, the condition mediated by a deficiency of myelin is selected from the group consisting of childhood leukodystrophies, lysosomal storage diseases, congenital demyelination, cerebral palsy, inflammatory demyelination, post-infectious and post-vaccination leukoencephalitis, demyelination due to radiation therapy or chemotherapy, and vascular demyelination.

[0031] In further embodiments, the condition mediated by a deficiency of myelin requires myelination. In another embodiment, the condition mediated by a deficiency of myelin requires remyelination. In some embodiments, the condition requiring remyelination is selected from the group consisting of multiple sclerosis, neuromyelitis optica, transverse osteomyelitis, optic neuritis, subcortical stroke, diabetic leukoencephalopathy, hypertensive leukoencephalopathy, age-related white matter disease, leukodementia, Binswanger's disease, spinal cord injury, radiation- or chemotherapy-induced demyelination, post-infectious and post-vaccination leukoencephalitis, periventricular leukomalacia, and cerebral palsy.

[0032] In further embodiments, the condition mediated by myelin deficiency is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is Huntington's disease. Huntington's disease is an autosomal dominant neurodegenerative disease characterized by relentless, progressive motor impairment accompanied by devastating psychiatric and cognitive deterioration. Huntington's disease is associated with consistent, severe neostriatal atrophy, which is linked to a significant loss of GABAergic medium spinous neurons, the main output neurons of the striatum. Huntington's disease is characterized by an abnormally long CAG repeat expansion in the first exon of the huntingtin gene. The encoded polyglutamine expansion of mutant huntingtin protein disrupts its normal function and protein-protein interactions, ultimately resulting in widespread neuropathology that is most rapidly manifested in the neostriatum.

[0033] Other neurodegenerative diseases that can be treated in accordance with the present application include frontotemporal dementia, Alzheimer's disease, Parkinson's disease, multiple system atrophy, and amyotrophic lateral sclerosis.

[0034] In one embodiment, the condition mediated by myelin deficiency is a neuropsychiatric disorder. In some embodiments, the neuropsychiatric disorder is schizophrenia. Schizophrenia is a severe mental illness that affects a person's thoughts, emotions, and behavior. Symptoms of schizophrenia are generally classified into three categories: (1) psychotic symptoms, including changes in perception; (2) negative symptoms, including loss of motivation, apathy, and lack of enjoyment; and (3) cognitive symptoms, including problems with attention, concentration, and memory. Other neuropsychiatric disorders that can be treated according to the present application include autism spectrum disorder and bipolar disorder.

[0035] The above-mentioned myelin-related disorders, which may be inherited, acquired, or age-related, affect millions of people and impose a heavy burden on affected individuals and their families. The pathological processes underlying many of these disorders remain poorly understood, and few disease-modifying therapies exist. There is an unmet need for therapeutic agents to treat these disorders. The present disclosure addresses these needs in several ways, including competitive replacement of senescent or older glial progenitor cells in the brain and rejuvenation of glial progenitor cells or their progeny.

[0036] Competitive replacement of glial progenitor cells in the adult brain Some aspects of the present disclosure relate to competitive replacement of glial progenitor cells. While competition between cell populations during development and tumorigenesis is well established, competition between cells within the adult brain has been poorly studied. Specifically, it is unclear whether allografted human glia can outcompete diseased cells to achieve therapeutic replacement within the adult human brain.

[0037] As disclosed herein, healthy, fluorophore-tagged wild-type (WT) hGPCs generated from human embryonic stem cells (hESCs) were engrafted into the striatum of adult mice that had been neonatally chimerized with spectrally distinct mutant HTT-expressing hGPCs generated from Huntington's disease (HD)-derived hESCs. WT hGPCs outcompeted their human HD counterparts, ultimately eliminating and repopulating the host striatum with healthy glia. Single-cell RNA-Seq revealed that WT donor hGPCs acquired a dominant competitor phenotype defined by YAP1 / MYC / E2F upon interaction with resident HD-derived glia. Competitive success was primarily dependent on the age difference between the competitor populations, in that adult-transplanted WT hGPCs outcompeted resident syngeneic WT cells that had been neonatally transplanted and were therefore older. These data demonstrate that aged, diseased human glia can be extensively replaced by younger, healthy hGPCs in the adult brain, suggesting that transplantation of newly generated glial progenitor cells could be used as a widespread therapeutic platform for the replacement of aged, diseased human glia.

[0038] Glial dysfunction is a causative factor in a wide range of neurological conditions. Astroglial and oligodendrocyte pathology is a contributing factor in amyotrophic lateral sclerosis (ALS). 1~4 , Huntington's disease (HD) 5~10 , and Parkinson's disease 11、12 , and schizophrenia and bipolar disorder 13~19 These cells are associated with the development and progression of several both neurodegenerative and neuropsychiatric disorders, including diverse conditions such as glaucoma, glaucoma, and glaucoma. In such conditions, replacement of diseased glia by healthy glial progenitor cells (hGPCs) offers real therapeutic benefit, given their ability to disperse and colonize their host while simultaneously generating new astrocytes and oligodendrocytes. 20 However, human GPC can outcompete and replace their murine counterparts in various experimental therapeutic models. 21~23On the other hand, it is unclear whether allogeneic transplanted human GPCs can replace other human cells, diseased or otherwise.

[0039] As disclosed herein, human glial chimeric mice 24 We used MRI to model competition between healthy and diseased human glia in vivo by engrafting healthy hGPCs into the striatum of adult mice that had been neonatally chimerized with hGPCs derived from subjects with HD, a prototypic monophenotype of neurodegenerative disease resulting from the expression of a mutant CAG repeat-expanded huntingtin (mHTT) gene. 25 .

[0040] Glial pathology is causally involved in synaptic dysfunction in HD, and replacement of mHTT-expressing mouse glia with transplanted healthy hGPCs was sufficient to delay disease progression and rescue key elements of function in a transgenic HD mouse model. 5Based on this, in this study, we used genetically tagged wild-type (WT) hGPCs and mHTT-expressing hGPCs derived from sibling lines of human embryonic stem cells (hESCs) to investigate whether healthy WT hGPCs could replace diseased HD hGPCs in vivo. When healthy hGPCs were delivered to the striatum of adult mice chimerized with HD hGPCs, we found that the healthy hGPCs outcompeted and replaced the existing HD hGPCs. However, we also conducted studies to investigate whether differences in cell age might have contributed to the competitive outcome, as the WT donor cells were effectively younger than the resident host glia they were replacing. This proved to be the case, in that healthy young hGPCs transplanted into adult mice neonatally engrafted with separately tagged glia derived from the same healthy line relentlessly replaced their older syngeneic counterparts. Single-cell RNA sequencing (scRNA-seq) of younger, winning, and older, losing hGPC populations revealed a set of differentially expressed pathways that overlapped with those in winning WT and losing HD hGPCs, suggesting a common transcriptional signature of competitively dominant GPCs. These data indicate that dynamic competition between clonally distinct glial populations can occur in the mature adult brain, whereby replacement of both existing and affected glia can be achieved by the introduction of young, healthy hGPCs.

[0041] In light of the contribution of glial pathology to a wide variety of neurodegenerative and neuropsychiatric disorders 36、37 Here, we sought to establish the relative suitability of both wild-type GPCs and aged, diseased human GPCs in vivo to assess the potential for allogeneic glial replacement as a therapeutic strategy. Some parts of this disclosure focus on Huntington's disease, given the well-described role of glial pathology in HD. 5、8、10、38、39When WT hGPCs that had already been chimerized with HD hGPCs were introduced into the brain, the WT cells were found to outcompete and ultimately replace resident HD glial progenitors. The selective expansion of healthy cells was associated with the active elimination of resident HD glia and was further supported by the proliferative advantage of healthy donor cells over their resident diseased counterparts.

[0042] Single-cell RNA sequencing revealed that the dominance of healthy WT hGPCs over HD glia in these adult chimeric mouse brains was associated with the expression of a transcriptional signature characteristic of competitively dominant cells in invertebrate systems. Surprisingly, however, when controlling for the relative age of the existing (older) and newly introduced (younger) donor hGPCs, we found that WT hGPCs transplanted into the adult neostriatum, neonatally chimerized with differentially tagged but otherwise syngeneic WT hGPCs, similarly outcompeted and replaced the older existing hGPCs. This observation suggested that cell youth is a key determinant of competitive success and the ability of the donor hGPC population to replace the host hGPC population. Thus, transplanted young WT hGPCs, whether exposed to already resident older HD hGPCs or to allogeneic syngeneic WT hGPCs, acquired the gene expression signature of a dominant competitor phenotype in vivo, and indeed, the analyses described herein suggested that cell youth is an even stronger determinant of competitive fitness than disease genotype.

[0043] These observations support the idea that cell replacement occurs when less fit clones are sensed by their more fit neighbors and eliminated from the tissue. 40~43 However, here we suggest that it is driven by the recapitulation of developmental cell competition, an evolutionarily conserved selection process that dynamically emerges in the adult brain. This process has been shown in a variety of systems to involve the active elimination of relatively slow-growing cells by faster-growing, more competitively fit neighbors. 44~48Within the adult brain, WT hGPCs were typically observed to expand from their transplantation site in ongoing waves of proliferation. These younger hGPCs largely eliminated their previously stably resident, older counterparts, regardless of whether the latter were mHTT-expressing HD cells or allogeneic syngeneic WT cells transplanted several months earlier. In both cases, the younger cells eventually recolonized the host brain with healthy new hGPCs (Figures 1 and 3). In both cases, the younger donor cells differentially expressed gene sets associated with competitive dominance (Figures 2, 4, and 5). Specifically, competitive dominance of hGPCs transplanted into younger adults was associated with increased levels of predicted YAP1, E2F, MYC, and MYCN pathway activity. These data provide a striking parallel to cell-cell competition in mouse embryos, where defective cells are eliminated by their neighbors following the acquisition of differential MYC expression during competitive exposure. 44、49、50 , YAP1 and MYC interact to determine the competitive outcome during cell-cell competition 51、52 Indeed, the simultaneous enrichment of YAP1 pathway members in "winner" WT hGPCs, including transcripts both upstream and downstream of YAP1, suggests that the Hippo pathway may be a particularly promising target for the regulation of glial replacement in the adult human brain. Indeed, these observations parallel the results of liver repopulation studies, where mouse fetal liver progenitors were found to drive faster and more extensive replacement when allografted into older than younger hosts. 53 , MYC and YAP1 activity were major determinants of competitive success. 54 Therefore, identifying YAP1 and MYC as key regulators of competition between hGPCs may enable strategies to further enhance the competitive advantage, rate, and extent of donor cell colonization after delivery of these cells to the brain.

[0044] The observed competitive replacement of resident glia by younger hGPCs resembles that of mouse glia by transplanted human GPCs, as their expansion in the mouse brain is also maintained by a relative proliferative advantage and proceeds by excluding their mouse counterparts upon contact. 22 Similar to the xenotransplantation setting, the winning population of young WT hGPCs appears to induce apoptotic death and local elimination of the resident losing population, whether composed of older syngeneic WT or sibling HD cells. The relative localization of apoptotic host cells to the advancing wavefront of younger WT cells suggests that the latter induce the death of already resident hGPCs, possibly via contact-dependent means. Potential mechanisms for such contact-dependent expression of relative cytocompatibility have been described in various models. 40 , which may be transduced by Piezo1-dependent regulation of YAP 57 , selective expression of Fwr isoforms 55、56 In addition, the selective elimination of both HD and allogeneic hGPCs when faced with younger hGPCs is consistent with the loss of ribosomal transcripts by the "loser" cells during cell competition. 58~59 , which was paralleled by their depletion of ribosomal-encoded transcripts, highlighting the contribution of ribosomal protein transcription to the regulation of cytocompatibility. 60~63 Taken together, these data suggest that transcriptional control of the translation machinery is as important in cell-cell competition in the adult brain as it is during development.

[0045] These observations suggest that the brain is a much more dynamic structural environment than previously recognized, and that intercellular competition between glial progenitor cells, and potentially the astrocytes from which they derive, plays a crucial role in maintaining the adult brain as well as during development. Indeed, this competitive advantage of young cells over older resident cells appears to largely mimic development, with successive waves of GPCs competing with each other, the oldest of which are largely eradicated from the brain by birth and replaced by younger successors. 64It is equally conceivable that in adulthood, somatic mutations among dividing glial progenitors could result in selective clonal advantage for one daughter lineage or the other, leading to the relentless replacement of the population by the descendants of the dominant daughter. This scenario is typical for the onset of carcinogenesis, but may also be involved in tumor suppression via competitive exclusion of tumor cells by their more fit non-tumorous neighbors. 65 Such a dynamic competition process between differentially adapted hGPCs may underlie glial-mediated non-tumor adult-onset brain disorders, such as some schizophrenia disorders. 13、14、16、17 , and the HD itself 5~10 It is particularly interesting to examine how glial pathology may be involved in the pathogenesis of neurodegenerative and neuropsychiatric disorders. Indeed, such mechanisms may contribute to the accelerated late stages of disease progression that are often noted in those neurodegenerative and neuropsychiatric disorders in which glial pathology is involved.

[0046] These data also have strong therapeutic implications, as they suggest that in the adult human brain, resident glia, whether diseased or simply aging, may be replaced after the introduction of younger, healthier GPCs, given the many neurodegenerative and neuropsychiatric disorders for which glial pathology is now recognized as a causally contributing factor. 1~19 The clinical implications of this observation are profound. It suggests that dysfunctional glia in diseased brains across a variety of disease etiologies and phenotypes may be effectively eliminated and replaced by intracerebral delivery of newly generated allogeneic hGPCs. The results suggest that glial progenitor cell delivery and glial replacement offer a viable and broadly applicable strategy for cell-based treatment of those diseases of the brain in which glial cells are causally involved.

[0047] Rejuvenation of glial progenitor cells or their progeny Some aspects of the present disclosure relate to the rejuvenation of glial progenitor cells or their progeny. Human glial progenitor cells emerge during the second trimester of pregnancy and colonize the brain, where they remain a parenchymal pool throughout adulthood. Fetal hGPCs are highly migratory and proliferative, but their expansion capacity declines with age and after demyelination-associated transformation.

[0048] Rejuvenation through upregulation In one aspect, the present disclosure provides methods for rejuvenating glial progenitor cells or their progeny. Methods for enhancing the developmental potential of glial progenitor cells or their progeny are also provided. Each of these methods comprises upregulating or increasing, in the glial progenitor cells or their progeny, (i) a transcription factor selected from the group consisting of CEBPZ, CTCF, E2F1, MYC, NFYB, and ETV4, or (ii) the level or activity of a target of the transcription factor.

[0049] Some examples of human CEBPZ, CTCF, E2F1, MYC, NFYB, and ETV4 proteins are shown below.

[0050] sp|P01106|MYC_HUMAN Myc proto-oncogene protein OS=Homo sapiens OX=9606 GN=MYC PE=1 SV=2 (SEQ ID NO: 1) MDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAP SEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDDNDGGGGSFSTADQLEMVTEL LGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPA RGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLS STESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAG GHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSP RSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILS VQAEEQKLISEEDLLRKRREQLKHKLEQLRNSCA sp|P01106-1|MYC_HUMAN Myc proto-oncogene protein isoform 1 OS=Homo sapiens OX=9606 GN=MYC (SEQ ID NO: 2) MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPP LSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPD DETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQD LSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVL HEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRC HVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHN VLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLL RKRREQLKHKLEQLRNSCA sp|P01106-3|MYC_HUMAN Myc proto-oncogene protein isoform 3 OS=Homo sapiens OX=9606 GN=MYC (SEQ ID NO: 3) MDFFRVVENQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPS EDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDDNDGGGGSFSTADQLEMVTELL GGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPAR GHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSS TESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGG HSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPR SSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSV QAEEQKLISEEDLLRKRREQLKHKLEQLRNSCA sp|P43268|ETV4_HUMAN ETS translocation variant 4 OS=Homo sapiens OX=9606 GN=ETV4 PE=1 SV=3 (sequence number 4) MERRMKAGYLDQQVPYTFSSKSPGNGSLREALIGPLGKLMDPGSLPPLDSEDLFQDLSHF QETWLAEAQVPDSDEQFVPDFHSENLAFHSPTTRIKKEPQSPRTDPALSCSRKPPLPYHH GEQCLYSSAYDPPRQIAIKSPAPGALGQSPLQPFPRAEQRNFLRSSGTSQPHPGHGYLGE HSSVFQQPLDICHSFTSQGGGREPLPAPYQHQLSEPCPPYPQQSFKQEYHDPLYEQAGQP AVDQGGVNGHRYPGAGVVIKQEQTDFAYDSDVTGCASMYLHTEGFSGPSPGDGAMGYGYE KPLRPFPDDVCVVPEKFEGDIKQEGVGAFREGPPYQRRGALQLWQFLVALLDDPTNAHFI AWTGRGMEFKLIEPEEVARLWGIQKNRPAMNYDKLSRSLRYYYEKGIMQKVAGERYVYKF VCEPEALFSLAFPDNQRPALKAEFDRPVSEEDTVPLSHLDESPAYLPELAGPAQPFGPKG GYSY sp|P43268-2|ETV4_HUMAN ETS translocation variant 4 isoform 2 OS=Homo sapiens OX=9606 GN=ETV4 (SEQ ID NO: 5) MDPGSLPPLDSEDLFQDLSHFQETWLAEAQVPDSDEQFVPDFHSENLAFHSPTTRIKKEP QSPRTDPALSCSRKPPLPYHHGEQCLYSSAYDPPRQIAIKSPAPGALGQSPLQPFPRAEQ RNFLRSSGTSQPHPGHGYLGEHSSVFQQPLDICHSFTSQGGGREPLPAPYQHQLSEPCPP YPQQSFKQEYHDPLYEQAGQPAVDQGGVNGHRYPGAGVVIKQEQTDFAYDSDVTGCASMY LHTEGFSGPSPGDGAMGYGYEKPLRPFPDDVCVVPEKFEGDIKQEGVGAFREGPPYQRRG ALQLWQFLVALLDDPTNAHFIAWTGRGMEFKLIEPEEVARLWGIQKNRPAMNYDKLSRSL RYYYEKGIMQKVAGERYVYKFVCEPEALFSLAFPDNQRPALKAEFDRPVSEEDTVPLSHL DESPAYLPELAGPAQPFGPKGGYSY sp|P43268-3|ETV4_HUMAN ETS translocation variant 4 isoform 3 OS=Homo sapiens OX=9606 GN=ETV4 (SEQ ID NO: 6) MYLHTEGFSGPSPGDGAMGYGYEKPLRPFPDDVCVVPEKFEGDIKQEGVGAFREGPPYQR RGALQLWQFLVALLDDPTNAHFIAWTGRGMEFKLIEPEEVARLWGIQKNRPAMNYDKLSR SLRYYYEKGIMQKVAGERYVYKFVCEPEALFSLAFPDNQRPALKAEFDRPVSEEDTVPLS HLDESPAYLPELAGPAQPFGPKGGYSY sp|P25208|NFYB_HUMAN Nuclear transcription factor Y subunit beta OS=Homo sapiens OX=9606 GN=NFYB PE=1 SV=2 (SEQ ID NO: 7) MTMDGDSSTTDASQLGISADYIGGSHYVIQPHDDTEDSMNDHEDTNGSKESFREQDIYLP IANVARIMKNAIPQTGKIAKDAKECVQECVSEFISFITSEASERCHQEKRKTINGEDILF AMSTLGFDSYVEPLKLYLQKFREAMKGEKGIGGAVTATDGLSEELTEEAFTNQLPAGLIT TDGQQQNVMVYTTSYQQISGVQQIQFS sp|Q01094|E2F1_HUMAN Transcription factor E2F1 OS=Homo sapiens OX=9606 GN=E2F1 PE=1 SV=1 (SEQ ID NO: 8) MALAGAPAGGPCAPALEALLGAGALRLLDSSQIVIISAAQDASAPPAPTGPAAPAAGPCD PDLLLFATPQAPRPTPSAPRPALGRPPVKRRLDLETDHQYLAESSGPARGRGRHPGKGVK SPGEKSRYETSLNLTTKRFLELLSHSADGVVDLNWAAEVLKVQKRRIYDITNVLEGIQLI AKKSKNHIQWLGSHTTVGVGGRLEGLTQDLRQLQESEQQLDHLMNICTTQLRLLSEDTDS QRLAYVTCQDLRSIADPAEQMVMVIKAPPETQLQAVDSSENFQISLKSKQGPIDVFLCPE ETVGGISPGKTPSQEVTSEEENRATDSATIVSPPPSSPPSSLTTDPSQSLLSLEQEPLLS RMGSLRAPVDEDRLSPLVAADSLLEHVREDFSGLLPEEFISLSPPHEALDYHFGLEEGEG IRDLFDCDFGDLTPLDF sp|P49711|CTCF_HUMAN Transcription repressor CTCF OS=Homo sapiens OX=9606 GN=CTCF PE=1 SV=1 (Accession number 9) MEGDAVEAIVEESETFIKGKERKTYQRRREGGQEEDACHLPQNQTDGGEVVQDVNSSVQM VMMEQLDPTLLQMKTEVMEGTVAPEAEAAVDDTQIITLQVVNMEEQPINIGELQLVQVPV PVTVPVATTSVEELQGAYENEVSKEGLAESEPMICHTLPLPEGFQVVKVGANGEVETLEQ GELPPQEDPSWQKDPDYQPPAKKTKKTKKSKLRYTEEGKDVDVSVYDFEEEQQEGLLSEV NAEKVVGNMKPPKPTKIKKKGVKKTFQCELCSYTCPRRSNLDRHMKSHTDERPHKCHLCG RAFRTVTLLRNHLNTHTGTRPHKCPDCDMAFVTSGELVRHRRYKHTHEKPFKCSMCDYAS VEVSKLKRHIRSHTGERPFQCSLCSYASRDTYKLKRHMRTHSGEKPYECYICHARFTQSG TMKMHILQKHTENVAKFHCPHCDTVIARKSDLGVHLRKQHSYIEQGKKCRYCDAVFHERY ALIQHQKSHKNEKRFKCDQCDYACRQERHMIMHKRTHTGEKPYACSHCDKTFRQKQLLDM HFKRYHDPNFVPAAFVCSKCGKTFTRRNTMARHADNCAGPDGVEGENGGETKKSKRGRKR KMRSKKEDSSDSENAEPDLDDNEDEEEPAVEIEPEPEPQPVTPAPPPAKKRRGRPPGRTN QPKQNQPTAIIQVEDQNTGAIENIIVEVKKEPDAEPAEGEEEEAQPAATDAPNGDLTPEM ILSMMDR sp|P49711-2|CTCF_HUMAN Transcriptional repressor CTCF isoform 2 OS=Homo sapiens OX=9606 GN=CTCF (SEQ ID NO: 10) MAFVTSGELVRHRRYKHTHEKPFKCSMCDYASVEVSKLKRHIRSHTGERPFQCSLCSYAS RDTYKLKRHMRTHSGEKPYECYICHARFTQSGTMKMHILQKHTENVAKFHCPHCDTVIAR KSDLGVHLRKQHSYIEQGKCCRYCDAVFHERYALIQHQKSHKNEKRFKCDQCDYACRQER HMIMHKRTHTGEKPYACSHCDKTFRQKQLLDMHFKRYHDPNFVPAAFVCSKCGKTFTRRN TMARHADNCAGPDGVEGENGGETKKSKRGRKRKMRSKKEDSSDSENAEPDLDDNEDEEEP AVEIEPEPEPQPVTPAPPPAKKRRGRPPGRTNQPKQNQPTAIIQVEDQNTGAIENIIVEV KKEPDAEPAEGEEEEAQPAATDAPNGDLTPEMILSMMDR sp|Q03701|CEBPZ_HUMAN CCAAT / Enhancer-binding protein zeta OS=Homo sapiens OX=9606 GN=CEBPZ PE=1 SV=3 (SEQ ID NO: 11) MAAVKEPLEFHAKRPWRPEEAVEDPDEEDEDNTSEAENGFSLEEVLRLGGTKQDYLMLAT LDENEEVIDGGKKGAIDDLQQGELEAFIQNLNLAKYTKASLVEEDEPAEKENSSKKEVKI PKINNKNTAESQRTSVNKVKNKNRPEPHSDENGSTTPKVKKDKQNIFEFFERQTLLLRPG GKWYDLEYSNEYSLKPQPQDVVSKYKTLAQKLYQHEINLFKSKTNSQKGASSTWMKAIVS SGTLGDRMAAMILLIQDDAVHTLQFVETLVNLVKKKGSKQQCLMALDTFKELLITDLPD NRKLRIFSQRPFDKLEQLSSGNKDSRDRRLILWYFEHQLKHLVAEFVQVLETLSHDTLVT TKTRALTVAHELLCNKPEEEKALLVQVVNKLGDPQNRIATKASHLLETLLCKHPNMKGVV SGEVERLLFRSNISSKAQYYAICFLNQMALSHEESELANKLITVYFCFFRTCVKKKDVES KMLSALLTGVNRAYPYSQTGDDKVREQIDTLFKVLHIVNNFNTSVQALMLLFQVMNSQQTI SDRYYTALYRKMLDPGLMTCSKQAMFLNLVYKSLKADIVLRRVKAFVKRLLQVTCQQMPP FICGALYLVSEILKAKPGLRSQLDDHPESDDEENFIDANDDEDMEKFTDADKETEIVKKL ETEETVPETDVETKKPEVASWVHFDNLKGGKQLNKYDPFSRNPLFCGAENTSLWELKKLS VHFHPSVALFAKTILQGNYIQYSGDPLQDFTLMRFLDRFVYRNPKPHKGKENTDSVVMQP KRKHFIKDIRHLPVNSKEFLAKEESQIPVDEVFFHRRYYKKVAVKEKQKRDADEESIEDVD DEEFEELIDTFEDDNCFSSGKDDMDFAGNVKKRTKGAKDNTLDEDSEGSDDELGNLDDDE VSLGSMDDEEFAEVDEDGGTFMDVLDDESESVPELEVHSKVSTKKSKRKGTDDFDFAGSF QGPRKKKKRNLNDSSLFVSAEEFGHLLDENMGSKFDNIGMNAMANKDNASLKQLRWEAERD DWLHNRDAKSIIKKKKHFKKKRIKTTQKTKKQRK

[0051] Each of the transcription factors described herein has a highly conserved protein domain that is conserved in several species, including humans, mice, rats, chickens, fish, and Drosophila. Thus, CEBPZ, CTCF, E2F1, MYC, NFYB, or ETV4 from non-human species can also be used in the expression cassettes, gene constructs, vectors, compositions, or methods disclosed herein.

[0052] The terms CEBPZ, CTCF, E2F1, MYC, NFYB, or ETV4 also encompass all alternative splice variants, isoforms, functional fragments, or derivatives that substantially retain the transcription factor activity of CEBPZ, CTCF, E2F1, MYC, NFYB, or ETV4 described herein. Typically, functional fragments or derivatives retain at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of their transcription factor activity. It is also intended that CEBPZ, CTCF, E2F1, MYC, NFYB, or ETV4 transcription factors can include conservative amino acid substitutions that do not substantially alter their activity. Suitable conservative amino acid substitutions are known to those of skill in the art and can usually be made without altering the biological activity of the resulting molecule. Those of skill in the art will recognize that, generally, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. Conservative and non-conservative amino acid substitutions are described herein.

[0053] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the activity of one of the above proteins. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are known in the art. A conservative modification or functional equivalent of a peptide, polypeptide, or protein disclosed herein refers to a polypeptide derivative of the peptide, polypeptide, or protein, such as a protein having one or more substitutions, point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof, that substantially retains activity relative to the parent peptide, polypeptide, or protein (such as those disclosed herein). Generally, a conservative modification or functional equivalent is at least 60% identical (e.g., any number between 60% and 100%, including, e.g., 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) to the parent (e.g., one of the human or non-human CEBPZ, CTCF, E2F1, MYC, NFYB, or ETV4 sequences disclosed herein).

[0054] Amino acid substitutions can be made, in some cases, by selecting substitutions that do not significantly differ in their effect on maintaining (a) the structure of the peptide backbone in the area of ​​substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. For example, naturally occurring residues can be divided into groups based on side chain properties: (1) hydrophobic amino acids (norleucine, methionine, alanine, valine, leucine, and isoleucine), (2) neutral hydrophilic amino acids (cysteine, serine, threonine, asparagine, and glutamine), (3) acidic amino acids (aspartic acid and glutamic acid), (4) basic amino acids (histidine, lysine, and arginine), (5) amino acids that affect chain orientation (glycine and proline), and (6) aromatic amino acids (tryptophan, tyrosine, and phenylalanine). Substitutions made within these groups can be considered conservative substitutions. Examples of substitutions include, but are not limited to, substitutions of valine with alanine, lysine with arginine, glutamine with asparagine, glutamic acid with aspartic acid, serine with cysteine, asparagine with glutamine, aspartic acid with glutamic acid, proline with glycine, arginine with histidine, leucine with isoleucine, isoleucine with leucine, arginine with lysine, leucine with methionine, leucine with phenylalanine, glycine with proline, threonine with serine, serine with threonine, tyrosine with tryptophan, phenylalanine with tyrosine, and / or leucine with valine. Exemplary substitutions are shown in the table below. Amino acid substitutions may be introduced into human CEBPZ, CTCF, E2F1, MYC, NFYB, or ETV4, and the products may be screened for retention of the biological activity of the parent protein. [Table 1]

[0055] The methods described herein can also be achieved by upregulating or increasing the level or activity of one or more targets of one or more of the above transcription factors in glial progenitor cells or progeny. Exemplary targets include RPL6, RPS27, RPS16, RPS21, DOHH, PCCB, UTP11, RPS8, RPL27A, EIF2A, UBLCP1, RPL32, GIN1, PATZ1, TNFRSF1A, MRPL10, RFXANK, BORCS8, ENOPH1, RPS16, SNHG11, SLC35A5, RAB1B, RPL23A, YBX1, TMEM129, DOHH, CCND1, MRPL24, RPL14, HMGA1, DCTPP1, ENOPH1, ZNF436, R PLP2, CCND1, TNFRSF1A, FBXL12, NTMT1, IMPDH2, MRPL18, LIMS1, CD82, POLR2H, LRRC8A, EXOSC5, RAN, DYNLT1, FDPS, ACTL6A, RPS5, DOLPP1 , GGCT, RPS2, SYCE1L, MRPL17, ZDHHC16, YBX1, RPLP0, ELK1, RPSA, NME2, RPS15, TSPAN33, B3GNT9, DCLRE1B, CMC1, RPLP1, ACAT2, RPL6, RPL2 8, RPL18A, CCDC51, RACK1, RPS14, RPS19, RPL12, RPS5, RPL19, RPL27A, RPS19, RPS21, POLR2H, RPS14, CCDC51, EIF2A, UBLCP1, SNHG19, RPL 32, ZNF579, RPS27, RPL6, GIN1, PATZ1, RACK1, LRRC8A, TNFRSF1A, RPL28, RPS18, MRPL10, RFXANK, BORCS8, FBXL12, PEF1, ENOPH1, PEX7, RP S16, SNHG11, RPL10, ZDHHC16, HMGA1, ZNF436, UTP11, DCLRE1B, RPS5, EXOSC5, MRPL17, RPL19, PRMT1, NME2, CCND1, NRN1, YBX1, LIMS1, RPL 23A, CD82, NTMT1, RPL18A, DOLPP1, GGCT, ELK1, ACTL6A, FDPS, MRPL18, RPLP0, ACAT2, SLC35A5, RAB1B, RAN, DYNLT1, TMEM129, RPSA, RPS15,Examples of genes that may be used include those listed in Table 1 and Figure 5, including, but not limited to, RPL13A, PCCB, DCTPP1, RPLP2, B3GNT9, IMPDH2, RPL14, MRPL24, RPS8, RPS2, SYCE1L, RPL12, CMC1, DOHH, RPLP1, BTBD17, TSPAN33, ACAT2, GGCT, FDPS, SNHG19, PRMT1, RPL13A, FBXL12, RPS19, RFXANK, NRN1, DCTPP1, ZNF579, YBX1, MRPL18, NTMT1, RPL14, PEF1, RPS21, PEX7, BTBD17, RPL10, and RPS18.

[0056] Similar to the transcription factors described above, one of these targets may also have a highly conserved protein domain that is conserved in several species, including humans, mice, rats, chickens, fish, and fruit flies. Thus, homologs of non-human species can also be used in the expression cassettes, gene constructs, vectors, compositions, or methods disclosed herein.

[0057] Thus, each of these targets also encompasses functional fragments or derivatives that substantially retain the respective activity of the targets described herein. Typically, a functional fragment or derivative retains at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of its parent's activity. It is also contemplated that a target may contain conservative amino acid substitutions that do not substantially alter its activity. As described herein, suitable conservative amino acid substitutions are known to those of skill in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art will recognize that, generally, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity.

[0058] Rejuvenation through inhibition In some embodiments, the methods disclosed herein can further include repressing in the glial progenitor cells or progeny a transcriptional repressor selected from the group consisting of E2F6, ZNF274, MAX, and IKZF3. Exemplary nucleic acid and amino acid sequences of these repressors include those described in PCT / US22 / 78344 and PCT / US22 / 78356, the contents of which are incorporated by reference.

[0059] In some instances, this inhibition can be achieved by administering one or more suppressors or inhibitors of transcriptional repressors to a subject in need of such inhibition or to a target cell in need of such inhibition. Such suppressors or inhibitors can include or be small molecule compounds, oligonucleotides, nucleic acids, peptides, polypeptides, CRISPR / Cas systems, or antibodies or antigen-binding portions thereof. Examples of suppressors / inhibitors include activators, agonists, or enhancers of related YAP or MYC pathway signaling pathways (e.g., the Hippo signaling pathway). Various activators for these signaling pathways are known in the art. In some embodiments, the suppressor is an inhibitory or interfering nucleic acid, such as an siRNA, shRNA, miRNA, antisense oligonucleotide (ASO), and / or a nucleic acid comprising one or more modified nucleic acid residues. Examples include those described in PCT / US22 / 78344 and PCT / US22 / 78356, the contents of which are incorporated by reference.

[0060] In one aspect, suppression or knockdown of one or more of the repressor genes described herein can also be achieved via CRISPR-Cas-guided nucleases using CRISPR-Cas systems and related methods known in the art, such as those described in PCT / US22 / 78344 and PCT / US22 / 78356, the contents of which are incorporated by reference.

[0061] Expression cassettes and expression vectors The present disclosure also provides an expression cassette comprising or consisting of a recombinant nucleic acid encoding the above-mentioned transcription factor or its target. If such a recombinant nucleic acid does not already contain a promoter, the expression cassette may further comprise a promoter. Thus, an expression cassette according to the present disclosure comprises, from 5' to 3', a promoter, a coding sequence, and optionally a terminator or other elements. The expression cassette allows for easy transfer of the nucleic acid sequence of interest into an organism, preferably a cell, preferably a diseased cell.

[0062] The expression cassette of the present disclosure is preferably contained in a vector. Thus, the vector of the present disclosure allows for transformation, transfection, transduction, infection, or introduction into a cell of a nucleic acid sequence of interest. Accordingly, the present disclosure provides a host cell comprising an expression cassette according to the present disclosure or a recombinant nucleic acid according to the present disclosure. The recombinant nucleic acid may also comprise a promoter or enhancer to allow expression of the nucleic acid sequence of interest.

[0063] Exogenous genetic material (e.g., nucleic acids, expression cassettes, or expression vectors encoding one or more therapeutic or inhibitory proteins or RNAs) can be introduced into a desired target cell in vivo by gene transfer methods such as transfection or transduction to provide a genetically modified cell. A variety of expression vectors (i.e., vehicles for facilitating delivery of exogenous genetic material to a target cell) are known to those skilled in the art. As used herein, "exogenous genetic material" refers to either natural or synthetic nucleic acids or oligonucleotides that are not naturally found within a cell, or, if it is naturally found within a cell, that are not transcribed or expressed at biologically significant levels by the cell. Thus, "exogenous genetic material" includes, for example, non-naturally occurring nucleic acids that can be transcribed into RNA.

[0064] As used herein, "cell transfection" refers to the acquisition of new genetic material by a cell through the incorporation of added nucleic acid (DNA, RNA, or a hybrid thereof) without the use of a viral delivery vehicle. Thus, transfection refers to the introduction of nucleic acid into a cell using physical or chemical methods. Several transfection techniques are known to those skilled in the art, including calcium phosphate nucleic acid coprecipitation, strontium phosphate nucleic acid coprecipitation, DEAE-dextran, electroporation, cationic liposome-mediated transfection, and tungsten particle-assisted microparticle bombardment. In contrast, "cell transduction" refers to the process of transferring nucleic acid into a cell using a DNA or RNA virus. RNA viruses (e.g., retroviruses) used to transfer nucleic acid into cells are referred to herein as transducing chimeric viruses. The exogenous genetic material contained in the virus can be integrated into the genome of the transduced cell. Cells transduced with a chimeric DNA virus (e.g., an adenovirus carrying DNA encoding a therapeutic agent) may not have the exogenous genetic material integrated into their genome, but may be capable of expressing exogenous genetic material carried extrachromosomally within the cell.

[0065] Typically, exogenous genetic material may include a heterologous gene (encoding a therapeutic RNA or protein) along with a promoter that controls transcription of the new gene. A promoter characteristically has a specific nucleotide sequence necessary to initiate transcription. Optionally, the exogenous genetic material further includes additional sequences (i.e., enhancers) necessary to obtain the desired gene transcription activity. The exogenous genetic material may be introduced into the cellular genome immediately downstream of the promoter such that the promoter and coding sequence are operably linked, allowing transcription of the coding sequence. Retroviral expression vectors may also include exogenous promoter elements to control transcription of the inserted exogenous gene. Such exogenous promoters include both constitutive and inducible promoters.

[0066] Naturally occurring constitutive promoters control the expression of essential cellular functions. As a result, genes under the control of a constitutive promoter are expressed under all conditions of cell growth. Exemplary constitutive promoters include the promoters of the following genes, which encode certain constitutive or "housekeeping" functions: hypoxanthine phosphoribosyltransferase, dihydrofolate reductase, adenosine deaminase, phosphoglycerol kinase, pyruvate kinase, phosphoglycerol mutase, actin promoter, ubiquitin, elongation factor-1, and other constitutive promoters known to those skilled in the art. In addition, many viral promoters function constitutively in eukaryotic cells. These include, among others, the early and late promoters of SV40, the long terminal repeats (LTRs) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Therefore, any of the above-mentioned constitutive promoters can be used to control the transcription of a heterologous gene insert.

[0067] Genes under the control of an inducible promoter are expressed only in the presence of, or are largely regulated by, an inducing agent (e.g., transcription under the control of the metallothionein promoter is greatly increased in the presence of certain metal ions). Inducible promoters contain response elements (REs) that stimulate transcription when their inducers bind. Examples include REs for serum factors, steroid hormones, retinoic acid, and cyclic AMP. Promoters containing specific REs can be selected to obtain an inducible response, and in some cases, REs themselves can be attached to different promoters, thereby conferring inducibility to the recombinant gene. Thus, by selecting the appropriate promoter (constitutive vs. inducible, strong vs. weak), it is possible to control both the presence and level of expression of a therapeutic agent in a genetically engineered cell. When a gene encoding a therapeutic agent is under the control of an inducible promoter, in situ delivery of the therapeutic agent can be triggered by exposing the genetically engineered cell in situ to conditions that allow transcription of the therapeutic agent, for example, by injecting a specific inducer of the inducible promoter that controls transcription of the agent. For example, in situ expression by genetically engineered cells of a therapeutic agent encoded by a gene under the control of a metallothionein promoter is enhanced by contacting the genetically engineered cells in situ with a solution containing the appropriate (i.e., inducing) metal ions.

[0068] Thus, the amount of therapeutic agent delivered in situ is regulated by controlling factors such as: (1) the nature of the promoter used to direct transcription of the inserted gene (i.e., whether the promoter is constitutive or inducible, strong or weak), (2) the number of copies of the exogenous gene inserted into the cells, (3) the number of transduced / transfected cells administered (e.g., implanted) into the patient, (4) the size of the implant (e.g., graft or encapsulated expression system), (5) the number of implants, (6) the length of time the transduced / transfected cells or implants remain in place, and (7) the rate of production of the therapeutic agent by the genetically modified cells. Selection and optimization of these factors for delivery of a therapeutically effective dose of a particular therapeutic agent is deemed to be within the skill of one in the art without undue experimentation, taking into account the factors disclosed above and the patient's clinical profile.

[0069] In addition to at least one promoter and at least one heterologous nucleic acid encoding a therapeutic agent, the expression vector may contain a selection gene, such as a neomycin resistance gene or a fluorescent protein gene, to facilitate selection of cells transfected or transduced with the expression vector. Alternatively, cells are transfected with two or more expression vectors, at least one vector containing a gene encoding a therapeutic agent and the other vector containing a selection gene. Selection of a suitable promoter, enhancer, selection gene, and / or signal sequence is deemed to be within the skill of one in the art without undue experimentation.

[0070] The coding sequences of the present disclosure can be inserted into any type of target or host cell. In the context of expression vectors, the vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0071] Polynucleotide Carriers / Delivery As disclosed herein, the above-described polynucleotides or nucleic acid molecules can be used to treat disorders in a subject. Accordingly, the present disclosure provides systems and methods for delivering polynucleotides to target cells or subjects.

[0072] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0073] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0074] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0075] The polynucleotides or nucleic acids described herein (e.g., protein-encoding nucleic acids, inhibitory nucleic acids, those encoding CRISPR-Cas systems, expression cassettes, and expression vectors) can be directly added, or complexed with cationic lipids, packaged into liposomes, or as recombinant plasmids or viral vectors, or otherwise delivered to target cells or tissues. Methods for delivering nucleic acid molecules are known in the art. See, e.g., U.S. Patent No. 6,395,713, WO 94 / 02595, Akhtar et al., 1992, Trends Cell Bio., 2, 139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140, Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137, 165-192, and Lee et al., 2000, ACS Symp. Ser., 752, 184-192. These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not limited to, encapsulation within liposomes, by iontophoresis, or by incorporation into other vehicles such as biodegradable polymers, hydrogels, cyclodextrins (see, e.g., Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074, WO 03 / 47518 and WO 03 / 46185), poly(lactic-co-glycolic) acid (PLGA) and PLCA microspheres (see, e.g., U.S. Pat. No. 6,447,796 and US 2002 / 130430), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (see, e.g., WO 00 / 53722).

[0076] In one aspect, the present application provides a carrier system containing a nucleic acid molecule described herein. In some embodiments, the carrier system is a lipid-based carrier system, a cationic lipid, or a liposome-nucleic acid complex, a liposome, a micelle, a virosome, a lipid nanoparticle, or a mixture thereof. In other embodiments, the carrier system is a polymer-based carrier system such as a cationic polymer-nucleic acid complex. In additional embodiments, the carrier system is a cyclodextrin-based carrier system such as a cyclodextrin polymer-nucleic acid complex. In further embodiments, the carrier system is a protein-based carrier system such as a cationic peptide-nucleic acid complex. Preferably, the carrier system is a lipid nanoparticle formulation. The lipid nanoparticle ("LNP") formulations described herein can be applied to any nucleic acid molecule (e.g., RNA molecule) or combination of nucleic acid molecules described herein.

[0077] In certain embodiments, the nucleic acid molecules described herein are formulated as lipid nanoparticle compositions such as those described in U.S. Patent Nos. 7,514,099 and 7,404,969. In some embodiments, the present application features a composition comprising a nucleic acid molecule formulated as any of the formulations described in U.S. Patent Nos. LNP-051, LNP-053, LNP-054, LNP-069, LNP-073, LNP-077, LNP-080, LNP-082, LNP-083, LNP-060, LNP-061, LNP-086, LNP-097, LNP-098, LNP-099, LNP-100, LNP-101, LNP-102, LNP-103, or LNP-104.

[0078] In other embodiments, the present disclosure features conjugates and / or complexes of the nucleic acid molecules described herein. Such conjugates and / or complexes can be used to facilitate delivery of nucleic acid molecules into biological systems, such as cells. The conjugates and complexes provided herein can impart therapeutic activity by transporting therapeutic compounds across cell membranes, altering pharmacokinetics, and / or modulating the localization of the nucleic acid molecules of the present disclosure. Non-limiting examples of such conjugates are described, for example, in U.S. Patent Nos. 7,833,992, 6,528,631, 6,335,434, 6,235,886, 6,153,737, 5,214,136, and 5,138,045.

[0079] In various embodiments, polyethylene glycol (PEG) can be covalently attached to the nucleic acid molecules described herein. The attached PEG can be of any molecular weight, preferably from about 100 to about 50,000 daltons (Da). Accordingly, the present disclosure features compositions or formulations comprising poly(ethylene glycol) lipids (PEG-modified or long-circulating liposomes or stealth liposomes) and surface-modified liposomes containing the nucleic acid molecules described herein. See, e.g., WO96 / 10391, WO96 / 10390, and WO96 / 10392).

[0080] In some embodiments, nucleic acid molecules also contain polyethyleneimine and its derivatives, such as polyethyleneimine-polyethylene glycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethylene glycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL). It may be formulated or complexed with a derivative. In one embodiment, the nucleic acid molecule may be formulated as described in US2003 / 0077829.

[0081] In other embodiments, the nucleic acid molecules described herein may be complexed with membrane disruptive agents, such as those described in US 2001 / 0007666. In yet other embodiments, the membrane disruptive agents and molecules may be complexed with cationic lipids or helper lipid molecules, such as those described in U.S. Patent No. 6,235,310.

[0082] In certain embodiments, the nucleic acid molecules described herein can be complexed with delivery systems described in U.S. Patent Application Publication Nos. 2003 / 077829, 2005 / 0287551, 2005 / 0164220, 2005 / 0191627, 2005 / 0118594, 2005 / 0153919, 2005 / 0085486, and 2003 / 0158133, as well as IWO00 / 03683 and WO02 / 087541.

[0083] In some embodiments, the liposomal formulations described herein are prepared in accordance with U.S. Patent Nos. 6,858,224, 6,534,484, 6,287,591, 6,835,395, 6,586,410, 6,858,225, 6,815,432, 6,586,001, 6,120,798, 6,977,223, 6,998,115, 5,981,501, 5,976,567, 5,705,385, and U.S. Patent Application Publication Nos. 2006 / 0019912, 2006 / 0019913, 2006 / 0019914, 2006 / 0019915, 2006 / 0019916, 2006 / 0019917, 2006 / 0019918, 2006 / 0019919 ... The nucleic acid molecules described herein may be formulated or complexed with compounds and compositions described in US Pat. Nos. 2006 / 0019258, 2006 / 0008909, 2005 / 0255153, 2005 / 0079212, 2005 / 0008689, 2003 / 0077829, 2005 / 0064595, 2005 / 0175682, 2005 / 0118253, 2004 / 0071654, 2005 / 0244504, 2005 / 0265961, and 2003 / 0077829.

[0084] As disclosed herein, the nucleic acid molecules described above can be used to treat disorders in a subject. The vectors described above (such as recombinant plasmids and viral vectors) can be used to deliver the therapeutic agents described herein. Delivery of the vector can be systemic, for example, by intravenous or intramuscular administration, by administration to target cells explanted from the subject and then reintroduced into the subject, or by any other means that would allow for introduction into the desired target cells. Such recombinant vectors can also be administered directly or in conjunction with a suitable delivery reagent, including, for example, Mirus Transit LT1 lipophilic reagent; Lipofectin; Lipofectamine; Cellfectin; polycations (e.g., polylysine); or liposome-lipid-based carrier systems, cationic lipids, or liposome-nucleic acid complexes, micelles, virosomes, or lipid nanoparticles.

[0085] viral vectors In some embodiments, a polynucleotide encoding an RNA molecule or protein may be inserted into or encoded by a vector, such as a plasmid or viral vector. Preferably, the polynucleotide is inserted into or encoded by a viral vector. The viral vector may be a herpesvirus (HSV) vector, a retroviral vector, an adenoviral vector, an AAV vector, a lentiviral vector, or the like. In some specific embodiments, the viral vector is an AAV vector. In some embodiments, the RNA may be encoded by a retroviral vector (see, e.g., U.S. Patent Nos. 5,399,346, 5,124,263, 4,650,764, and 4,980,289, the contents of each of which are incorporated herein by reference in their entirety).

[0086] Lentiviral vectors Lentiviruses, such as HIV, are "slow viruses." Lentivirus-derived vectors can be expressed in host cells for long periods of time after several administrations to patients, for example, via ex vivo transduction of stem or progenitor cells. For most diseases and disorders, including genetic disorders, cancer, and neurological disorders, long-term expression is essential for successful treatment. Regarding lentiviral vector safety, several strategies for eliminating the replication capacity of lentiviral vectors are currently known in the art. See, for example, US2021 / 0401868 and US2021 / 0403517, each of which is incorporated herein by reference in its entirety. For example, deletion of promoter and enhancer elements from the U3 region of the long terminal repeat (LTR) would result in no LTR-directed transcription. The resulting vector is called a "self-inactivating" (SIN) vector.

[0087] Lentiviral vectors are particularly suitable for achieving long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have the additional advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, in that they can transduce non-proliferating cells, such as CNS cells. They also have the additional advantage of being less immunogenic. Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction nuclease sites, and one or more selectable markers (e.g., WO01 / 96584 and WO01 / 29058, and U.S. Patent No. 6,326,193). Several vector promoter sequences are available for transgene expression. One example of a suitable promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong, constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is EF1a. However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Inducible promoters include, but are not limited to, the metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0088] The present disclosure provides recombinant lentiviruses capable of infecting dividing and non-dividing cells, such as oligodendrocytes, astrocytes, or glial progenitor cells. The viruses are useful for transferring and expressing nucleic acid sequences in vivo and ex vivo. The lentiviral vectors of the present disclosure can be lentiviral transfer plasmids or infectious lentiviral particles. Constructions of lentiviral vectors, helper constructs, envelope constructs, and the like for use in lentiviral transfer systems are described, for example, in US2021 / 0401868 and US2021 / 0403517, each of which is incorporated herein by reference in its entirety.

[0089] Adenovirus Adenovirus is a eukaryotic DNA virus that can be modified to efficiently deliver nucleic acid to various cell types in vivo, and is widely used in gene therapy protocols, including targeting genes to neuronal and glial cells.A variety of replication-defective adenoviruses and minimal adenovirus vectors have been described for nucleic acid therapeutic agents (see, for example, PCT Patent Publication Nos. 1994 / 26914, 1995 / 02697, 1994 / 28152, 1994 / 12649, 1995 / 02697, and 1996 / 22378, the contents of each of which are incorporated by reference in their entirety).Such adenovirus vectors can also be used to deliver therapeutic molecules of the present disclosure to cells.

[0090] Adeno-associated virus Adeno-associated viruses are widely used gene therapy vectors due to their clinical safety record, non-pathogenicity, ability to infect non-dividing cells (e.g., neurons), and ability to provide long-term gene expression after a single administration. Currently, many human and non-human primate AAV serotypes have been identified. AAV vectors have demonstrated safety in hundreds of clinical trials worldwide, and clinical efficacy has been shown in trials for hemophilia B, spinal muscular atrophy, alpha-1 antitrypsin, and Leber's congenital anmyelopathy.

[0091] Due to their safety, non-pathogenic nature, and ability to infect neurons, AAVs, such as AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, and AAV9, are commonly used gene therapy vectors for CNS applications. However, after direct CNS injection, these serotypes exhibit predominantly neuronal tropism and low expression in oligodendrocytes, especially when gene expression is driven by a constitutive promoter, limiting their potential use in the treatment of white matter diseases. AAV1 / 2, AAV2, and AAV8 have been shown to transduce oligodendrocytes. The reliance on cell-specific promoters for expression specificity allows for nonselective cellular uptake and the potential for leaky expression of transgenes via cryptic promoter activity in non-oligodendrocyte lineage cells.

[0092] The approach described herein to alleviate these problems involves using AAV serotypes with high tropism for oligodendrocytes, astrocytes, or glial progenitor cells. Recently, using DNA shuffling and directed evolution, a chimeric AAV capsid, AAV / Olig001, with strong preference for oligodendrocytes was described (Powell et al., 2016, Gene Ther 23:807-814). AAV / Olig001 was subsequently shown to transduce neonatal oligodendrocytes in a mouse model of Canavan disease (Francis et al., 2021, Mol Ther Methods Clin Dev 20:520-534). Other approaches, such as random mutagenesis and peptide library insertion, can be used to generate capsid libraries that can be screened for tropism and selectivity for oligodendrocytes, astrocytes, or glial progenitor cells.

[0093] As mentioned above, the terms "adeno-associated virus" and / or "AAV" refer to parvoviruses and their variants that have a linear, single-stranded DNA genome. The term encompasses all subtypes and both naturally occurring and recombinant forms, unless otherwise required. Parvoviruses, including AAV, are useful as gene therapy vectors because they can enter cells and deliver nucleic acids (e.g., transgenes) to the nucleus. In some embodiments, the delivered nucleic acid (e.g., rAAV vector genome) forms circular concatemers that persist episomally in the nucleus of transduced cells. In some embodiments, the transgene is inserted into a specific site in the host cell genome. Site-specific integration, as opposed to random integration, is believed to be more likely to result in a predictable long-term expression profile. The insertion site of AAV into the human genome is designated AAVS1. Once delivered to a cell, the RNA or polypeptide encoded by the nucleic acid can be expressed by the cell. Because AAV is not associated with any pathogenic disease in humans, nucleic acids delivered by AAV can be used to express therapeutic RNAs or polypeptides for the treatment of diseases, disorders, and / or conditions in human subjects.

[0094] Multiple serotypes of AAV exist in nature, and at least 15 wild-type serotypes have been identified in humans (i.e., AAV1-AAV15). Naturally occurring and variant serotypes are distinguished from other AAV serotypes by possessing serologically distinct protein capsids. Examples include AAV1, AAV2, AAV, AAV3 (including AAV3A and AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV12, AAVrhlO, AAVrh74 (see WO2016 / 210170), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, as well as recombinantly produced variants (e.g., capsid variants with insertions, deletions, and substitutions), such as the variants designated AAV2i8, NP4, NP22, NP66, DJ, DJ / 8, DJ / 9, LK3, RHM4-1, among others. For example, "primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, and "bovine AAV" refers to AAV that infects bovine mammals.

[0095] Serotype uniqueness is determined based on the lack of cross-reactivity between antibodies against one AAV compared to another. Such differences in cross-reactivity are usually due to differences in capsid protein sequences and antigenic determinants (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). However, some naturally occurring AAVs or artificial AAV variants (e.g., recombinant AAVs) may not exhibit serological differences from any of the currently known serotypes. These viruses may then be considered subgroups of the corresponding type, or more simply, variant AAVs. Therefore, as used herein, the term "serotype" refers to both serologically distinct viruses and viruses that are not serologically distinct but may be within a subgroup or variant of a given serotype.

[0096] A comprehensive list and alignment of the amino acid sequences of the capsids of known AAV serotypes is provided by Marsic et al. (2014) Molecular Therapy 22(11):1900-1909. The genomic sequences of various AAV serotypes, as well as the sequences of the native ITRs, rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Nos. NC_002077 (AAV1), AF063497 (AAV1), NC_001401 (AAV2), AF043303 (AAV2), NC_001729 (AAV3), NC_001863 (AAV3B), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), NC_001862 (AAV6), AF513851 (AAV7), AF513852 (AAV8), and NC_006261 (AAV8), the disclosures of which are incorporated herein by reference. For example, Srivistava et al. (1983) J. Virology 45:555, Chiorini et al. (1998) J. Virology 71:6823, Chiorini et al. (1999) J. Virology 73:1309, Bantel-Schaal et al. (1999) J. Virology 73:939, Xiao et al. al. (1999) J. Virology 73:3994, Muramatsu et al. (1996) Virology 221:208, Shade et al. (1986) J. Virol. 58:921, Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99:11854, Morris et al. (2004) Virology 33:375-383, International Patent Publication Nos. 00 / 28061, 99 / 61601, 98 / 11244, 2013 / 063379, 2014 / 194132, 2015 / 121501, and U.S. Patent Nos. 6,156,303 and 7,906,111.

[0097] As discussed herein, "recombinant adeno-associated virus" or "rAAV" is distinguished from wild-type AAV by replacing all or part of the endogenous viral genome with non-native sequences. The incorporation of non-native sequences into the virus defines the viral vector as a "recombinant" vector, and therefore an "rAAV vector." rAAV vectors can contain a heterologous polynucleotide encoding a desired RNA or protein or polypeptide (e.g., an RNA molecule disclosed herein). The recombinant vector sequence may be encapsidated or packaged into an AAV capsid and is referred to as an "rAAV vector," "rAAV vector particle," "rAAV viral particle," or simply "rAAV."

[0098] The present disclosure provides rAAV vectors containing polynucleotide sequences not of AAV origin (e.g., polynucleotides heterologous to AAV). The heterologous polynucleotide may be flanked by at least one, and optionally two, AAV terminal repeat sequences (e.g., inverted terminal repeats). The heterologous polynucleotide flanked by ITRs, also referred to herein as the "vector genome," typically encodes an RNA or polypeptide of interest, or a gene of interest, e.g., a target for therapeutic treatment. Delivery or administration of the rAAV vector to a subject (e.g., a patient) provides the encoded RNA / protein / peptide to the subject. Thus, rAAV vectors can be used to transcribe / deliver heterologous polynucleotides for expression, for example, to treat various diseases, disorders, and conditions.

[0099] rAAV vector genomes generally retain 145-base ITRs in cis relative to heterologous nucleic acid sequences that replace the viral rep and cap genes. Such ITRs are useful for producing recombinant AAV vectors, although modified AAV ITRs and non-AAV terminal repeats containing partially or completely synthetic sequences can also serve this purpose. The ITRs form hairpin structures and function, for example, as primers for host cell-mediated synthesis of complementary DNA strands after infection. ITRs also play a role in viral packaging, integration, and the like. ITRs are the only AAV viral elements required in cis for AAV genome replication and packaging into rAAV vectors. rAAV vector genomes contain two ITRs, typically located at the 5' and 3' ends of the vector genome, that optionally contain heterologous sequences (e.g., a transgene encoding a gene of interest, or a nucleic acid sequence of interest, including, but not limited to, antisense and siRNA, and CRISPR molecules, among many others). The 5' and 3' ITRs may both contain the same sequence, or each may contain a different sequence. The AAV ITRs can be derived from any AAV, including but not limited to serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, or any other AAV.

[0100] The rAAV vectors of the present disclosure may contain ITRs from an AAV serotype (e.g., wild-type AAV2, a fragment, or a variant thereof) that is different from the capsid serotype (e.g., AAV8, Olig001). Such rAAV vectors that contain at least one ITR from one serotype but a capsid from a different serotype may be referred to as hybrid viral vectors (see U.S. Patent No. 7,172,893). The AAV ITRs may include the entire wild-type ITR sequence or may be a variant, fragment, or modification thereof, while retaining functionality.

[0101] In some embodiments, the rAAV vector genome is linear, single-stranded, and flanked by AAV ITRs. Prior to transcription and translation of the heterologous gene, the free 3'-OH of one of the self-priming ITRs must be used by a DNA polymerase (e.g., a DNA polymerase within the transduced cell) to convert the approximately 4700 nucleotide single-stranded DNA genome into a double-stranded form and prime second-strand synthesis. In some embodiments, full-length single-stranded vector genomes (i.e., sense and antisense) anneal to generate full-length double-stranded vector genomes. This can occur when multiple rAAV vectors carrying genomes of opposite polarity (i.e., sense or antisense) simultaneously transduce the same cell. Regardless of how they are produced, once the double-stranded vector genomes are formed, the cell can transcribe and translate the double-stranded DNA and express the heterologous gene.

[0102] The efficiency of transgene expression from rAAV vectors can be hindered by the need to convert single-stranded rAAV genomes (ssAAV) to double-stranded DNA before expression. This step can be circumvented by using self-complementary AAV genomes (scAAV), which can package inverted repeat genomes that can fold into double-stranded DNA without the need for DNA synthesis or base pairing between multiple vector genomes. See, for example, U.S. Patent No. 8,784,799; McCarty, (2008) Molec. Therapy 16(10):1648-1656; McCarty et al., (2001) Gene Therapy 8:1248-1254; McCarty et al., (2003) Gene Therapy 10:2112-2118.

[0103] The viral capsid of the rAAV vector may be a wild-type AAV or a mutant AAV, such as AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAVrh74 (see WO2016 / 210170), AAV12, AAV2i8, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, RHM4-1 (WO2015 / 013313), RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, RHM15-6, AAV The AAV vector can be from hu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9,45, AAV2i8, AAV29G, AAV2,8G9, AVV-LK03, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, snake AAV, caprine AAV, shrimp AAV, ovine AAV, and variants thereof (see, e.g., Fields et al., VIROLOGY, volume 2, chapter 69(4)). th (ed., Lippincott-Raven Publishers). The capsid can be derived from several AAV serotypes disclosed in U.S. Pat. No. 7,906,111, Gao et al. (2004) J. Virol. 78:6381, Morris et al. (2004) Virol. 33:375, WO 2013 / 063379, WO 2014 / 194132, including the true AAV (AAV-TT) variant disclosed in WO 2015 / 121501, and RHM4-1, RHM15-1 through RHM15-6, and variants thereof, disclosed in WO 2015 / 013313. The full complement of AAV cap proteins includes VP1, VP2, and VP3. An ORF comprising a nucleotide sequence encoding an AAV VP capsid protein may contain less than the full complement of AAV Cap proteins, or the full complement of AAV Cap proteins may be provided.

[0104] In some embodiments, an rAAV vector comprising capsid proteins encoded by nucleotide sequences from two or more AAV serotypes (e.g., wild-type AAV serotype, variant AAV serotype) is referred to as a "chimeric vector" or "chimeric capsid" (see U.S. Pat. No. 6,491,907, the entire disclosure of which is incorporated herein by reference). In some embodiments, the chimeric capsid proteins are encoded by nucleic acid sequences from two, three, four, five, six, seven, eight, nine, ten, or more AAV serotypes. In some embodiments, the recombinant AAV vector comprises a capsid sequence derived from, for example, AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh74, AAVrh10, AAV2i8, or a variant thereof, resulting in a chimeric capsid protein comprising a combination of amino acids from any of the aforementioned AAV serotypes (see Rabinowitz et al. (2002) J. Virology 76(2):791-801). Alternatively, the chimeric capsid may comprise a VP1 from one serotype, a VP2 from a different serotype, a VP3 from yet another serotype, and a mixture of combinations thereof. For example, the chimeric viral capsid may comprise an AAV1 cap protein or subunit and at least one AAV2 cap protein or subunit. The chimeric capsid can include, for example, an AAV capsid having one or more B19 cap subunits, e.g., an AAV cap protein or subunit can be replaced with a B19 cap protein or subunit. For example, in one embodiment, the VP3 subunit of an AAV capsid can be replaced with a VP2 subunit of B19. In some embodiments, the chimeric capsid is an Olig001 capsid, as described in WO2021 / 221995 and WO2014 / 052789, which are incorporated herein by reference.

[0105] In some embodiments, the chimeric vector is engineered to exhibit altered tropism or tropism for a specific tissue or cell type. The term "tropism" refers to the preferential entry of a virus into a particular cell (e.g., oligodendrocyte) or tissue type and / or preferential interactions with the cell surface that facilitate entry into a particular cell or tissue type. AAV tropism is generally determined by the specific interactions between different viral capsid proteins and their cognate cellular receptors (Lykken et al. (2018) J. Neurodev. Disord. 10:16). Preferably, upon entry of the virus or viral vector into a cell, sequences (e.g., heterologous sequences such as transgenes) carried by the vector genome (e.g., rAAV vector genome) are expressed.

[0106] A "tropism profile" refers to the pattern of transduction of one or more target cells in various tissues and / or organs. For example, a chimeric AAV capsid may have a tropism profile characterized by efficient transduction of oligodendrocytes, astrocytes, or oligodendrocyte precursor cells with only low transduction of neurons and other CNS cells. See WO 2014 / 052789, incorporated herein by reference. Such chimeric capsids may be considered specific for oligodendrocytes, astrocytes, or glial precursor cells, exhibiting tropism for oligodendrocytes, astrocytes, or glial precursor cells, referred to herein as "glialtropism," and, when administered directly to the CNS, preferentially transduce oligodendrocytes, astrocytes, or oligodendrocyte precursor cells over neurons and other CNS cell types. In some embodiments, at least about 80% of the cells transduced with the oligodendrocyte or oligodendrocyte precursor cell-specific capsids are oligodendrocytes or oligodendrocyte precursor cells, e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the transduced cells are oligodendrocytes or oligodendrocyte precursor cells.

[0107] Gene Therapy and Cell Therapy The nucleic acids, gene constructs, expression cassettes, expression vectors, and cells described herein can be used for gene therapy treatment and / or prevention of diseases, disorders, or conditions. Specifically, by increasing expression of a transcription factor or its target, it can be used to treat or prevent diseases, disorders, or conditions associated with oligodendrocyte or myelin deficiency or dysfunction, as well as any other condition and / or disease in which increasing protein expression can provide a therapeutic benefit or improvement, e.g., a disease, disorder, or condition mediated by or associated with reduced protein level or function compared to that in otherwise healthy individuals.

[0108] As used herein, the terms myelin disorder, myelin disease, myelin-related disorder, myelin-related disease, myelin disorder, disorder mediated by myelin deficiency, and myelin disease are used interchangeably. These include conditions mediated by loss of white matter / oligodendrocytes / astrocytes, and any disease, condition (e.g., those resulting from traumatic spinal cord injury and cerebral infarction) or disorder associated with demyelination, insufficient myelination and remyelination, or hypomyelination in a subject. Such disorders may be hereditary, acquired, or both. They may result from myelination-related disorders or demyelination due to various neurotoxic insults. As used herein, "demyelination" refers to the demyelination process, or loss of the myelin sheath that insulates nerves, and is a hallmark of several neurodegenerative autoimmune diseases, including multiple sclerosis, transverse myelitis, chronic inflammatory demyelinating polyneuropathy, and Guillain-Barré syndrome. Leukodystrophies are caused by inherited enzyme deficiencies that result in the abnormal formation, destruction, and / or abnormal turnover of myelin sheaths within the CNS white matter. Both acquired and inherited myelin disorders have poor prognoses that lead to severe disability. Accordingly, some embodiments of the present disclosure may include methods for treating neurodegenerative autoimmune diseases in a subject. Remyelination of neurons requires oligodendrocytes. As used herein, the term "remyelination" refers to the regeneration of the myelin sheath of nerves by replacing or restoring the function of myelin-producing cells.

[0109] Myelin-related diseases or disorders that can be treated or ameliorated by the methods of the present disclosure include diseases, disorders, or injuries associated with hypomyelination or demyelination in brain cells, e.g., CNS neurons, of a subject. Such diseases include, but are not limited to, diseases and disorders in which the myelin sheath surrounding neurons is absent, incomplete, improperly formed, or deteriorated. Such diseases include, but are not limited to, multiple sclerosis (MS), neuromyelitis optica (NMO), progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelinolysis (CPM), adrenoleukodystrophy, Alexander disease, Pelizaeus-Merzbacher disease (PMD), Wallerian degeneration, optic neuritis, transverse osteomyelitis, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, post-radiation injury, neurological complications of chemotherapy, stroke, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, Bassen-Kornzweig syndrome, Marchiafava-Bignami disease, metachromatic leukodystrophy, trigeminal neuralgia, acute disseminated encephalitis, Guillain-Barré syndrome, Marie-Charcot-Tooth disease, and Bell's palsy.

[0110] Myelin-related diseases or disorders that can be treated or ameliorated by the methods of the present disclosure include diseases or disorders characterized by myelin deficiency. Insufficient myelination in the central nervous system is involved in a wide range of neurological disorders. Among these are forms of cerebral palsy, in which congenital deficits in forebrain myelination contribute to neurological morbidity in children with periventricular leukomalacia (Goldman et al., 2008). Goldman, SA, Schanz, S., and Windrem, MS (2008). Stem cell-based strategies for treating pediatric disorders of myelin. Hum Mol Genet. 17, pp. 76-83. At the other end of the age spectrum, myelin loss and ineffective repair may contribute to aging-related cognitive decline (Kohama et al., 2011). Kohama, SG, Rosene, DL, and Sherman, LS (2011) Age (Dordr). Age-related changes in human and non-human primate white matter: from myelination disturbances to cognitive decline. It is therefore contemplated that effective compositions and methods that enhance myelination and / or remyelination could have substantial therapeutic benefit in halting disease progression and restoring function in a wide range of myelin-related disorders.

[0111] Thus, one aspect of the present disclosure provides a method for treating a condition mediated by white matter loss, oligodendrocyte loss, or astrocyte loss. The method comprises administering to a subject in need thereof (a) a therapeutically effective amount of (i) a transcription factor selected from the group consisting of CEBPZ, CTCF, E2F1, MYC, NFYB, and ETV4, or (ii) an agent that increases the level or activity of a target of the transcription factor, or (b) a therapeutically effective amount of a cell or progeny thereof prepared according to the methods described herein. The target is selected from the group listed in Table 1 and Figure 5.

[0112] Such agents can include or be small molecule compounds, oligonucleotides, nucleic acids, gene constructs, peptides, polypeptides, CRISPR / Cas systems, or antibodies or their antigen-binding portions. Examples of agents include activators, agonists, or enhancers of the related CEBPZ, CTCF, E2F1, MYC, NFYB, and ETV4 signaling pathways. Various activators for this signaling pathway are known in the art.

[0113] In one embodiment, the agent comprises or is (i) a CEBPZ, CTCF, E2F1, MYC, NFYB, or ETV4 polypeptide or target thereof (e.g., those listed in Table 1 and Figure 5), or (ii) a nucleic acid, gene construct, or vector encoding the polypeptide.

[0114] In some embodiments, the agent may be or may comprise (i) a suppressor of a transcriptional repressor selected from the group consisting of E2F6, ZNF274, MAX, and IKZF3, or (ii) a nucleic acid, genetic construct, or vector encoding the suppressor.

[0115] In one embodiment, one or more genetic constructs may activate transcription of one of the genes described herein via CRISPR-Cas9-guided nucleases (Gimenez et al., "CRISPR-on System for the Activation of the Endogenous Human INS Gene," Gene Therapy 23:543-547 (2016); Wiedenheft et al., "RNA-Guided Genetic Silencing Systems in Bacteria and Archaea," Nature 482:331-338 (2012); Zhang et al., "Multiplex Genome Engineering Using CRISPR / Cas Systems," Science 339(6121):819-23 (2013); and Gaj et al., "ZFN, TALEN, and CRISPR / Cas-based Methods for Genome Engineering," Cell 31(7):397-405 (2013), which are incorporated herein by reference in their entireties). CRISPR-Cas9 is a genetic technology that allows sequence-specific control of gene expression in prokaryotic and eukaryotic cells by guided nuclease double-stranded DNA breaks. It is based on the CRISPR (clustered regularly interspaced palindromic repeats) pathway derived from the bacterial immune system.

[0116] In the embodiments described above, one or more gene constructs can be packaged into a suitable delivery vehicle or carrier for delivery to a subject. Suitable delivery vehicles include, but are not limited to, viruses, virus-like particles, bacteria, bacteriophages, biodegradable microspheres, microparticles, nanoparticles, exosomes, liposomes, collagen minipellets, and cochleates. These and other biological gene delivery vehicles are well known to those skilled in the art (see, e.g., Seow and Wood, "Biological Gene Delivery Vehicles: Beyond Viral Vectors," Mol. Therapy 17(5):767-777 (2009) (incorporated herein by reference in its entirety)).

[0117] In one embodiment, the gene construct is packaged into a therapeutic expression vector to facilitate delivery. Suitable expression vectors are well known in the art and include, but are not limited to, viral vectors such as adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, or herpes viral vectors.

[0118] The viral vector or other suitable expression vector contains a sequence encoding the genetic construct of the present application and any suitable promoter and / or enhancer for expressing the genetic construct. Suitable promoters include, but are not limited to, U6 or HI RNA pol III promoter sequences and cytomegalovirus promoters. The selection of other suitable promoters is within the skill of the art. The expression vector may also contain an inducible or regulatable promoter for expression of the inhibitory nucleic acid molecule in a tissue- or cell-specific manner.

[0119] Gene therapy vectors carrying therapeutic gene constructs or nucleic acid molecules are administered to a subject, for example, by intravenous injection, local administration (U.S. Patent No. 5,328,470 to Nabel et al., incorporated herein by reference in its entirety), or by stereotactic injection (see, e.g., Chen et al., "Gene Therapy for Brain Tumors: Regression of Experimental Gliomas by Adenovirus Mediated Gene Transfer In vivo," Proc. Nat'l. Acad. Sci. USA 91:3054-3057 (1994), incorporated herein by reference in its entirety). Pharmaceutical preparations of therapeutic vectors can include the therapeutic vector in an acceptable diluent, or can comprise a slow-release matrix in which the therapeutic delivery vehicle is imbedded. Alternatively, where the complete therapeutic delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the therapeutic delivery system. Gene therapy vectors typically utilize constitutive regulatory elements that are responsive to endogenous transcription factors.

[0120] Another suitable approach for delivery of the genetic constructs of the present disclosure involves the use of liposomal or nanoparticle delivery vehicles.

[0121] In another embodiment of the present application, the delivery vehicle is a nanoparticle. A variety of nanoparticle delivery vehicles are known in the art and are suitable for delivering the genetic constructs of the present application (see, e.g., van Vlerken et al., "Multi-functional Polymeric Nanoparticles for Tumor-Targeted Drug Delivery," Expert Opin. Drug Deliv. 3(2):205-216 (2006) (incorporated herein by reference in its entirety)).Suitable nanoparticles include poly(beta amino ester) (Sawicki et al., "Nanoparticle Delivery of Suicide DNA for Epithelial Ovarian Cancer Cell Therapy," Adv. Exp. Med. Biol. 622:209-219 (2008) (incorporated herein by reference in its entirety)), polyethyleneimine-alt-poly(ethylene glycol) copolymer (Park et al., "Degradable Polyethylenimine-alt-Poly(ethylene glycol) Copolymers As Novel Gene Carriers," J. Control Release 105(3):367-80 (2005) and Park et al., "Intratumoral Administration of Anti-KITENIN shRNA-Loaded PEI-alt-PEG Nanoparticles Suppressed Colon Carcinoma Established Subcutaneously in Mice," J. Nanosci. Nanotechnology 10(5):3280-3(2010) (incorporated herein by reference in its entirety), poly(d,l-lactide-coglycolide) (Chan et al., "Antisense Oligonucleotides: From Design to Therapeutic Application," Clin. Exp. Pharm. Physiol. 33:533-540(2006) (incorporated herein by reference in its entirety)), and liposome-entrapped siRNA nanoparticles (Kenny et al., "Novel Multifunctional Nanoparticle Mediates siRNA Tumor Delivery, Visualization and Therapeutic Tumor Reduction In vivo," J. Control Release 149(2):111-116(2011) (incorporated herein by reference in its entirety)).Other nanoparticle vehicles suitable for use in the present application include the microcapsule nanotube devices disclosed in U.S. Patent Publication No. 2010 / 0215724 to Prakash et al., which is incorporated herein by reference in its entirety.

[0122] In another embodiment, the gene construct is contained in a liposome delivery vehicle. The term "liposome" refers to a vesicle composed of a spherical bilayer or bilayer-arranged amphiphilic lipids. Liposomes are unilamellar or multilamellar vesicles with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes have the advantage of being able to fuse with the cell wall. Non-cationic liposomes cannot fuse efficiently with the cell wall but are taken up by macrophages in vivo.

[0123] Some advantages of liposomes include their biocompatibility and biodegradability, their ability to incorporate a wide range of water- and lipid-soluble drugs, and their protection of encapsulated molecules from metabolism and degradation. Important considerations in the preparation of liposomal formulations are the lipid surface charge, vesicle size, and the aqueous volume of the liposomes.

[0124] Liposomes are useful for transporting and delivering active ingredients to the site of action. Because liposome membranes are structurally similar to biological membranes, when liposomes are applied to tissues, they begin to combine with cell membranes, and as the combination of liposomes with cells progresses, the contents of the liposomes are released into the cells where the active agent can act.

[0125] Methods for preparing liposomes include those disclosed in Bangham et al., "Diffusion of Univalent Ions Across the Lamellae of Swollen Phospholipids," J. Mol. Biol. 13:238-52 (1965), U.S. Patent No. 5,653,996 to Hsu, U.S. Patent No. 5,643,599 to Lee et al., U.S. Patent No. 5,885,613 to Holland et al., U.S. Patent No. 5,631,237 to Dzau et al., and U.S. Patent No. 5,059,421 to Loughrey et al., which are incorporated by reference in their entireties.

[0126] As disclosed herein, in another embodiment, the genetic construct, expression cassette, or expression vector may be administered in association with a glial progenitor cell-targeting fusogen or glial progenitor cell-selective surface-binding moiety, e.g., the genetic construct, expression cassette, or expression vector may be present in or associated with a fusosome.

[0127] As used herein, "fusogen" refers to an agent or molecule that creates an interaction between two membrane-bound lumens. In embodiments, the fusogen facilitates membrane fusion. In other embodiments, the fusogen creates a connection, e.g., a pore, between two lumens (e.g., the lumen of a liposome and the cytoplasm of a target cell, or the lumen of a viral vector and the cytoplasm of a target cell). In some embodiments, the fusogen comprises a protein or a complex of two or more proteins having a targeting domain or binding moiety. In some examples, the targeting domain or binding moiety specifically targets or binds to a molecule on a glial progenitor cell or a glial progenitor cell. Examples of molecules include, but are not limited to, CD140a, NG2 / CSPG4, A2B5 ganglioside, O4 sulfatide, or CD133. The targeting domain or binding moiety can be a receptor ligand, peptide / polypeptide, antibody, or antigen-binding portion thereof that specifically binds to a molecule or marker on a glial progenitor cell or a glial progenitor cell. Non-limiting examples of human and non-human fusogens are described, for example, in US2021 / 0198698 and US2021 / 0137839, which are incorporated by reference in their entireties.

[0128] As used herein, "fusosome" refers to a bilayer of amphiphilic lipids that surrounds a lumen or cavity and a fusogen that interacts with the amphiphilic lipid bilayer. In some embodiments, fusosomes contain nucleic acids. In some embodiments, fusosomes are membrane-encapsulated preparations. In some embodiments, fusosomes are derived from a source cell.

[0129] Fusosomes can take a variety of forms. For example, in some embodiments, the fusosomes described herein are derived from source cells. Fusosomes can be or include, for example, extracellular vesicles, microvesicles, nanovesicles, exosomes, microparticles, or any combination thereof. In some embodiments, fusosomes are naturally released from source cells, and in some embodiments, the source cells are treated to enhance fusosome formation. In some embodiments, fusosomes are about 10-10,000 nm in diameter, e.g., about 30-100 nm in diameter. In some embodiments, fusosomes include one or more synthetic lipids.

[0130] In some embodiments, the fusosome is or comprises a virus, e.g., a retrovirus, e.g., a lentivirus. For example, in some embodiments, the amphiphilic lipid bilayer of the fusosome is or comprises a viral envelope. The viral envelope can comprise a fusogen, e.g., a fusogen endogenous to the virus, or a pseudotyped fusogen. In some embodiments, the lumen or cavity of the fusosome comprises viral nucleic acid, e.g., a retroviral nucleic acid, e.g., a lentiviral nucleic acid. The viral nucleic acid can be a viral genome. In some embodiments, the fusosome further comprises one or more viral nonstructural proteins, e.g., in its cavity or lumen.

[0131] Fusosomes can have a variety of structures or properties that facilitate delivery of a payload to a target cell. For example, in some embodiments, the fusosome and the source cell together contain sufficient nucleic acid to create a particle capable of fusing with a target cell. In embodiments, these nucleic acids encode proteins with one or more (e.g., all) of the following activities: gag polyprotein activity, polymerase activity, integrase activity, protease activity, and fusogenic activity.

[0132] In some embodiments, compositions of the present disclosure may be administered to subjects who do not have and / or are not suspected of having a myelin-related disorder to enhance or promote myelin-dependent processes.

[0133] In some embodiments, the compositions described herein can be administered to a subject to promote myelination of CNS neurons to enhance cognition, which is known to be a myelin-dependent process in cognitively healthy subjects. In certain embodiments, the compositions described herein can be administered in combination with a cognitive-enhancing (nootropic) agent. Exemplary agents include any drug, supplement, or other substance that improves cognitive function in healthy individuals, particularly executive function, memory, creativity, or motivation. Non-limiting examples include racetams (e.g., piracetam, oxiracetam, and aniracetam), dietary supplements (e.g., bacopa monnieri, panax ginseng, ginkgo biloba, and GABA), stimulants (e.g., amphetamine medications, methylphenidate, eugeroics, xanthines, and nicotine), L-theanine, tolcapone, levodopa, atomoxetine, and desipramine.

[0134] The overall dosage of a therapeutic agent (e.g., a protein, a polynucleotide encoding a protein, or a vector such as an rAAV vector or cells) will be therapeutically effective, depending on several factors, including the subject's overall health, the subject's disease state, the severity of the condition, the observed improvement, and the selected formulation and route of administration of the agent. Determining a therapeutically effective amount is within the ability of one skilled in the art. The exact formulation, route of administration, and dosage can be chosen by the individual physician, taking into account the subject's condition.

[0135] Cell Replacement Therapy Host cells containing the above-described gene constructs, cassettes, or expression vectors, or progeny cells of the host cells, are also within the scope of the present disclosure. The host cells may be stem cells or progenitor cells. Examples of stem cells include embryonic stem cells, ES-like stem cells, fetal stem cells, adult stem cells, pluripotent stem cells, induced pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells, etc.

[0136] In some embodiments, the host cells are glial progenitor cells, e.g., oligodendrocyte progenitor cells. The glial progenitor cells described herein can be derived from any suitable source of pluripotent stem cells, including, but not limited to, human induced pluripotent stem cells (iPSCs) and embryonic stem cells, as described in more detail below. In one example, the glial progenitor cells can be rejuvenated from the glial progenitor cells described herein or their progeny. The host cells or their progeny can be used as therapeutic cells or drugs to treat the disorders or conditions described herein.

[0137] One aspect of the present application relates to a method for reducing the adverse effects of oligodendrocyte loss, astrocyte loss, or white matter loss in the CNS (e.g., brain) of an adult subject. The loss can be age-related loss. The method includes identifying a subject, e.g., an adult subject, who suffers from the adverse effects of oligodendrocyte loss, astrocyte loss, or white matter loss in the CNS (e.g., brain), and providing a population of isolated glial progenitor cells. The isolated population of glial progenitor cells is then introduced into the CNS (e.g., brain and / or brainstem) of the selected subject to at least partially replace cells in the subject's brain in locations that suffer from the adverse effects of oligodendrocyte loss, astrocyte loss, or white matter loss.

[0138] Glial cells are a population of non-neuronal cells that provide support and nutrition, maintain homeostasis, either form myelin or promote myelination, and participate in signaling in the nervous system. As used herein, "glial cells" encompass fully differentiated cells of the glial lineage, such as oligodendrocytes or astrocytes, as well as glial progenitor cells. Glial progenitor cells are cells that have the potential to differentiate into cells of the glial lineage, such as oligodendrocytes and astrocytes. In some embodiments, to treat a subject in need thereof, the glial progenitor cells or rejuvenated cells are young glia or glial progenitor cells, or are younger than their counterparts in the subject to be treated.

[0139] As used herein, the term "young" glia or glial progenitor cells refers to cells induced to begin differentiation into glial progenitor cells in an in vitro environment (approximately 105 days after cell isolation from fetal donor tissue). In some embodiments, the term "young glial cells" refers to differentiated glial progenitor cells ready for transplantation into an animal (approximately 160 days after cell isolation from fetal donor tissue). In some embodiments, the term "young glial cells" refers to glial progenitor cells or their progeny within 1 to 20 weeks of transplantation. The term "older glial cells" is used in comparison to "young glial cells." Compared to older glial cells, young glial cells may have one or more of the following characteristics: (i) they grow or proliferate or divide more rapidly; (ii) they have lower levels of senescence-associated transcripts encoding CDKN1A (p21Cip1) and CDKN2 / p16 (INK4) and p14 (ARF) than older ones; and (iii) they have longer telomeres or higher telomerase activity or both.

[0140] In some embodiments, the older glial cells are glial cells derived from glial progenitor cells transplanted into a host for 5, 10, 20, 30, or 40 weeks. In some embodiments, the older glial cells are glial cells cultured from differentiated glial progenitor cells for an additional 5, 10, 20, 30, or 40 weeks (e.g., about 160 days from initial tissue harvest). In some embodiments, the older glial cells are glial cells cultured from induced differentiation for an additional 5, 10, 20, 30, or 40 weeks (e.g., about 105 days from initial tissue harvest).

[0141] The glial progenitor cells described herein can be derived from any suitable source of pluripotent stem cells, including iPSCs, which are pluripotent cells derived from non-pluripotent cells, such as somatic cells. For example, but not limited to, iPSCs can be derived from tissue, peripheral blood, umbilical cord blood, and bone marrow (see, e.g., Cai et al., J. Biol. Chem. 285(15):112227-11234 (2110); Giorgetti et al., Nat. Protocol. 5(4):811-820 (2010); Streckfuss-Bomeke et al., Eur. Heart J. doi:10.1093 / eurheartj / ehs203 (July 12, 2012); Hu et al., Blood doi:10.1182 / blood-2010-07-298331 (February 4, 2011); Sommer et al., J. Vis. Exp. 68:e4327 doi:10.3791 / 4327(2012), which are incorporated herein by reference in their entireties. Somatic cells can be reprogrammed to an embryonic stem cell-like state using genetic manipulation. Exemplary somatic cells suitable for forming iPSCs include fibroblasts (see, e.g., Streckfuss-Bomeke et al., Eur. Heart J. doi:10.1093 / eurheartj / ehs203(2012), which are incorporated herein by reference in their entireties), such as dermal fibroblasts obtained by skin sample or biopsy, synoviocytes from synovial tissue, keratinocytes, mature B cells, mature T cells, pancreatic beta cells, melanocytes, hepatocytes, foreskin cells, cheek cells, or lung fibroblasts.

[0142] Methods for producing induced pluripotent stem cells are known in the art and typically involve expressing a combination of reprogramming factors in somatic cells. Suitable reprogramming factors that promote and induce iPSC generation include one or more of Oct4, Klf4, Sox2, c-Myc, Nanog, C / EBPα, Esrrb, Lin28, and Nr5a2. In certain embodiments, at least two reprogramming factors are expressed in somatic cells to normally reprogram the somatic cells. In other embodiments, at least three reprogramming factors are expressed in somatic cells to normally reprogram the somatic cells.

[0143] iPSCs can be derived by methods known in the art, including using integrative viral vectors (e.g., lentiviral vectors, inducible lentiviral vectors, and retroviral vectors), excisable vectors (e.g., transposon and floxed lentiviral vectors), and non-integrative vectors (e.g., adenoviral and plasmid vectors) that deliver genes that promote cell reprogramming (e.g., Takahashi and Yamanaka, Cell 126:663-676 (2006); Okita et al., Nature 448:313-317 (2007); Nakagawa et al., Nat. Biotechnol. 26:101-106 (2007); Takahashi et al., Cell 131:1-12 (2007); Meissner et al., Nat. Biotech. 25:1177-1181 (2007); Yu et al., Science 318:1917-1920 (2007), Park et al. Nature 451:141-146 (2008), and U.S. Patent Application Publication No. 2008 / 0233610, which are incorporated by reference herein in their entireties.Other methods for generating iPS cells include those described in WO2007 / 069666, WO2009 / 006930, WO2009 / 006997, WO2009 / 007852, WO2008 / 118820, U.S. Patent Application Publication Nos. 2011 / 0200568 to Ikeda et al., 2010 / 0156778 to Egusa et al., 2012 / 0276070 to Musick, and 2012 / 0276636 to Nakagawa, Shi et al., Cell Stem Cell 3(5):568-574 (2008), Kim et al., Nature 454:646-650 (2008), Kim et al., Cell 136(3):411-419 (2009), Huangfu et al., Nat. Biotechnol. 26:1269-1275 (2008), Zhao et al., Cell Stem Cell 3:475-479 (2008), Feng et al., Nat. Cell Biol. 11:197-203 (2009), and Hanna et al. Cell 133(2):250-264 (2008), which are incorporated herein by reference in their entireties.

[0144] The above-described methods for generating iPSCs can be modified to include small molecules that enhance reprogramming efficiency or even replace reprogramming factors. These small molecules include, but are not limited to, epigenetic modulators such as the DNA methyltransferase inhibitor 5'-azacytidine, the histone deacetylase inhibitor VPA, and the G9a histone methyltransferase inhibitor BIX-01294 along with BayK8644, and L-type calcium channel agonists. Other small molecule reprogramming factors include those that target signal transduction pathways, such as TGF-β inhibitors and kinase inhibitors (e.g., kempaullone) (see review by Sommer and Mostoslavsky, Stem Cell Res. Ther. 1:26 doi:10.1186 / scrt26 (August 10, 2010) which is incorporated herein by reference in its entirety).

[0145] For methods of obtaining highly enriched preparations of glial progenitor cells from iPSCs suitable for generating the non-human mammalian models described herein, see WO2014 / 124087 by Goldman and Wang, and Wang et al., Cell Stem Cell 12(2):252-264 (2013), which are incorporated herein by reference in their entireties.

[0146] In another embodiment of the present application, the glial progenitor cells are derived from embryonic stem cells. Embryonic stem cells are derived from totipotent cells of early mammalian embryos and are capable of unlimited undifferentiated proliferation in vitro. As used herein, the term "embryonic stem cells" refers to cells isolated from an embryo, placenta, or umbilical cord, or immortalized versions of such cells, i.e., embryonic stem cell lines. Suitable embryonic stem cell lines include, but are not limited to, lines WA-01 (H1), WA-07, WA-09 (H9), WA-13, and WA-14 (H14) (see Thomson et al., Science 282(5391):1145-47 (1998) and U.S. Patent No. 7,029,913 to Thomson et al., which are incorporated herein by reference in their entireties). Other suitable embryonic stem cell lines include the HAD-C100 cell line (Tannenbaum et al., PLoS One 7(6):e35325 (2012), which is incorporated herein by reference in its entirety), the WIBR4, WIBR5, WIBR6 cell lines (Lengner et al., Cell 141(5):872-83 (2010), which is incorporated herein by reference in its entirety), and the human embryonic stem cell line (HUES) lines 1-17 (Cowan et al., N. Engl. J. Med. 350:1353-56 (2004), which is incorporated herein by reference in its entirety).

[0147] Human embryonic stem cells provide a virtually unlimited source of cloned / genetically modified cells potentially useful for tissue replacement therapy. Methods for obtaining highly enriched preparations of glial progenitor cells from embryonic cells suitable for generating the non-human mammalian models of the present disclosure are described herein as disclosed in Wang et al., Cell Stem Cell 12:252-264 (2013), which is incorporated herein by reference in its entirety.

[0148] Briefly, glial progenitor cells are derived from pluripotent cell populations, i.e., iPSCs or embryonic stem cells, using a protocol that directs pluripotent cells through successive stages of neural and glial progenitor differentiation. Each stage of lineage restriction is characterized and identified by the expression of certain cellular proteins. Step 1 of this process involves culturing the pluripotent cell population under conditions effective to induce embryoid body formation. As described herein, the pluripotent cell population can be maintained in embryonic stem cell (ESC) medium (e.g., DMEM / F12 containing appropriate serum replacement and bFGF) in co-culture with other cells, such as embryonic fibroblasts. Pluripotent cells are passaged before reaching 100% confluence, e.g., 80% confluence, when colonies are approximately 250-300 μm in diameter. The pluripotent state of the cells is easily assessed using markers for SSEA4, TRA-1-60, OCT-4, NANOG, and / or SOX2.

[0149] To generate embryoid bodies (EBs), complex three-dimensional cellular aggregates of pluripotent stem cells (Stage 2), pluripotent cell cultures are dissociated when they achieve approximately 80% confluence with colony diameters of 250-300 µm or approximately 250-300 µm. EBs are first cultured in suspension in ESC medium without bFGF and then switched to neural induction medium supplemented with bFGF and heparin. To induce neuroepithelial differentiation (Stage 3), EBs are plated and cultured in neural induction medium supplemented with bFGF, heparin, and laminin and then switched to neural induction medium supplemented with retinoic acid. Neuroepithelial differentiation is assessed by coexpression of PAX6 and SOX1, which characterize central neural stem and progenitor cells.

[0150] To induce differentiation of pre-oligodendrocyte precursor cells ("pre-OPC"), neuroepithelial cell colonies can be cultured in the presence of additional factors, including retinoic acid, B27 supplement, and sonic hedgehog (shh) agonists (e.g., palmofamine). The emergence of pre-OPC colonies is assessed by the presence of OLIG2 and / or NKX2.2 expression. While both OLIG2 and NKX2.2 are expressed by central oligodendrocyte precursor cells, NKX2.2 is a more specific indicator of oligodendrocyte differentiation. Thus, the early pre-oligodendrocyte precursor cell stage is characterized by OLIG2 expression. + / NKX2.2 - Characterized by cell colonies. + / NKX2.2 - Early pre-OPCs are transformed into late OLIGs by replacing retinoic acid with bFGF. + / NKX2.2 + At the end of stage 5, a significant proportion of cells express OLIG2 + / NKX2.2 + Pre-OPCs, as indicated by their expression profiles.

[0151] Pre-OPCs can be further differentiated into bipotential glial progenitor cells by culturing them in glial induction medium supplemented with growth factors such as triiodothyronine (T3), neurotrophin 3 (NT3), insulin-like growth factor (IGF-1), and platelet-derived growth factor-AA (PDGF-AA) (stage 6). These culture conditions can be extended for 3-4 months or longer, if desired, to maximize the production of myelinogenic glial progenitor cells. Cell preparations suitable for transplantation into appropriate subjects express PDGFRα. + Identified as containing glial precursor cells.

[0152] The population of glial progenitor cells used in performing the methods of the present application can comprise at least about 80% glial progenitor cells, including, for example, about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% glial cells. The selected preparation of glial progenitor cells can be relatively devoid of other cell types, such as neurons and neuronal progenitor cells (e.g., containing less than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%). Optionally, the cell population can be a substantially pure population of glial progenitor cells.

[0153] The subject treated according to the method of the present application may be an adult suffering from age-related white matter / oligodendrocyte / astrocytosis loss in the brain. The method alleviates the adverse effects of this condition, which may occur as part of the normal aging process.

[0154] As used herein, "treating" or "treatment" refers to any indicator of success in ameliorating an injury, pathology, or condition, including any objective or subjective parameter, for example, relief; remission; a decrease in symptoms or making the injury, pathology, or condition more tolerable to the patient; a slowing or reduction in the rate of degeneration; making the end point of degeneration less debilitating; or an improvement in the physical or mental health of the subject. Treatment or amelioration of symptoms may be based on objective or subjective parameters, including the results of a physical examination, neurological examination, and / or psychiatric evaluation.

[0155] "Treating" can include administration of glial progenitor cells and / or other agents to prevent or delay the onset, alleviate, or arrest or inhibit the onset of symptoms or conditions associated with a disease, condition, or disorder. A "therapeutic effect" refers to the reduction, elimination, or prevention of a disease, disease symptom, or side effect of a disease, condition, or disorder in a subject. Treatment can be prophylactic (to prevent or delay the onset or worsening of a disease, condition, or disorder, or to prevent the manifestation of clinical or subclinical symptoms thereof), or therapeutic suppression or alleviation of symptoms after the manifestation of a disease, condition, or disorder.

[0156] One condition resulting from age-related white matter loss, oligodendrocyte loss, or astrocyte loss in the brain that can be treated by the methods of the present application is subcortical dementia.

[0157] As used herein, the term "white matter" relates to components of the central nervous system in the brain and superficial spinal cord, which are largely composed of glial cells and myelinated axons that transmit signals from one region of the cerebrum to another, and between the cerebrum and lower brain centers.

[0158] Glial progenitor cells can be introduced into a subject in need of alleviation of adverse effects by a variety of known techniques, including, but not limited to, injection, deposition, and transplantation techniques described herein.

[0159] In one embodiment, glial progenitor cells can be transplanted bilaterally into multiple sites in a subject, as described in U.S. Patent No. 7,524,491 to Goldman, Windrem et al., Cell Stem Cell 2:553-565 (2008), Han et al., Cell Stem Cell 12:342-353 (2013), and Wang et al., Cell Stem Cell 12:252-264 (2013), which are incorporated by reference in their entireties. Methods for transplanting neural tissue and cells into a host brain are described by Bjorklund and Stenevi (eds), Neural Grafting in the Mammalian CNS, Ch. 3-8, Elsevier, Amsterdam (1985), U.S. Patent No. 5,082,670 to Gage et al., and U.S. Patent No. 6,497,872 to Weiss et al., which are incorporated by reference in their entireties. Typical procedures include intraparenchymal, intracallosal, intraventricular, intrathecal, and intravenous implantation.

[0160] Intraparenchymal transplantation can be achieved by injecting or depositing tissue within the host brain so that it is apposed to the brain parenchyma at the time of transplantation. The two main procedures for intraparenchymal transplantation are as follows: (1) injecting donor cells into the host brain parenchyma, or (2) preparing a cavity by surgical means to expose the host brain parenchyma and then depositing the graft into the cavity (Bjorklund and Stenevi (eds), Neural Grafting in the Mammalian CNS, Ch. 3, Elsevier, Amsterdam (1985)), the entire contents of which are incorporated herein by reference). Both methods provide parenchymal apposition between the donor cells and the host brain tissue at the time of transplantation, and both promote anatomical integration between the graft and the host brain tissue. This is important if the donor cells are to become an integral part of the host brain and survive throughout the host's lifetime.

[0161] Glial progenitor cells can also be delivered into the corpus callosum, as described in U.S. Patent Application Publication No. 2003 / 0223972 to Goldman, which is incorporated herein by reference in its entirety. Glial progenitor cells can also be delivered directly to the subcortical forebrain, specifically to the anterior and posterior anlage of the corpus callosum. Glial progenitor cells can also be delivered to the cerebellar peduncle white matter to gain access to the main cerebellum and brainstem. Glial progenitor cells can also be delivered to the spinal cord.

[0162] Alternatively, cells may be placed in a ventricle, such as a cerebral ventricle. Transplanting cells into the ventricle can be achieved by injecting donor cells or by growing the cells in a matrix, such as 30% collagen, to form a solid tissue plug that can be subsequently implanted into the ventricle to prevent dislocation of the transplanted cells. For subdural implantation, cells may be injected around the surface of the brain after making a slit in the dura.

[0163] Suitable techniques for cell delivery are described above. In one embodiment, the preparation of glial progenitor cells is administered to the striatum, forebrain, brainstem, and / or cerebellum of a subject.

[0164] Delivery of cells to a subject can involve either a single-step or multiple-step injection directly into the nervous system. For focal disorders such as demyelination of the optic nerve, a single injection can be used. Adult and fetal oligodendrocyte precursor cells are widely distributed within the brain of transplant recipients, but for widespread disorders, multiple injection sites can be used to optimize treatment. Injections are optionally directed to regions of the central nervous system, such as white matter tracts like the corpus callosum (e.g., anterior and posterior anlage), dorsal columns, cerebellar peduncles, cerebral peduncles, etc. Such injections can be performed unilaterally or bilaterally using precise localization methods such as stereotaxic surgery, optionally with accompanying imaging methods (e.g., high-resolution MRI imaging). Those skilled in the art will recognize that brain regions vary by species; however, they will also recognize equivalent brain regions across mammalian species.

[0165] The cell transplant can be optionally injected as dissociated cells, or can be provided by localized placement of undissociated cells. In either case, the cell transplant optionally includes an acceptable solution. Such acceptable solutions include solutions that avoid undesired biological activity and contamination. Suitable solutions include an appropriate amount of pharmaceutically acceptable salts to make the formulation isotonic. Examples of pharmaceutically acceptable solutions include, but are not limited to, saline, Ringer's solution, dextrose solution, and culture medium. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5.

[0166] The injection of the dissociated cell transplant can be a streaming injection across the inlet, outlet, or both inlet and outlet pathways of an injection device (e.g., a cannula, needle, or tube). Automation can be used to provide uniform inlet and outlet rates and injection rates and volumes.

[0167] The number of glial progenitor cells administered to a subject may range from approximately 10 to 100 cells per administration (e.g., injection site), depending on the size and species of the recipient and the volume of tissue requiring cell replacement.2 ~10 8 A single administration (e.g., injection) dose can range from 10 to 20 mg / kg for a transplant recipient patient. 3 ~10 5 , 10 4 ~10 7 , and 10 5 ~10 8 The number of cells can range from 1 to 10, or any number in total.

[0168] Because the CNS is an immunologically privileged site, administered cells, including xenogeneic cells, can survive, and optionally, immunosuppressants or typical immunosuppressant regimens are not used in the treatment method. However, optionally, immunosuppressants may also be administered to the subject. Immunosuppressants and their administration regimens are known to those skilled in the art and include drugs such as azathioprine, azathioprine sodium, cyclosporine, daltroban, gusperimus trihydrochloride, sirolimus, and tacrolimus. The dose range and duration of the regimen may vary depending on the disorder being treated, the degree of rejection, the activity of the specific immunosuppressant used, the subject's age, weight, general health, sex, and diet, the time of administration, the route of administration, the excretion rate of the specific immunosuppressant used, the duration and frequency of treatment, and concomitant medications. Those skilled in the art can determine the acceptable dose and duration of immunosuppression. The administration regimen can be adjusted by an individual physician in the event of contraindications or changes in the subject's condition.

[0169] In one embodiment, one or more immunosuppressive agents can be administered to a subject beginning 10 weeks prior to administration of the cells. In one embodiment, one or more immunosuppressive agents are administered to a subject beginning 9, 8, 7, 6, 5, 4, 3, 2, 1, 7, 6, 5, 4, 3, 2, 1, 24 hours prior to administration of the cells. In one embodiment, one or more immunosuppressive agents are administered to a subject beginning on the day of administration of the cells and continuing for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration. In one embodiment, one or more immunosuppressive agents are administered to a subject for more than one year after administration.

[0170] Suitable subjects for treatment with the methods described herein include any mammalian subject suffering from age-related white matter loss. Exemplary mammalian subjects include humans, mice, rats, guinea pigs and other small rodents, dogs, cats, sheep, goats, and monkeys. In one embodiment, the subject is a human.

[0171] The above-described rejuvenation therapy and cell therapy can be used in combination. For example, the above-described nucleic acid molecules, inhibitory molecules, CRISPR / Cas systems, expression cassettes, or expression vectors can be used as therapeutic reagents in ex vivo applications. To achieve this goal, the reagents can be introduced into tissues or cells that are then transplanted into a subject for therapeutic effect. The cells and / or tissues can be derived from the organism or subject that will subsequently receive the explant (e.g., allogeneic or autologous), or can be derived from another organism or subject (e.g., a relative, sibling, or HLA-matched donor) before transplantation (e.g., heterologous, xenogenic, allogeneic, or allogeneic). The reagents can be used to regulate the expression of one or more genes in cells or tissues so that the cells or tissues acquire a desired phenotype or function when transplanted in vivo. In one embodiment, certain target cells from a patient are extracted or isolated. These isolated cells are contacted with a reagent that targets a specific nucleotide sequence within the cells under conditions suitable for uptake of the reagent by the cells (e.g., using a delivery reagent such as a cationic lipid, liposome, or using a technique such as electroporation to facilitate delivery of the reagent to the cells), and the cells are then reintroduced into the same or another patient.

[0172] For therapeutic use, a pharmaceutically effective dose of a therapeutic reagent or pharmaceutical composition can be administered to a subject. A pharmaceutically effective dose is that required to prevent, inhibit, or treat (alleviate some symptoms, preferably all symptoms) the development of a disease state. One skilled in the art can easily determine the therapeutically effective dose of a reagent to be administered to a given subject by considering factors such as the subject's size and weight, the extent of disease progression or penetration, the subject's age, health, and sex, the route of administration, and whether the administration is local or systemic. Generally, an amount of 0.1 mg / kg to 100 mg / kg body weight / day of the active ingredient is administered, depending on the potency of the negatively charged polymer. The therapeutic reagent or pharmaceutical composition can be administered in a single dose or multiple doses.

[0173] In certain embodiments, the cell, protein, or nucleotide compositions described herein increase the amount of one or more myelin proteins (e.g., MBP, MAG, MOG, MOBP, PLP1, GPR37, ASPA, CNP, MYRF, BCAS1, PLP1, UGT8, TF, LPAR1, and FA2H) by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 8 The compound may be administered in an amount effective to enhance myelin production in the CNS of a subject by increasing myelin production by 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000%.

[0174] In other embodiments, the composition may be administered in an amount effective to promote survival of CNS neurons in a subject by increasing the number of surviving neurons by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% compared to untreated CNS neurons or the number of surviving neurons in the subject.

[0175] Another strategy for treating a subject suffering from a myelin-related disorder is to administer a therapeutically effective amount of a cell or nucleotide composition described herein together with a therapeutically effective amount of an oligodendrocyte differentiation and / or proliferation inducer and / or an anti-neurodegenerative disease agent. Examples of anti-neurodegenerative disease agents include L-dopa, cholinesterase inhibitors, anticholinergics, dopamine agonists, steroids, and immunomodulators, including interferons, monoclonal antibodies, and glatiramer acetate. Thus, in a further aspect of the present disclosure, the compositions described herein can be administered as part of a combination therapy with an adjunctive therapy for treating neurodegenerative and myelin-related disorders.

[0176] The phrase "combination therapy" encompasses the administration of an oligodendrocyte precursor differentiation-inducing composition described herein and a therapeutic agent as part of a specific treatment regimen intended to provide a beneficial effect from the combined action of these therapeutic agents. When administered in combination, the oligodendrocyte precursor differentiation-inducing compound and the therapeutic agent can be formulated as separate compositions. The administration of these combined therapeutic agents typically occurs over a defined period of time (usually minutes, hours, days, or weeks, depending on the combination selected).

[0177] The genetic nucleic acids, genetic constructs, expression cassettes, expression vectors, and cells of the present application may be administered by intracerebral delivery, intrathecal delivery, intranasal delivery, or via direct injection into the ventricles of the brain.

[0178] Pharmaceutical Composition The present disclosure provides pharmaceutical compositions or medicaments for preventing or treating inherited or acquired disorders of myelin. In some embodiments, the pharmaceutical compositions comprise one or more of the above protein molecules, polynucleotides, expression cassettes, expression vectors (e.g., viral vector genomes, expression vectors, rAAV vectors), systems (e.g., CRISPR / Cas systems, or nucleic acids encoding components of the systems), and host cells or their progeny.

[0179] The pharmaceutical composition further comprises a pharmaceutically acceptable carrier, adjuvant, diluent, excipient, and / or other agent. A pharmaceutically acceptable carrier, adjuvant, diluent, excipient, or other agent is not biologically or otherwise undesirable, e.g., the material can be administered to a subject without causing undesirable biological effects that outweigh the beneficial biological effects of the material. Any suitable pharmaceutically acceptable carrier or excipient can be used to prepare a pharmaceutical composition according to the present disclosure (see, e.g., Remington The Science and Practice of Pharmacy, Adeboye Adejare (Editor), Academic Press, November 2020).

[0180] Pharmaceutical compositions are typically sterile, pyrogen-free, and stable under the conditions of manufacture and storage. Pharmaceutical compositions may be formulated as a solution (e.g., water, saline, dextrose solution, buffer solution, or other pharmaceutically sterile fluid), microemulsion, liposome, or other ordered structure suitable for accommodating high product (e.g., viral vector particle, microparticle, or nanoparticle) concentrations.

[0181] In some embodiments, pharmaceutical compositions comprising the above-described proteins, polynucleotides, expression cassettes, expression vectors, vector genomes, cells, or rAAV vectors of this disclosure are formulated in water or buffered saline. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In some embodiments, it may be preferable to include isotonicity agents, such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, such as monostearate salts and gelatin. In some embodiments, the nucleic acids, vectors, and / or host cells of this disclosure may be administered in compositions containing controlled-release formulations, such as slow-release polymers or other carriers that protect the product against rapid release, including implants and microencapsulated delivery systems.

[0182] In some embodiments, the pharmaceutical compositions of the present disclosure are parenteral pharmaceutical compositions, including compositions suitable for intravenous, intraarterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intraarticular, intraparenchymal (IP), intrathecal (IT), intracerebroventricular (ICV), and / or intracisternal (ICM) administration. In some embodiments, the pharmaceutical compositions of the present disclosure are formulated for administration by ICV injection. In some embodiments, the vector (e.g., a viral vector such as AAV) may be formulated in 350 mM NaCl and 5% D-sorbitol in PBS.

[0183] Administration The above-described molecules, or polynucleotides, or vectors (e.g., vector genomes, rAAV vectors), or systems (e.g., CRISPR / Cas systems, or nucleic acids encoding components of the systems), or cells may be administered to a subject (e.g., a patient) or target cells to treat the subject. The administration of the vector to a human subject or animal in need thereof can be carried out by any means known in the art for administering vectors. Examples of target cells include cells of the CNS, preferably oligodendrocytes, astrocytes, or their precursor cells.

[0184] The vectors can be administered in addition to and as an adjunct to standard care treatments. That is, the vectors can be co-administered with another agent, compound, drug, treatment, or treatment regimen at the same time, contemporaneously, or at predetermined dosing intervals, as determined by one of skill in the art using routine methods. The uses disclosed herein include administration of the rAAV vectors of the present disclosure according to dosing schedules in addition to and / or concurrently with standard care for the disease known in the art.

[0185] In some embodiments, the combination composition comprises one or more immunosuppressants. In some embodiments, the combination composition comprises an rAAV vector comprising a transgene (e.g., a polynucleotide encoding an RNA molecule disclosed herein) and one or more immunosuppressants. In some embodiments, the method comprises administering or delivering to a subject an rAAV vector comprising the transgene, and administering to the subject an immunosuppressant prophylactically prior to administration of the vector or after administration of the vector (i.e., before or after the vector and / or protein are provided and thereby symptoms of a response thereto become apparent).

[0186] In one embodiment, a vector (e.g., an rAAV vector) of the present disclosure is administered systemically. Exemplary methods of systemic administration include, but are not limited to, intravenous (e.g., portal vein), intra-arterial (e.g., femoral artery, hepatic artery), intravascular, subcutaneous, intradermal, intraperitoneal, transmucosal, intrapulmonary, intralymphatic, and intramuscular administration, as well as direct tissue or organ injection. One skilled in the art will appreciate that systemic administration can deliver nucleic acids to all tissues. In some embodiments, direct tissue or organ administration includes administration to areas directly affected by oligodendrocyte deficiency (e.g., the brain and / or central nervous system). In some embodiments, vectors of the present disclosure and pharmaceutical compositions thereof are administered to the brain parenchyma (i.e., by intraparenchymal administration), the spinal canal or subarachnoid space to reach the cerebrospinal fluid (CSF) (i.e., by intrathecal administration), the ventricles of the brain (i.e., by intraventricular administration), and / or the cisterna magna of the brain (i.e., by intracisternal administration).

[0187] Thus, in some embodiments, vectors of the present disclosure are administered by direct injection into the brain (e.g., into the parenchyma, ventricle, cisterna magna, etc.) and / or CSF (e.g., into the spinal canal or subarachnoid space) to treat myelin disorders. Target cells of vectors of the present disclosure include cells located in the cortex, subcortical white matter of the corpus callosum, striatum, and / or cerebellum. In some embodiments, target cells of vectors of the present disclosure are oligodendrocytes or their precursor cells. Additional routes of administration may also include local application of vectors under direct visualization, e.g., superficial cortical application, or other stereotaxic application.

[0188] In some embodiments, the vectors of the present disclosure are administered by at least two routes. For example, the vector is administered systemically and also directly to the brain. When administered via at least two routes, the administration of the vectors can be, but need not be, simultaneous or contemporaneous. Alternatively, the administration via different routes can be separate, with a time interval between each administration.

[0189] The above-described proteins, or polynucleotides encoding the proteins, or vector genomes, or vectors containing the polynucleotides (e.g., rAAV vectors) can be used for ex vivo transduction of cells or for direct administration to a subject (e.g., direct administration to the CNS of a patient with a disease). In some embodiments, the transduced cells (e.g., host cells) are administered to a subject to treat or prevent a disease, disorder, or condition (e.g., cell therapy for a disease). For example, rAAV vectors containing therapeutic nucleic acids (e.g., encoding proteins) can be administered in a biologically effective amount, preferably to oligodendrocytes, astrocytes, or their precursor cells.

[0190] The dosage of the vector depends, for example, on the mode of administration, the disease or condition being treated, the stage and / or aggressiveness of the disease, the condition of the individual subject (age, sex, weight, etc.), the particular viral vector, the stability of the expressed protein, the host immune response to the vector, and / or the gene being delivered. Generally, the dose should be at least 1 x 10 per kg of subject body weight to achieve a therapeutic effect. 8 or more, e.g., 1 x 10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , or more vector genomes (vg).

[0191] In some embodiments, a polynucleotide encoding a protein described herein can be administered as a component of a DNA molecule (e.g., a recombinant nucleic acid) having appropriate regulatory elements (e.g., a promoter) for expression in target cells (e.g., oligodendrocytes, astrocytes, or their precursor cells). The polynucleotide can be administered as a component of a plasmid or viral vector, e.g., an rAAV vector. The rAAV vector can be administered in vivo by delivering the vector directly to a patient in need of treatment (e.g., directly to the CNS). The rAAV vector can also be administered ex vivo to a patient by in vitro administration of the vector to cells from a donor patient in need of treatment, followed by reintroduction of the transduced cells into the donor (e.g., cell therapy).

[0192] kit The present disclosure provides kits comprising packaging materials and one or more of the components described therein. The kits typically include a label or insert containing a description of the components or instructions for in vitro, in vivo, or ex vivo use of the components therein. The kits may contain a collection of such components, such as the polynucleotides, nucleic acids, expression cassettes, expression vectors (e.g., viral vector genomes, expression vectors, rAAV vectors), and cells described above, and optionally a second active agent, such as a compound, therapeutic agent, drug, or composition.

[0193] A kit refers to a physical structure containing one or more components of the kit. The packaging material can maintain the components sterilized and can be made of materials commonly used for such purposes (e.g., paper, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0194] The label or package insert may include identification of one or more components therein, dosage, mechanism of action, pharmacokinetics, and pharmacodynamics of the active ingredients. The label or package insert may include information identifying manufacture, lot number, location and date of manufacture, expiration date. The label or package insert may include information regarding the disease for which the kit components may be used (e.g., inherited, acquired, or age-related disorders of myelin, such as HD). The label or package insert may include instructions for a clinician or subject for using one or more of the kit components in a method, use, or treatment protocol or regimen. The instructions may include dosage, frequency of duration, and instructions for practicing any of the methods, uses, treatment protocols, or prophylactic or therapeutic regimens described herein.

[0195] The label or package insert may include information about potential side effects, complications or reactions, for example, warnings to the subject or clinician about situations in which it is not appropriate to use a particular composition.

[0196] definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the disclosure. As used in the description of this disclosure and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. The following terms have the following meanings:

[0197] As used herein, the term "about" or "approximately" refers to a measurable value such as the amount of biological activity, polynucleotide or polypeptide sequence homology or length, dosage, time, temperature, etc., and is intended to encompass a variation of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or even 0.1% in either direction of the specified amount (greater than or less than), unless otherwise specified, apparent from the context, or unless such number exceeds 100% of the possible value.

[0198] The term "transgene" refers to a heterologous polynucleotide that can be introduced into a cell, transcribed into RNA, and optionally translated and / or expressed under appropriate conditions. In an aspect, it confers a desired characteristic on the cell into which it is introduced, or otherwise produces a desired therapeutic or diagnostic result. In another aspect, it can be transcribed into a molecule that mediates RNA interference, such as miRNA, siRNA, or shRNA.

[0199] As used herein, the terms "homologous" or "homology" refer to two or more reference entities (e.g., nucleic acid or polypeptide sequences) that share at least partial identity over a given region or portion. For example, if an amino acid position in two peptides is occupied by the same amino acid, the peptides are homologous at that position. In particular, homologous peptides retain an activity or function associated with the unmodified or reference peptide, and modified peptides generally have an amino acid sequence that is "substantially homologous" to the amino acid sequence of the unmodified sequence. When referring to a polypeptide, nucleic acid, or fragment thereof, "substantial homology" or "substantial similarity" means that there is at least about 70% to 99% sequence identity between two sequences when optimally aligned with another polypeptide, nucleic acid (or its complementary strand), or fragment thereof, with appropriate insertions or deletions. The degree of homology (identity) between two sequences can be ascertained using computer programs or mathematical algorithms known in the art. Such algorithms that calculate percent sequence homology (or identity) generally account for sequence gaps and mismatches over the region or area of ​​comparison.

[0200] Nucleic acid or polynucleotide refers to a DNA molecule (e.g., cDNA or genomic DNA), an RNA molecule (e.g., mRNA), or a DNA or RNA analog. DNA or RNA analogs can be synthesized from nucleotide analogs. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA.

[0201] An isolated or recombinant nucleic acid refers to a nucleic acid whose structure is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid. Thus, the term encompasses, for example, (a) DNA that has the sequence of a portion of a naturally occurring genomic DNA molecule but is not flanked by both sequences that flank that portion of the molecule in the genome of the naturally occurring organism; (b) nucleic acid that is incorporated into a vector or into the genomic DNA of a prokaryotic or eukaryotic organism in such a way that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) separate molecules such as cDNA, genomic fragments, fragments produced by polymerase chain reaction (PCR), or restriction fragments; and (d) recombinant nucleotide sequences that are part of a hybrid gene, i.e., a gene encoding a fusion protein. The nucleic acids described above can be used to express the proteins of the present disclosure. To this end, the nucleic acids can be operably linked to appropriate regulatory sequences to generate expression vectors.

[0202] A "recombinant nucleic acid" is a combination of nucleic acid sequences that are joined together using recombinant techniques and procedures used to join nucleic acid sequences together.

[0203] The terms "heterologous" DNA molecule and "heterologous" nucleic acid, as used herein, refer to molecules that are derived from a source foreign to a particular host cell, or, if derived from the same source, that have been modified from their original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but that has been modified, for example, through the use of shuffling or recombination. When used to describe two nucleic acid segments, these terms mean that the two nucleic acid segments are not from the same gene, or, if they form the same gene, one or both of them have been modified from their original form. These terms also include non-naturally occurring multiple copies of a naturally occurring DNA molecule. Thus, these terms refer to a nucleic acid segment that is foreign or heterologous to the cell, or that is homologous to the cell but is located in a location within the host cell nucleic acid where the element is not normally found. An exogenous DNA segment is expressed to produce an exogenous RNA or polypeptide. A "homologous DNA molecule" is a DNA molecule that is naturally associated with the host cell into which it is introduced.

[0204] "Regulatory sequences" include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include sequences that direct constitutive expression of nucleotide sequences, as well as tissue-specific regulatory sequences and / or inducible sequences. The design of an expression vector may depend on factors such as the choice of host cell to be transformed and the level of expression of the desired protein or RNA. The expression vector can be introduced into host cells to produce the desired RNA or polypeptide. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one that initiates RNA at a high frequency.

[0205] A "promoter" is a nucleotide sequence that initiates and regulates transcription of a polynucleotide. Promoters can include inducible promoters (expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. The terms "promoter" or "control element" are intended to include full-length promoter regions and functional (e.g., transcription or translation controlling) segments of these regions.

[0206] "Operably linked" refers to an arrangement of elements such that the components so described are configured to perform their normal functions. Thus, a given promoter operably linked to a nucleic acid sequence is capable of affecting the expression of that sequence when the proper enzymes are present. The promoter need not be contiguous with the sequence, so long as it functions to direct its expression. Thus, for example, intervening untranslated but transcribed sequences can be present between the promoter sequence and the nucleic acid sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence. Thus, the term "operably linked" is intended to encompass any spacing or orientation of the promoter element and DNA sequence of interest that allows initiation of transcription of the DNA sequence of interest upon recognition of the promoter element by a transcription complex.

[0207] As used herein, the terms "gene construct" or "nucleic acid construct" refer to a non-naturally occurring nucleic acid molecule resulting from the use of recombinant DNA technology (e.g., recombinant nucleic acid). A gene or nucleic acid construct is a nucleic acid molecule, either single-stranded or double-stranded, that has been modified to contain segments of nucleic acid sequences combined and arranged in a manner not found in nature. A nucleic acid construct may be a "cassette" or "vector" (e.g., a plasmid, rAAV vector genome, expression vector, etc.), i.e., a nucleic acid molecule designed to deliver exogenously produced DNA into a host cell.

[0208] As used herein, "expression cassette" refers to a nucleic acid sequence capable of directing the expression of a specific nucleotide sequence in an appropriate host cell, which may include a promoter operably linked to the nucleotide sequence of interest, which may be operably linked to a termination signal. It may also include sequences necessary for proper translation of the nucleotide sequence. The coding region typically encodes the desired RNA or protein. The expression cassette containing the nucleotide sequence of interest may be chimeric. The expression cassette may also be naturally occurring but obtained in a recombinant form useful for heterologous expression. Expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or a regulatable promoter that initiates transcription only when the host cell is exposed to some specific stimulus. In the case of multicellular organisms, the promoter may also be specific to a particular tissue or organ, or developmental stage.

[0209] A vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. A vector may or may not be capable of autonomous replication, or may or may not be capable of integrating into host DNA. Examples include plasmids, viruses (e.g., rAAV), cosmids, or other vehicles that can be manipulated by the insertion or incorporation of a nucleic acid (e.g., a recombinant nucleic acid). Vectors can be used for a variety of purposes, including, for example, genetic engineering (e.g., cloning vectors), to introduce / transfect nucleic acids into cells and transcribe or translate the inserted nucleic acid within the cell. In some embodiments, the vector nucleic acid sequence contains at least an origin of replication for propagation in a cell. In some embodiments, the vector nucleic acid comprises a heterologous nucleic acid sequence, an expression control element (e.g., a promoter, an enhancer), a selectable marker (e.g., antibiotic resistance), a polyadenosine (polyA) sequence, and / or an ITR. In some embodiments, the nucleic acid sequence propagates upon delivery to a host cell. In some embodiments, when delivered to a host cell either in vitro or in vivo, the cell expresses a polypeptide encoded by the heterologous nucleic acid sequence. In some embodiments, when delivered to a host cell, the nucleic acid sequence or a portion of the nucleic acid sequence is packaged into a capsid. The host cell may be an isolated cell or a cell within a host organism. In addition to the nucleic acid sequence encoding the RNA or polypeptide or protein (e.g., a transgene), additional sequences (e.g., regulatory sequences) may be present within the same vector (i.e., in cis with the gene) and adjacent to the gene. In some embodiments, the regulatory sequences may be present on a separate (e.g., second) vector that acts in trans to regulate expression of the gene. Plasmid vectors may be referred to herein as "expression vectors."

[0210] As used herein, the term "vector genome" refers to a recombinant nucleic acid sequence that is packaged or encapsidated to form a rAAV vector. Typically, a vector genome includes heterologous polynucleotide sequences, such as transgenes, regulatory elements, and ITRs not originally present in the capsid. When a recombinant plasmid is used to construct or produce a recombinant vector (e.g., an rAAV vector), the vector genome does not include the entire plasmid, but rather only the sequences intended for delivery by the viral vector. This non-vector genome portion of the recombinant plasmid is typically referred to as the "plasmid backbone," and is important for the cloning, selection, and amplification of the plasmid, processes required for the propagation of recombinant viral vector production, but is not itself packaged or encapsidated into the rAAV vector.

[0211] As used herein, the term "viral vector" generally refers to a viral particle that functions as a nucleic acid delivery vehicle and contains a vector genome (e.g., containing a transgene in place of nucleic acids encoding AAV rep and cap) packaged within the viral particle (i.e., capsid), including, for example, lentiviruses and parvoviruses, including AAV serotypes and variants (e.g., rAAV vectors). Recombinant viral vectors do not contain a vector genome containing the rep and / or cap genes.

[0212] As used herein, the terms "overexpressing," "overexpress," "overexpressed," or "overexpression," when referring to the production of a nucleic acid or protein in a host cell, mean that the nucleic acid or protein is produced in an amount that is greater than the amount that is produced in its naturally occurring environment. The term is intended to encompass the overexpression of endogenous, as well as exogenous or heterologous, nucleic acids and proteins. Thus, the term and the like are intended to encompass increasing the expression of a nucleic acid or protein in a cell to a level that is higher than the level that the cell naturally contains. In certain embodiments, the expression level or amount of a nucleic acid or protein in a cell is increased by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% compared to the level or amount naturally contained in the cell.

[0213] Terms such as "overexpressing," "overexpress," "overexpressed," and "overexpression," in the context of mutant or diseased cells, are intended to encompass increasing the expression of a nucleic acid or protein to a level greater than that contained in a mutant, diseased, wild-type, or non-diseased cell. In certain embodiments, the expression level or amount of a nucleic acid or protein in a mutant or diseased cell is increased by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% compared to the level or amount present in a mutant, diseased, wild-type, or non-diseased cell.

[0214] " Antisense " refers to the nucleic acid sequence that is complementary to the coding strand or mRNA of nucleic acid sequence, regardless of length.Antisense RNA can be introduced into individual cells, tissues, or organoids.Antisense nucleic acid can contain modified backbone, for example, phosphorothioate, phosphorodithioate, or other modified backbone known in the art, or can contain non-natural internucleoside bond.

[0215] As referred to herein, a "complementary nucleic acid sequence" is a nucleic acid sequence that can hybridize with another nucleic acid sequence consisting of complementary nucleotide base pairs. "Hybridizing" refers to pairing of complementary nucleotide bases to form a double-stranded molecule under suitable stringent conditions (e.g., in DNA, adenine (A) base pairs with thymine (T), and guanine (G) base pairs with cytosine (C)). (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0216] A "suppressor" or "inhibitor" refers to an agent that causes a decrease in the expression or activity of a target gene or protein, respectively.

[0217] The terms "inhibit," "down-regulate," or "reduce" refer to a reduction in the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, below the expression or level observed in the absence of an inhibitor, suppressor, or repressor, such as an inhibitory nucleic acid molecule (e.g., siRNA) described herein. Down-regulation can be associated with post-transcriptional silencing, e.g., RNAi-mediated cleavage, or changes in DNA methylation patterns or DNA chromatin structure.

[0218] As used herein, an "inhibitory nucleic acid" is a double-stranded RNA, RNA interference, miRNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that, when administered to a mammalian cell, results in a reduction in expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule or its ortholog, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. Typically, expression of the target gene is reduced by 10%, 25%, 50%, 75%, or even 90-100%.

[0219] As used herein, the term "siRNA" refers to a double-stranded RNA molecule that interferes with the post-transcriptional expression of a specific gene or multiple genes. In some embodiments, siRNA functions to interfere with or inhibit gene expression using the RNA interference pathway. Similar interference or inhibition effects can be achieved with one or more short hairpin RNAs (shRNAs), microRNAs (mRNAs), and / or nucleic acids (such as siRNAs, shRNAs, or miRNAs) containing one or more modified nucleic acid residues, such as peptide nucleic acids (PNAs), locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), or triazole-linked DNA. Optimally, siRNAs are 18, 19, 20, 21, 22, 23, or 24 nucleotides in length and have a two-base overhang at their 3' end. These dsRNAs can be introduced into individual cells or culture systems. Such siRNAs are used to downregulate mRNA levels or promoter activity.

[0220] As used herein, the terms "treat," "treating," or "treatment" refer to administering a therapy that partially or completely relieves, improves, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition.

[0221] As used herein, the term "amelioration" refers to a detectable or measurable improvement in a subject's disease, disorder, or condition, or its symptoms, or underlying cellular response. Detectable or measurable improvement includes a subjective or objective decrease, reduction, inhibition, suppression, limitation, or control of the occurrence, frequency, severity, progression, or duration of the disease, disorder, or condition, or the causes of complications due thereto or associated therewith, an improvement in the symptoms thereof, or a reversal thereof.

[0222] As used herein, the term "associated with" refers to a relationship between two entities when the presence, level, and / or form of one correlates with the presence, level, and / or form of the other. For example, a particular entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) is considered to be associated with a particular disease, disorder, or condition if its presence, level, and / or form correlates with the incidence and / or susceptibility of the disease, disorder, or condition (e.g., across a relevant population).

[0223] As used herein, the term "prevent" or "prevention" refers to a delay in the onset and / or a reduction in the frequency and / or severity of one or more signs or symptoms of a particular disease, disorder, or condition (e.g., a myelin disease). In some embodiments, prevention is assessed on a population basis, such that an agent is considered to "prevent" a particular disease, disorder, or condition if a statistically significant reduction in the onset, frequency, and / or intensity of one or more signs or symptoms of the disease, disorder, or condition is observed in a population susceptible to the disease, disorder, or condition. Prevention may be considered complete if the onset of the disease, disorder, or condition is delayed for a predetermined period of time.

[0224] As used herein, the term "therapeutically effective amount" refers to an amount that produces the desired therapeutic effect for which it is administered. In some embodiments, the term refers to an amount sufficient to treat a disease, disorder, or condition when administered according to a therapeutic dosing regimen to a population suffering from or susceptible to the disease, disorder, or condition. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of, and / or delays the onset of, one or more symptoms of the disease, disorder, and / or condition. Those skilled in the art will understand that the term "therapeutically effective amount" does not actually require that successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount can be an amount that, when administered to patients in need of such treatment, provides a specific, desired pharmacological response in a significant number of subjects.

[0225] A "population" of cells refers to any number of cells greater than 1, but at least 1 x 10 3 cells, at least 1 x 10 4 cells, at least 1 x 10 5 cells, at least 1 x 10 6 cells, at least 1 x 10 7 cells, at least 1 x 10 8 cells, at least 1 x 10 9 cells, or at least 1 x 10 10 Each cell is an individual cell.

[0226] As used herein, the term "stem cell" refers to a cell that has the ability to both replace itself and differentiate into more specialized cells. Their self-renewal capacity generally persists for the lifespan of an organism. Pluripotent stem cells can give rise to all of the body's various cell types. Multipotent stem cells can give rise to a limited subset of cell types. For example, hematopoietic stem cells can give rise to the various cell types found in the blood, but cannot give rise to other cell types. Multipotent stem cells can also be referred to as somatic stem cells, tissue stem cells, lineage-specific stem cells, and adult stem cells. Non-stem cell descendants of multipotent stem cells are progenitor cells (also referred to as restricted progenitor cells). Progenitor cells can give rise to fully differentiated cells, but give rise to a more restricted set of cell types than stem cells. Progenitor cells also have a relatively limited self-renewal capacity; as they divide and differentiate, they eventually wear out and are replaced by new progenitor cells derived from their upstream multipotent stem cells.

[0227] "Induced pluripotent stem cells," commonly abbreviated as iPS cells or iPSCs, refer to a type of pluripotent stem cell that is artificially prepared from non-pluripotent cells, typically adult somatic cells, or terminally differentiated cells such as fibroblasts, hematopoietic cells, muscle cells, neurons, or epithelial cells, by introducing certain factors called reprogramming factors.

[0228] "Pluripotent" refers to stem cells that have the potential to differentiate into all cells that make up one or more tissues or organs, particularly any of the three germ layers: endoderm (stomach lining, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital tract), or ectoderm (epidermal tissue and nervous system). As used herein, "pluripotent stem cells" refer to cells that can differentiate into cells derived from any of the three germ layers, e.g., the direct descendants of totipotent cells or induced pluripotent cells.

[0229] As used herein, "therapeutic cells" refers to a population of cells that ameliorate a patient's condition, disease, and / or injury. Therapeutic cells can be autologous (i.e., derived from the patient), allogeneic (i.e., derived from an individual of the same species but different from the patient), or xenogeneic (i.e., derived from a species different from the patient). Therapeutic cells can be homogeneous (i.e., composed of a single cell type) or heterogeneous (i.e., composed of multiple cell types). The term "therapeutic cells" includes both therapeutic active cells and progenitor cells that can differentiate into therapeutic active cells.

[0230] The term "autologous" refers to any material derived from the same subject or individual that is later reintroduced. For example, the methods of autologous cell therapy described herein involve the collection of glial cells or their progenitor cells from a donor, e.g., a patient, which are then engineered, e.g., to express a transgene, and then re-administered to the same donor, e.g., patient.

[0231] The term "heterologous" refers to any material (e.g., a cell or tissue scaffold) that is derived from a different subject or individual. As used herein, "heterologous" or "non-endogenous" or "exogenous" also refers to any material (e.g., a gene, protein, compound, molecule, cell, or tissue or tissue component) or activity that is not native to the host cell or host subject, or any gene, protein, compound, molecule, cell, tissue, or tissue component that is native to the host or host cell but that has been altered or mutated such that the structure, activity, or both differ between the native version and the mutated version.

[0232] The term "allogeneic" refers to any material (e.g., cells or tissue) derived from one individual that is then introduced into another individual of the same species, e.g., allogeneic cell transplantation. For example, cells can be obtained from a first subject, modified ex vivo according to the methods described herein, and then administered to a second subject to treat a disease. In such embodiments, the cells administered to the subject are allogeneic and heterologous cells. The term "xenogenic" refers to any material (e.g., cells or tissue) derived from an individual of a different species. The term "allogeneic" refers to any material (e.g., cells or tissue) that is characterized by essentially the same genes.

[0233] As used herein, the term "subject" refers to an organism, e.g., a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, an experimental animal, a mouse, a rat, a hamster, a gerbil, a cat, a dog). In some embodiments, the subject is a non-human disease model. In some embodiments, the human subject is an adult, an adolescent, or a pediatric subject. In some embodiments, the subject is suffering from a disease, disorder, or condition, e.g., a disease, disorder, or condition that can be treated as provided herein. In some embodiments, the subject is suffering from a disease, disorder, or condition associated with deficient or dysfunctional myelin. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, a susceptible subject is prone to and / or exhibits an increased risk (compared to the average risk observed in a reference subject or population) of developing the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms of the disease, disorder, or condition. In some embodiments, the subject does not exhibit specific symptoms (e.g., clinical symptoms of the disease) or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a human patient. In some embodiments, the subject is an individual to whom and / or has been administered a diagnosis and / or therapy.

[0234] As used herein, the term "therapeutically effective amount" refers to an amount that produces the desired therapeutic effect for which it is administered. In some embodiments, the term refers to an amount sufficient to treat a disease, disorder, or condition when administered according to a therapeutic dosing regimen to a population suffering from or susceptible to the disease, disorder, or condition. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of, and / or delays the onset of, one or more symptoms of the disease, disorder, and / or condition. Those skilled in the art will understand that the term "therapeutically effective amount" does not actually require that successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount can be an amount that, when administered to patients in need of such treatment, provides a specific, desired pharmacological response in a significant number of subjects.

[0235] The following examples are intended to illustrate the practice of embodiments of the present disclosure, but are not intended to limit its scope in any way. [Example]

[0236] Example 1 Materials and Methods This example describes the materials and methods used in Examples 2-10 below.

[0237] Human embryonic stem cell lines and culture conditions Sibling human embryonic stem cell (hESC) lines GENEA019 (WT: 18; 15 CAG) and GENEA020 (HD: 48; 17 CAG) (both female) were obtained from GENEA, Inc. (Sydney, Australia). hESCs were cultured at 0.55 μg / cm with mTeSR1 medium (StemCell Technologies, Cat. No. 85850). 2hESCs were regularly cultured under feeder-free conditions on cell culture flasks coated with human recombinant 521 (Biolamina, catalog no. LN521). Daily medium changes were performed. hESCs were routinely passaged at 80% confluency onto freshly coated flasks. Passage was performed using ReLeSR (StemCell Technologies, catalog no. 05872). All hESC and differentiation cultures were maintained in a 37°C, 5% CO2 incubator and regularly checked for contamination and mycoplasma-free status. The karyotypes of the source lines, both before and after reporter insertion (see below), were analyzed on metaphase spreads by G-banding (Institut fur Medizinishche Genetik und Angewandte Genomik, Universitatsklinikum Tubingen). All hESC lines had normal karyotypes. Additionally, acquired copy number variants (CNVs) and loss of heterozygosity (LOH) were assessed by array CGH (Cell Line Genetics). No CNVs were observed that are known to or might be expected to affect the outcome of competitive interactions between clones.

[0238] Generation of fluorescent reporter hESCs For universal differential fluorescent labeling of WT and HD cells, reporter constructs driving expression of either mCherry or EGFP were used. 26 A modified version of the CRISPR-Cas9-mediated strategy previously described in was used to insert the plasmid into the AAVS1 safe harbor locus of WT GENEA019 and HD GENEA020 hESCs. To prepare hESCs for plasmid delivery by electroporation, hESCs were harvested as single-cell suspensions after dissociation with Accutase (StemCell Technologies, catalog no. 07920), washed in medium, and counted with an automated cell counter, NucleoCounter NC-200 (ChemoMetec). A total of 1.5 × 10 cells were counted per electroporation. 6Cells were mixed with 5 μg of the AAVS1 targeting CRISPR-Cas9 plasmid (pXAT2) and 5 μg of reporter donor plasmid (pAAVS1-P-CAG-mCherry or pAAVS1-P-CAG-EGFP). pXAT2 (Addgene Plasmid No. 80494), pAAVS1-P-CAG-mCherry (Addgene Plasmid No. 80491), and pAAVS1-P-CAG-EGFP (Addgene Plasmid No. 80492) were gifts from Knut Woltjen. Electroporation was performed using an Amaxa 4D-Nucleofector (Lonza) with a P3 Primary Cell Kit (Lonza, catalog no. V4XP-3024) according to the manufacturer's guidelines. After nucleic acid excision, the electroporated hESC suspension was transferred to a 10 cm cell culture dish and cultured in mTeSR1 supplemented with 10 μM Y-27632 (Tocris, catalog no. 1254) for the first 24 hours. The electroporated hESCs were grown for 48–72 hours and then treated with 0.5 μg / μL puromycin (ThermoFisher, catalog no. A1113803). The electroporated hESC culture was maintained under puromycin until individual colonies were large enough to be manually picked. Colonies were assessed by fluorescence microscopy and transferred to 96-well plates based on the uniformity of reporter expression. After expansion, individual clones were split for further expansion and genotyping. For genotyping, DNA was extracted using the prepGEM Tissue DNA Extraction Kit (Zygem). Precisely targeted transgenic integration at the AAVS1 locus was detected by PCR using the following primers: dna803: 5'-TCGACTTCCCCTCTTCCGATG-3' (SEQ ID NO: 12) and dna804: 5'-CTCAGGTTCTGGGAGAGGGTAG-3' (SEQ ID NO: 13), while zygosity of the integrations was determined by the presence or absence of the WT allele using additional primers: (dna803 and dna183: 5'-GAGCCTAGGGCCGGGATTCTC-3', SEQ ID NO: 14).hESCs with correctly targeted insertions were cryopreserved in Pro-Freeze CDM (Lonza, catalog no. BEBP12-769E) and then expanded for karyotyping and array competitive genomic hybridization (aCGH) before generating hGPCs.

[0239] Induction of hGPCs from reporter WT hESCs and HD hESCs Human GPCs were obtained from both reporter WT hESCs and HD hESCs using the protocol described by Wang et al., Cell Stem Cell 12, 252–264 (2013), with minor modifications to the embryoid body (EB) generation step. Cells were collected for transplantation at 150–200 DIV, at which point cultures derived from both WT-mCherry / EGFP and HD-EGFP hESCs expressed primarily PDGFRα. + / CD44 + It consisted of a bipotential GPC.

[0240] xenograft Cell preparation: To prepare cells for transplantation, glial cultures were cultured in Ca 2+ / Mg 2+ -free Hank's Balanced Salt Solution (HBSS) (- / -) The cells were collected in a 10 mL syringe (ThermoFisher, catalog number 14170112) and then gently mechanically dissociated into small clusters and counted in a hemocytometer. The cell suspension was then spun and counted in a 10 mL syringe. 5 Cold HBSS at cells / μL (- / -) and kept on ice until transplantation.

[0241] Neonatal immunodeficiency Rag1 mice were transplanted to generate human-mouse chimeras carrying mHTT-expressing human glia (HD chimeras). (- / -) puppy 66Pups were cryoanesthetized, immobilized on custom-baked clay platforms, and injected bilaterally with 100,000 HD glia (50,000 per hemisphere) into the presumed striatum within 48 hours of birth. Cells were delivered to a depth of 1.2–1.4 mm using a 10 μL syringe with a pulled glass pipette (Hamilton, catalog no. 7653-01). Pups were then returned to their mothers until weaning.

[0242] Adult Grafts. To assess the ability of transplanted healthy human glia to replace their diseased counterparts, 36-week-old HD glial chimeras were anesthetized with ketamine / xylazine and immobilized in a stereotaxic frame. 200,000 WT glia were delivered bilaterally to the humanized striatum using a 10 μL syringe and metal needle (AP: +0.8 mm; ML: ±1.8 mm; DV: -2.5 to -2.8 mm, all from bregma). To minimize injury, cells were injected at a controlled rate of 175 nL / min using a controlled micropump system (World Precision Instruments). The needle was left in place for an additional 5 minutes to minimize backflow. Experimental animals were compared with HD chimeric littermates that did not receive WT glia and naive rag1 mice that received WT glia at 36 weeks of age following this exact procedure. (- / -) compared with mice.

[0243] Human glial striatal isografts. To assess the influence of cell age as a determinant of competitive advantage among human glia, we performed neonatal Rag1 transplants following the same perinatal transplantation protocol described above. (- / -) Mice were injected with WT-mCherry glia, but instead received glia derived from WT-mCherry glia, to generate human-mouse chimeras bearing WT human glia (WT chimeras). Then, at 40 weeks of age, WT chimeras were injected according to the same adult transplantation procedure described above, but instead received allogeneic WT-EGFP glia. Similarly, experimental animals were compared with WT chimera littermates that did not receive WT-EGFP glia, and with naive rag1 littermates that received WT-EGFP glia at 40 weeks of age according to this exact procedure. (- / -) compared with mice.

[0244] Aseptic technique was used for all xenotransplantations. All mice were housed in a pathogen-free environment with free access to food and water, and all procedures were performed in accordance with protocols approved by the University of Rochester Committee on Animal Resources.

[0245] Tissue processing and immunostaining Experimental animals were perfused with HBSS (ThermoFisher, catalog no. 24020117) followed by 4% PFA. Brains were removed, post-fixed in 4% PFA for 2 hours, and rinsed three times with PBS. They were then incubated in 30% sucrose solution (Sigma-Aldrich, catalog no. S9378) until equilibration. At that point, they were embedded in OCT in the sagittal orientation (Sakura, catalog no. 4583), frozen in 2-methylbutane (Fisher Scientific, catalog no. 11914421) at -60°C to -70°C, and transferred to a -80°C freezer. The resulting blocks were then cut into 20 μm sections on a CM1950 cryostat (Leica), serially collected onto adhesive slides, and stored at -20°C until further use.

[0246] Identification and phenotyping of human cells was achieved by immunostaining for their respective fluorescent reporters along with phenotypic markers, including Olig2 (GPCs and oligodendrocytes), GFAP (astrocytes), or Ki67 (proliferating cells). Gene expression fluorescent reporters were used as markers for human cells because expression of the fluorescent reporters remained stable throughout the life of the animals. In animals receiving a 1:1 mixture of WT-mCherry human glia and WT-untagged human glia, the latter were identified by expression of human nuclear antigen (hN) and the lack of fluorescent reporter expression.

[0247] Immunolabeled sections were rehydrated with PBS and then incubated for 2 hours in permeabilization / blocking buffer (PBS + 0.1% Triton-X (Sigma-Aldrich, Cat. No. T8787) + 10% Normal Goat Serum (ThermoFisher, Cat. No. 16210072)). Sections were then incubated with primary antibody overnight at 4°C. The following day, sections were rinsed with PBS, and secondary antibody was applied for 1 hour. After rinsing again with PBS, a second primary antibody, this time against a fluorescent reporter, was applied to the sections overnight at 4°C. These were then rinsed the following day with PBS, and sections were incubated with secondary antibody for 1 hour. Slides were again thoroughly washed with PBS and embedded with Vibrance (Vector Labs, Cat. No. H-1800).

[0248] Apoptosis assay Identification of apoptotic cells within human cell populations was achieved by terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) and immunostaining for their respective fluorescent reporters. TUNEL was performed using the Click-iT TUNEL Alexa Fluor 647 Imaging Assay (Invitrogen, catalog no. C10247) according to the manufacturer's instructions, except that samples were incubated in proteinase K solution at room temperature for 20 minutes. To confirm efficient TUNEL staining in fixed-frozen brain cryosections, positive control sections were treated with DNase I according to the manufacturer's instructions. After TUNEL, sections were immunolabeled for the fluorescent reporters according to the previously described immunostaining protocol.

[0249] quantitative histology Transplantation Mapping and 3D Reconstruction: To map human cell distribution, a whole-brain montage of 15 equally spaced 160 μm sagittal sections spanning the entire striatum was acquired using a Nikon Ni-E Eclipse microscope equipped with a DS-Fi3 camera at 10x magnification and stitched together using NIS-Elements imaging software (Nikon). The striatum within each section was outlined, and immunolabeled human cells were identified and mapped within the outlined striatum using Stereo Investigator (MicroBrightField Bioscience). Where applicable, adult injection sites were mapped as reference points for volumetric quantification of human cell distribution. The mapped sections were then aligned using the lateral ventricle as a reference to generate a 3D reconstructed model of the humanized mouse striatum. After 3D reconstruction, the Cartesian coordinates of each human cell marker, injection site, and striatal outline were exported for further analysis.

[0250] To map the distribution and proportion of mitotically active cells within each human donor cell population, human cells expressing Ki67 immunoreactivity were mapped at every third section of the 15 equidistant sections used to generate the three-dimensional reconstructions, thus resulting in Ki67 sampling every (160 μm × 3 = 480 μm).

[0251] Volumetric distribution analysis: To quantify the spatial distribution of HD glia in HD chimeras (Figure 6), the volume of each mapped striatal section was calculated by multiplying the section thickness (20 μm) by the section area. The cell density of each section was then calculated by dividing the number of mapped cells in each section by their respective volumes.

[0252] To quantify the spatiotemporal dynamics of competing human glia, we developed a program to calculate the volumetric distribution of each cell population as a function of distance to the WT glial delivery site in three-dimensional reconstruction datasets (Figures 1 and 3, 6 and 8). To achieve this, for each quantified section, the striatum contour was represented as two identical polygons separated from each other by the section thickness (20 μm), defining upper and lower boundaries z. u , z l Then, because the depth position of each cell marker within each individual section is unknown, the mapped cells within each section were represented as a uniform point probability function with a constant probability across the section, i.e., z l From z u Each cell marker in the slices up to has a probability function.

number

[0253] The spatial distribution of each cell population was then measured by counting the number of mapped cells within concentric spherical shells radiating from the WT glial delivery site at radial increments of 125 μm (for control HD or WT chimeras, the average of the coordinates of the WT glial delivery site was used). Mapped cells were counted as 1 if each representative line segment was completely inside, 0 if it was completely outside, or partially if it intersected the spherical shell at either the upper or lower boundary of its corresponding segment. The density of each cell population, ρ, was then calculated. a,b (a, b represent the minimum and maximum radii of the spherical shell) was calculated by dividing the number of mapped cells within the spherical shell by the combined section volume within the shell,

number

[0254] Human cell phenotyping. Quantification of each human cell phenotype (except Ki67 and TUNEL) was performed using an optical fractionator on five equidistant sagittal sections separated by 480 μm across the entire striatum. 67 This was performed using a Nikon Ni-E Eclipse microscope equipped with a DS-Fi3 camera at 20x magnification. A whole-brain z-stack montage was first acquired using a Nikon Ni-E Eclipse microscope equipped with a DS-Fi3 camera at 20x magnification and stitched together in NIS-Elements imaging software. Each z-stack tile was acquired using a 0.9 μm step size. The montage was then loaded into Stereo Investigator, and the striatum was delineated. A series of 200 × 200 μm counting frames were systematically randomly placed by the software within a 400 × 400 μm grid covering the outlined striatum in each cross section. Counts were performed across the entire cross section height (without guard zones), and cells were counted based on their immunolabeling in the optical section in which they were initially focused. A representative image showing the entire striatum was generated from the whole-brain montage using the "crop" function in NIS-Elements imaging software and adjusting the "min / max" levels.

[0255] TUNEL +To assess the distribution and proportion of apoptotic cells within each human cell pool, a Nikon Ni-E Eclipse microscope equipped with a DS-Fi3 camera at 10x magnification was used to acquire whole-striatal montages of five equally spaced 480 μm sagittal sections spanning the entire striatum, which were stitched together using NIS-Elements imaging software. The striatum was outlined within each section, and immunolabeled human cells were identified and mapped based on their TUNEL labeling within the outlined striatum using Stereo Investigator.

[0256] Representative images showing the entire humanized striatum were generated from previously acquired whole-brain montages using the "crop" function in NIS-Elements imaging software and adjusting the "min / max" levels. Representative images of the human glial competition interface were then acquired as large-field z-stack montages using a Nikon Ti-E C2+ confocal microscope equipped with 488 nm, 561 nm, and 640 nm laser lines and a standard PMT detector. Images were acquired at 40x or 60x magnification using an oil immersion objective and stitched together in NIS-Elements. Maximum intensity projections were then generated and the "min / max" levels adjusted in NIS-Elements. Similarly, representative images of human cell phenotypes were acquired, imaged, and processed as z-stacks using a Nikon Ti-E C2+ confocal and the same laser lines.

[0257] Fluorescence-activated cell sorting (FACS) of human glia from chimeric mice To isolate human cells for scRNA-seq, experimental chimeras were performed as previously described. 68The dissected striatum and dissociated tissue were perfused intracardially with HBSS. Briefly, mice were euthanized with euthasol, perfused transcardially with sterile Hank's balanced salt solution (HBSS) containing magnesium chloride and calcium chloride, and their brains were removed. The brains were immersed in ice-cold sterile HBSS for approximately 5 minutes to facilitate microdissection. Under a dissecting microscope, the striatum from each mouse was dissected and placed in sterile HBSS on ice. The striatal tissue was transferred to a Petri dish containing sterile HBSS without magnesium chloride and calcium chloride, cut into small pieces using a sterile disposable scalpel, transferred to a sterile tube, and then incubated in papain / DNase dissociation solution at 37°C for 50 minutes. Ovomucoid dissolved in EBSS was then added to inactivate the papain. The tissue was triturated by repeated pipetting to obtain a single-cell suspension. The cells were then pelleted, resuspended in MEM, and filtered for flow cytometry. Single-cell suspensions were isolated based on their expression of mCherry or EGFP, or their absence, using a BD FACSAria Fusion (BD Biosciences). To exclude dead cells, 4',6-diamidino-2-phenylindole (DAPI; ThermoFisher, catalog no. D1306) was added at 1 μg / mL.

[0258] Single-cell RNA sequencing analysis Primary Data Acquisition: Isolated cells were acquired for scRNA-seq on a 10X Genomics Chromium Controller (v3.1 chemistry). Libraries were generated according to the manufacturer's instructions and sequenced on an Illumina NovaSeq 6000 at the University of Rochester Genomics Center. scRNA-seq libraries were aligned with STARsolo using a custom two-pass strategy. 69、70First, annotated chimeric GRCh38 and GRCm38 references were generated using the human and mouse annotations from Ensembl 102, adding mCherry and EGFP. STARsolo was then run with the following parameters: twopassMode=basic, limitSjdbInsertNsj=3000000, and soloUMIfiltering=MultiGeneUMI. BAM files were then split by species, and cross-species multi-mapping reads were assigned to both the human BAM and the mouse BAM. FASTQ files were regenerated from either the mouse or human BAM files and realigned to the single-species reference. STARsolo was run again with the following parameters: twopassMode=basic, limitSjdbInsertNsj=2000000, and soloUMIfiltering=MultiGeneUMI.

[0259] Differential expression analysis of human data using Seurat 72 Imported into R 71 Cells were filtered (>250 unique genes and <15 mitochondrial gene ratio). Cells were then further filtered for expression of mCherry or EGFP. Counts were imported into Python for integration using scvi, where the 4,000 most variable features were used. 73 The model was trained for integration using mouse samples and cell lines, along with the number of unique genes and mitochondrial gene expression rates. The latent representation was then used for dimensionality reduction via UMAP and Louvain community detection. Smaller populations of cells were classified into six major types of glia based on marker expression. The data was then re-imported into Seurat and analyzed using MAST. 74Differential expression was performed. A gene was considered differentially expressed if its expression was detected in at least 3% of all GPCs. Model design for differential expression utilized the number of unique genes in the cells and the experimental group (cell line / age of cells, and whether the cells were in the presence of opposing clones). Significance of differential expression was P<0.05, and the log2 fold change was at least 0.15. Ingenuity Pathway Analysis (QIAGEN) was used for functional analysis of each differentially expressed gene list.

[0260] Cell cycle analysis: The G2M scores for each experimental group were calculated using the CellCycleScoring function in Seurat. Statistical comparisons between experimental groups for each model were then calculated using Dunn's test with Benjamini-Hochberg multiple comparison adjustment.

[0261] Identification of transcription factor-associated regulons. Genes were first filtered to retain only those expressed with at least 3 counts in at least 1% of cells. All 10,410 cells were used in this analysis. The filtered raw matrix was then analyzed using pySCENIC 75 Using the standard pipeline of cv.glmnet as input, each transcription factor in the dataset and its putative downstream targets were identified. These gene sets, referred to as regulons, were assigned an "area under the curve" (AUC) value to represent their activity in each cell, with higher values ​​indicating stronger enrichment of such regulons. The resulting AUC matrix was then used to search for significant transcription factors. Within the GPC subpopulations in both syngeneic and allograft models, cells from young WT samples were assigned a value of 1, and cells from aged WT or aged HD samples were assigned a value of 0. Next, using glmnet, a logistic regression was performed with the AUC of all TFs as predictors, with a given 0 / 1 outcome. The lambda for the logistic regression was automatically defined in cv.glmnet.

[0262] We isolated TFs with positive coefficients and further filtered them based on their average activity per group, such that the average TF activity in young WTs should be higher than that in their aged counterparts. The final step was to perform gene set enrichment analysis (GSEA) on the previously identified regulons. 76 to determine whether genes that were differentially upregulated in young WT cells were enriched compared to senescent HD and WT cells (adjusted p < × 10 -3 , NES>0).

[0263] Identifying co-expressed gene sets with competitive advantages We filtered to exclude genes with less than 1 count across all cells and used the resulting matrix to perform DCA. 77 Data were denoised using Weighted Gene Co-expression Network Analysis (WGCNA). 35 was performed on the denoised data of the GPC subset. Signature network adjacencies were calculated with a soft thresholding power of 19. Modules were detected after hierarchical clustering of genes on differences based on a topological overlap matrix and dynamic tree cutting. We then performed GeneOverlap (adjusted p<10 -2 ) 78 We identified modules whose gene members represent significant overlap with the important TF targets identified above using the relaimpo package. 79 The relative contribution of linear independent covariates, age (young, aged) and genotype (HD, WT) to the additional explanation for each module eigengene (e.g., ME ~ age + genotype) was calculated by the lmg method, implemented in .

[0264] Network representation: Functional annotation of gene targets of transcription factors using IPA 80 To generate a representative network, we focused on the MYC regulon and its shared targets with other important TFs. 81The network was constructed using

[0265] Statistical analysis and reproducibility Samples exhibiting artifacts related to technical issues from the experimental procedure, such as mistarget injection or obvious surgical damage, were excluded from this study. Statistical tests were performed using GraphPad Prism 9, and all tests used are listed in the figure captions. For comparisons between three or more groups, one-way analysis of variance with Tukey's or Sidak's test for multiple comparisons was applied. For comparisons between two groups with three or more factors, two-way analysis of variance with Sidak's multiple comparison test was applied. When comparing two unmatched groups, an unpaired two-tailed t-test was applied. Significance was defined as P<0.05. Whenever possible, the respective P values ​​are noted in the figures; otherwise, ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. The number of replicates is indicated in the figure legends, and n indicates the number of independent experiments. Data are expressed as the mean ± standard error of the mean (SEM).

[0266] Data availability The sequencing dataset reported here can be accessed through GEO accession number GSE206322.

[0267] Code Availability A program for quantification of mapped cell populations in 3D tissue reconstructions is publicly available through GitLab (dtu.dk) at the following link: QIM / Tools / Thick Section Point Density.

[0268] All code used to analyze and generate figures for the genomics datasets can be accessed on Github at https: / / github.com / CTNGoldmanLab / HD_Competition_2022.

[0269] Example 2 Generation of distinct color-tagged human glia from WT and HD hESCs To assess the ability of healthy glia to replace diseased counterparts in vivo, we first generated fluorescently tagged reporter lines of WT and HD human embryonic stem cells (hESCs) to enable the production of spectrally distinct GPCs of each genotype whose growth in vivo could be independently monitored using a CRISPR-Cas9-mediated knock-in strategy. 26 was first used to integrate EGFP and mCherry reporter cassettes into the AAVS1 locus of matched female sibling wild-type (WT, GENEA019) hESCs and mHtt-expressing (HD, GENEA020) hESCs. 27、28 It was then verified that the reporter cassette was stably integrated into each of these clones and that editing did not affect the self-renewal, pluripotency, or karyotypic stability of the tagged hESCs. From these spectrally distinct tagged lines, differentiation protocols were used to differentiate hESCs. 5 Using this method, we were able to generate color-coded human glial progenitor cells (hGPCs) from each line and compare their in vivo behavior, both alone and in competition. We verified the ability of each line to maintain EGFP or mCherry expression after maturation as astrocytes or oligodendrocytes, as well as the absence of any significant differentially expressed oncogenic mutations or copy number variants (CNVs) that could bias growth. Both WT and mHTT-expressing hGPCs colonized the mouse host brain when injected alone (Figures 6A-6B and 7A-7B).

[0270] Protocol for generating hGPCs from both WT-mCherry hESCs and HD-EGFP hESCs 21 We performed assays to assess both their ability to differentiate into glia and the stability of their reporter expression during the acquisition of a glial fate. By 150 days in vitro (DIV), glial cultures derived from both WT-mCherry and HD-EGFP expressed PDGFRα, comprising nearly half of the cells in the culture. + / CD44 + Two-potential GPC 29 (P=0.78) were equally enriched, and the remaining immature A2B5 + GPC 30 and PDGFRα - / CD44 + Astrocytes and their precursor cells 31 Importantly, virtually all immunophenotypic cells derived from WT-mCherry and HD-EGFP hESCs, including mature astrocytes and GPCs, continued to express their respective fluorescent reporters, indicating that transgene expression remained stable upon acquisition of final glial identity both in vitro and after subsequent transplantation in vivo.

[0271] Example 3 Establishment of human HD glial chimeric mice These spectrally distinct WT and HD hGPCs were used to investigate whether resident mHTT-expressing HD glia are less compatible and therefore potentially replaceable by their healthy counterparts. To this end, we generated mice in which the striatum was substantially chimerized with tagged mHTT-expressing glia, by culturing hGPCs derived from EGFP-tagged HD hESCs in immunodeficient Rag1 mice. (- / -) HD GPCs were generated by neonatal injection into the neostriatum of mice (Figure 6A). After transplantation, HD glia rapidly infiltrated the striatum of these mice, initially migrating and expanding within the striatal white matter tracts, and then progressively replacing their mouse counterparts from the striatal neuropil (Figure 6B). Consequently, by 36 weeks, the mouse striatum was substantially humanized by HD glia (Figure 6B, Figure 6F, and Figure 6G). Colonization of the host striatum by human HD GPCs was driven by the mitotic expansion of HD hGPCs, the total number of which typically doubled between 12 and 36 weeks (Figure 6C, P = 0.0032). In contrast, as these cells reached their final density in their host, their proliferative cell pool (Ki67 + ) progressively decreased ( Fig. 6D , P = 0.0036), and their rate of expansion slowed over time.

[0272] Most HD glia express Olig2 + GPCs (72.7±1.9%) expanded, which persisted as a new resident pool after replacing their mouse counterparts. A small fraction of these (4.8±0.9%) further expanded into GFAP + These affected astrocytes differentiated into astrocytes (Figures 6I-6J). Astrocyte differentiation was observed primarily within the striatal white matter tract. These affected astrocytes, as previously reported, 8 , lacking the structural complexity typically observed in their healthy counterparts and exhibiting abnormal fiber structure ( Fig. 6J ).

[0273] Example 4 Healthy WT hGPCs infiltrate and outcompete resident glia in HD chimeric adult striatum Using established chimeras in which striatal glia were predominantly mHTT-expressing and human, we performed assays to examine how these resident HD human glia responded to the introduction of healthy hGPCs. To this end, we engrafted hGPCs derived from WT hESCs engineered to express mCherry into the striatum of 36-week-old HD chimeras and monitored their expansion histologically as they competed with the already resident HD glia (Figure 1A and Figure 7A-B).

[0274] After engraftment, WT glia infiltrated the previously humanized striatum and gradually replaced their HD counterparts as they expanded from their transplantation site (Figure 1B). This process was slow but sustained, resulting in substantial repopulation of the HD striatum with WT glia over time (Figure 1B, G, and H1; 54 weeks: P<0.0001; 72 weeks: P<0.0001). Notably, the expansion of WT glia was paralleled by the simultaneous elimination of HD glia from the tissue (Figure 1B, G, and H2; 54 weeks: P<0.0001; 72 weeks: P<0.0001). This was typically characterized by a distinct, advancing front, behind which only a few HD glia were found (Figure 1C). Consequently, following this competition between HD and WT GPCs, mutually exclusive domains formed (Figure 1D). Notably, within regions dominated by WT glia, HD astrocytes, defined by GFAP, were often found to persist, primarily within white matter tracts (Figure 1E). Astrocyte replacement proceeded more slowly than hGPCs, such that most WT donor cells were still Olig2+ when assessed at 72 weeks. + (80.1±4.7%), while only a small proportion (4.0±1.5%) expressed GFAP + hGPCs differentiated as astrocytes (Figure 7A-H). Thus, astrocyte replacement appears to depend on astrocyte turnover by replacement from locally dominant parenchymal hGPCs, rather than from competition between mature astrocytes, and dynamic competition within the time frame studied appeared to be primarily characteristic of hGPCs.

[0275] Notably, naive adult Rag1 (- / -)While WT hGPCs transplanted into mice expanded more rapidly and extensively throughout the host striatum than WT hGPCs transplanted into adult HD chimeras, allogeneic replacement of hGPCs by other hGPCs proceeded at a slower rate than xenogeneic replacement of mice by hGPCs (Figure 8A-D; 54 weeks: P = 0.006; 72 weeks: P = 0.0009). These results indicate that competitive glial replacement develops with different kinetics between xenografts and allogeneic grafts, with allogeneic cells offering greater (but easily overcome) competitive resistance.

[0276] Parallel control studies confirmed that these diverse observations were not artifacts of off-target effects of either gene editing or fluorescent reporter toxicity. WT-mCherry-derived co-engrafted hGPCs and their unmodified counterparts (WT-untagged) inhibited the growth of naive adult Rag1 cells. (- / -) Tagged and untagged WT cells, which expanded comparably within the striatum of both HD mice and HD chimeric mice and were otherwise allogeneic, intermingled freely, resulting in similar glial repopulation in each (Fig. 9A–D, F; 54 weeks: P = 0.50; 72 weeks: P = 0.15).

[0277] Example 5 Human WT Glia Enjoy a Proliferative Advantage Compared to Resident HD Glia Because striatal repopulation by HD glia slowed over time as the proportion of proliferative HD hGPCs decreased (Figure 6D), we performed an assay to determine whether the selective expansion of younger WT glia within the HD striatum was sustained by differential proliferative capacity between the two populations. To do so, we assessed Ki67 expression in both WT and HD glial populations as competitive striatal repopulation unfolded. At both 54 and 72 weeks of age, the mitotic fraction of transplanted WT human glia was significantly greater than that of resident HD-derived human glia (Figures 1I and 1J; 54 weeks: P < 0.0001; 72 weeks: P = 0.009). These data suggest that repopulation of the HD glial chimeric host striatum by WT glia was mediated, in part, by selective expansion of a differentially proliferating donor pool. Notably, while the proliferative advantage of adult-engrafted WT glia over resident HD glia became less pronounced as the mice, and therefore the cells, aged, it was maintained at least until 72 weeks of age, suggesting a sustained competitive advantage of WT glia beyond the experimental time point observed. Interestingly, throughout the 72-week observation period, we found that the leading edge of neonatally engrafted human HD glia continued to slowly expand far beyond the striatum into the basal forebrain, even as the leading edge of adult-engrafted WT hGPCs effectively displaced the striatal population from behind (Figure 10).

[0278] Example 6 Human WT Glia Assume a Dominant Competitor Profile When Encountering HD Glia Because transplanted WT hGPCs effectively colonized the HD glial chimeric striatum at the expense of resident mHTT-expressing glia, we performed assays to define the molecular signals underlying their competitive advantage. To achieve this goal, we used single-cell RNA sequencing (scRNA-seq; 10X Genomics, v3.1 chemistry) to analyze the transcriptional profiles of WT and HD human glia isolated from the striatum of chimeras in which the two cell populations co-resident and competed, and from their respective controls in which one or the other was transplanted without the other (Figure 2A). After integration of all captures and alignment to the human and mouse mixed-species genomes, Leiden community detection revealed six major populations of human glia, including hGPCs, circulating hGPCs, immature oligodendrocytes (iOLs), neural progenitor cells (NPCs), astrocytes, and their intermediate progenitor cells (astrocytic progenitor cells, APCs) (Figure 2B-D). Within these populations, cell cycle analysis predicted higher G2 / M scores in competing WT hGPCs compared to their HD counterparts (Figure 2E), consistent with histological observations (Figure 1J). To proceed, we focused on hGPCs as the primary competing population in the model. Pairwise differential expression revealed distinct sets of differentially expressed genes across groups (Figure 2F), and subsequent functional analysis using Ingenuity pathway analysis (IPA) within the hGPC population revealed numerous significant terms related to their competition (Figure 2G).

[0279] During competition, WT GPC was found to activate pathways that drive protein synthesis, while HD GPC was predicted to downregulate them. The predicted activation of upstream transcription factors is thought to be conserved master regulators of cell growth and proliferation. 32~34We identified YAP1, MYC, and MYCN as significantly modulated across experimental groups. Importantly, YAP1 and MYC targets were found to be selectively downregulated in competing HD GPCs compared with their controls (Figure 2G). Notably, this downregulation was accompanied by a significant repression of ribosomal-encoding genes (Figure 2I). Conversely, competing WT hGPCs showed upregulation of both YAP1 and MYC targets, as well as ribosomal-encoding genes, compared with controls (Figure 2G-H). Thus, these data suggest that transplanted WT hGPCs, upon contact with their HD counterparts, actively assumed a competitively dominant phenotype, promoting their own expansion and colonization while driving the local elimination of the latter.

[0280] Example 7 Age Differences Drive Competitive Human Glial Repopulation Because WT cells transplanted into adult hosts were fundamentally younger than the resident host cells they replaced, we performed assays to examine whether differences in cell age, in addition to disease status, might have contributed to the competitive success of late donor cells. To this end, we engrafted the striatum of 40-week-old adult glial chimeras with newly generated hGPCs from WT hESCs engineered to express EGFP, and these chimeras were perinatally engrafted with hGPCs derived from mCherry-tagged but otherwise syngeneic WT hESCs (Figure 3A). The expansion of the transplanted cells was then monitored histologically to map the relative fitness and competitive performance of these pools of syngeneic but otherwise distinctly senescent hGPCs.

[0281] The expansion of transplanted WT glia within the striatum of WT chimeras was remarkably similar to that of HD chimeras (Figure 1). After engraftment, younger WT glia rapidly infiltrated the previously humanized striatum and progressively replaced their senescent counterparts as they expanded from the transplantation site, ultimately resulting in substantial recolonization of the tissue (Figures 3B-D and 3E, P<0.0001). Their expansion was paralleled by the localized elimination of senescent WT glia (Figures 3B-D and 3F, P<0.0001), which was also characterized by a distinct advancing front behind which few resident WT glia were observed (Figure 3C). Accordingly, the mitotic fraction of transplanted WT glia was significantly greater than that of their resident senescent counterparts (Figures 3G-I, P=0.018). Taken together, these data indicated that repopulation of the human WT glial chimeric striatum with younger syngeneic hGPCs was accompanied by replacement of older cells by their younger counterparts and was driven, in part, by the relative expansion of the younger, more mitotically active cell population.

[0282] Example 8 Young cells replace their older counterparts through the induction of apoptosis Because younger glia appeared to exert a clear competitive advantage over their older counterparts, we performed assays to examine whether the elimination of older glia by younger cells occurred passively as a result of the higher proliferation rate of younger cells, leading to the relative attrition of older, resident counterparts during normal turnover, or whether replacement was actively driven by the induction of programmed cell death in older cells by more fit younger cells. To address this question, we used TUNEL assays to compare the rates of apoptosis in aged and young WT glial populations as they competed at baseline in the host striatum, as well as in single-transplant controls. We found that as competitive repopulation unfolded, aged WT glia underwent apoptosis at a significantly higher rate than their young counterparts (Figure 11A-C, P<0.0001). Importantly, the increased apoptosis of older resident glia appeared to be driven by interactions with younger cells, as a significantly higher proportion of senescent glia was found to be apoptotic in chimeras transplanted with younger cells as adults than in controls that did not receive later adult injections (Figure 11C, P = 0.0013). These data suggest that senescent resident glia confronted with their younger counterparts are actively eliminated, at least in part, through apoptosis induced by encounter with younger hGPCs, and that the higher relative fitness of hGPCs allowed them to repopulate the chimeric host striatum.

[0283] Example 9 Young hGPCs acquire a dominant signature when exposed to older syngeneic cells To confirm whether the molecular signals underlying the competitive advantage of younger WT glia over senescent WT glia are similar to those underlying their advantage over HD glia, we used scRNA-seq to analyze the transcriptional signatures of competing young and senescent WT glia and their respective controls (Figure 4A). Within the sequenced hGPC population (Figure 4B-D), we observed that the G2 / M scores of competing senescent WT cells were significantly lower than those of their younger counterparts (Figure 4E), consistent with the histological data (Figure 3I). Differential expression analysis revealed distinct sets of differentially expressed genes between competing young and senescent WT GPCs (Figure 4F and H), and subsequent IPA analysis of these gene sets revealed a similar signature to that observed between donor (young) WT and already resident (senescent) HD GPCs in the competing allograft model (Figure 4G). Specifically, genes functionally related to protein synthesis, including ribosomal genes and E2F family members, as well as upstream MYC and MYCN signaling, were all activated in young, competing WT GPCs compared with their senescent counterparts (Figure 4G). However, despite these similarities, senescent WT GPCs responded differently to newly transplanted WT GPCs than HD GPCs in other respects. In contrast to HD GPCs, senescent WT cells confronted with younger isogenic competitors upregulated both MYC and MYCN targets, with a concomitant upregulation of ribosomal genes (Figure 4I), compared with their uncompeted counterparts (Figure 4G). This difference in their profiles may represent an intrinsic ability to respond competitively when exposed, which HD hGPCs expressing mHTT lack. Nevertheless, this upregulation was insufficient to match the higher fitness of their younger counterparts, which similarly (but to a relatively greater extent) revealed selective upregulation of MYC targets as well as ribosomal genes compared to their uncompeted controls (Figure 4G-H).Taken together, these data indicate that determinants of relative cytofitness can be conserved across different scenarios of exposure, and that the outcome of the resulting competition is greatly influenced by the relative age of the competing populations.

[0284] Example 10 Competitive advantage is associated with distinct sets of transcription factors We performed assays to determine which gene signature defines the competitive advantage of newly transplanted human GPCs over resident cells. To this end, we applied a multistep analysis using lasso-adjusted logistic regression (Figure 5A), which identified six TFs (CEBPZ, CTCF, E2F1, MYC, NFYB, and ETV4), whose activity significantly explained the advantage of young WT GPCs over both senescent HD and senescent WT GPCs (Figure 5E). These six TFs and their putative targets comprised a gene set (regulon) upregulated in young WT cells in both allograft and syngeneic transplant models (normalized enrichment score NES > 0, adjusted p < 10). -3 ) (Figure 5C and Table 1). We also found that although their activity varied in the absence of competition (aged HD, aged WT, or young WT alone), they were higher in the dominant young WT cells, especially MYC, in both allograft (vs. HD) and syngeneic (vs. older allogeneic syngeneic autologous) paradigms (Figure 5E). [Table 2] TIFF2026505771000006.tif249170TIFF2026505771000007.tif249170TIFF2026505771000008.tif249170TIFF2026505771000009.tif216170

[0285] Next, we performed an assay to identify cohorts of genes with defined expression patterns as well as significant overlap with the six prioritized regulons described above. We first used weighted gene co-expression network analysis (WGCNA) to identify genes with significant overlap with the six prioritized regulons. 35 We detected a total of 25 modules in the GPC dataset (Figure 5A). Only one module (blue) harbored genes with significant overlap with targets in the TF cohort. We also found that the expression of genes in the blue module was driven by competition, as they were upregulated under competitive conditions compared to non-competitive conditions (Figure 5B).

[0286] We then performed assays to determine whether the expression patterns of prioritized modules could be explained by the age of the cells (young vs. old), their genotype (HD vs. WT), or both. WGCNA defined a module eigengene as the first principal component of the gene cohort, thereby representing the general expression pattern of all genes within that module. We therefore constructed a linear model in which the module eigengene was a response described by both age and genotype. In contrast to the other modules identified from WGCNA, we observed that the blue module was primarily influenced by age (Figure 5D).

[0287] MYC, whose regulated pathway activation has previously been speculated to confer a competitive advantage (Figures 2 and 4), was also one of the six prioritized TFs. Therefore, we performed further analysis to characterize the MYC regulon and its downstream targets, and found that these downstream targets were also regulated by other prioritized TFs (Figure 5F). Interestingly, MYC is part of the blue module and regulates these blue module genes, whose expression levels were higher in the competition paradigm compared to the non-competition paradigm (Figure 5B). This pattern suggests that the blue signature was not activated unless the cells were under a competitive environment. Furthermore, we noticed lower TF activity of MYC in aged HD compared to aged WT hGPCs (Figure 5E), potentially highlighting the inherently higher competitive ability of WT cells. This is consistent with our previous observation that aged WT hGPCs respond differently from HD hGPCs when exposed to newly engrafted WT hGPCs. Importantly, the blue module eigengenes could be described using age, indicating that the competitive advantage associated with MYC signaling was primarily driven by cell age. Accordingly, targets within this network were enriched for pathways regulating cell proliferation (TP53, YAP1, RICTOR), transcription (MYCN, MLXIPL), and protein synthesis (LARP1), each of which were previously mentioned as differentially expressed in each competition scenario (Figures 2 and 4). Thus, the output of this competition-induced regulatory network appeared to confer a competitive advantage to young WT hGPCs when introduced into the adult brain, regardless of whether they were faced with older HD-derived or allogeneic hGPCs.

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[0289] The foregoing examples and description of preferred embodiments should be construed as illustrating, but not limiting, the present disclosure, which is defined by the claims. As will be readily understood, numerous variations and combinations of the features described above can be utilized without departing from the present disclosure as set forth in the claims. Such variations will not be considered a departure from the scope of the present disclosure, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference in their entirety.

Claims

1. 1. A method of rejuvenating or enhancing the developmental potential of a glial progenitor cell or its progeny, said method comprising increasing in said glial progenitor cell or said progeny (i) the level or activity of a transcription factor selected from the group consisting of CEBPZ, CTCF, E2F1, MYC, NFYB, and ETV4, or (ii) a target of said transcription factor; The target is RPL6, RPS27, RPS16, RPS21, DOHH, PCCB, UTP11, RPS8, RPL27A, EIF2A, UBLCP1, RPL32, GIN1, PATZ1, TNFRSF1A, MRPL10, RFXANK, BORCS8, ENOPH1, RPS16, SNHG11, SLC35A5, RAB1B, RPL23A, YBX1, TMEM129, DOHH, CCND1, MRPL24, RPL14, HMGA1, DCTPP1, ENOPH1, ZNF436, RPLP2, CCND1, TNFRSF1A, FBXL12, NTM T1, IMPDH2, MRPL18, LIMS1, CD82, POLR2H, LRR C8A, EXOSC5, RAN, DYNL T1, FDPS, ACTL6A, RPS5, DOLPP1, GGCT, RPS2, SYCE1L, MRPL17, ZDHHC16, YBX1, RPLP0, ELK1, RPSA, NME2, RPS15, TSPAN33, B3GNT9, DCLRE1B, CMC1, RPLP1, ACAT2, RPL6, RPL28, RPL18A, CCDC51, RACK1, RPS14, RPS19, RPL12, RPS5, RPL19, RPL27A, RPS19, RPS21, POLR2H, RPS14, CCDC51, EIF2A, UBLCP1, SNHG19, RPL32, ZNF579, RPS27, RPL6, GIN1, PATZ1, RACK1, LRR C8A, TNFRSF1A, RPL28, RPS18, MRPL10, RFXANK, BORCS8, FBXL12, PEF1, ENOPH1, PEX7, RPS16, SNHG11, RPL10, ZDHHC16, HMGA1, ZNF436, UTP11, DCLRE1B, RPS5, EXOSC5, MRPL17, RPL19, PRMT1, NME2, CCND1, NRN1, YBX1, LIMS, RPL, RPL23A, CD82, NTM T1, RPL18A, DOLPP1, GGCT, ELK1, ACTL6A, FDPS, MRPL18, RPLP0, ACAT2, SLC35A5, RAB1B, RAN, DYNL T1, TMEM129, RPSA, RPS15, RPL13A, PCCB, DCTPP1, RPLP2, B3GNT9, IMPDH2, RPL14, MRPL24, RPS8, RPS2, SYCE1L, RPL12, CMC1, DOHH, RPLP1, BTBD17TSPAN33, ACAT2, GGCT, FDPS, SNHG19, PRMT1, RPL13A, FBXL12, RPS19, RFXANK, NRN1, DCTPP1, ZNF579, YBX1, MRPL18, NTMT1, RPL14, PEF1, RPS21, PEX7, BTBD17, RPL10, and RPS18.

2. The method of claim 1 , wherein the glial progenitor cells are senescent glial progenitor cells.

3. 10. The method of any one of the preceding claims, wherein the progeny are oligodendrocytes or astrocytes.

4. The method of any one of claims 1 to 3, wherein the increasing step comprises expressing or introducing said transcription factor or said target in said glial precursor cells or said progeny.

5. 4. The method of any one of claims 1 to 3, wherein the increasing step comprises contacting the glial progenitor cells or the progeny with an agent that increases the level or activity of the transcription factor or target.

6. 6. The method of any one of claims 1 to 5, further comprising suppressing in said glial precursor cells or said progeny a transcriptional repressor selected from the group consisting of E2F6, ZNF274, MAX, and IKZF3.

7. A cell prepared according to the method of any one of claims 1 to 6, or a progeny thereof.

8. 1. A method of treating a condition mediated by white matter loss, oligodendrocyte loss, or astrocyte loss, said method comprising administering to a subject in need thereof: (a) a therapeutically effective amount of (i) a transcription factor selected from the group consisting of CEBPZ, CTCF, E2F1, MYC, NFYB, and ETV4, or (ii) an agent that increases the level or activity of a target of said transcription factor; or (b) administering a therapeutically effective amount of cells prepared according to the method of any one of claims 1 to 6, or progeny thereof; The target is RPL6, RPS27, RPS16, RPS21, DOHH, PCCB, UTP11, RPS8, RPL27A, EIF2A, UBLCP1, RPL32, GIN1, PATZ1, TNFRSF1A, MRPL10, RFXANK, BORCS8, ENOPH1, RPS16, SNHG11, SLC35A5, RAB1B, RPL23A, YBX1, TMEM129, DOHH, CCND1, MRPL24, RPL14, HMGA1, DCTPP1, ENOPH1, ZNF436, RPLP2, CCND1, TNFRSF1A, FBXL12, NTMT1, IMPDH2, MRPL18, LIMS1, CD82, POLR2H, LRRRC8A, EXOSC5, RAN, DYNLT1, FDPS, ACTL6A, RPS5, DOLPP1, GGCT, RPS2, SYCE1L, MRPL17, ZDHHC16, YBX1, RPLP0, ELK1, RPSA, NME2, RPS15, TSPAN33, B3GNT9, DCLRE1B, CMC1, RPLP1, ACAT2, RPL6, RPL28, RPL18A, CCDDC51, RACK1, RPS14, RPS19, RPL12, RPS5, RPL19, RPL27A, RPS19, RPS21, POLR2H, RPS14, CCDDC51, EIF2A, UBLCP1, SNHG19, RPL32, ZNF579, RPS27, RPL6, GIN1, PATZ1, RACK1, LRRRC8A, TNFRSF1A, RPL28, RPS18, MRPL10, RFXANK, BORCS8, FBXL12, PEF1, ENOPH1, PEX7, RPS16, SNHG11, RPL10, ZDHHC16, HMGA1, ZNF436, UTP11, DCLRE1B, RPS5, EXOSC5, MRPL17, RPL19, PRMT1, NME2, CCND1, NRN1, YBX1, LIMS1, RPL23A, CD82, NTMT1, RPL18A, DOLPP1, GGCT, ELK1, ACTL6A, FDPS, MRPL18, RPLP0, ACAT2, SLC35A5, RAB1B, RAN, DYNLT1, TMEM129, RPSA, RPS15, RPL13A, PCCB, DCTPP1, RPLP2, B3GNT9, IMPDH2, RPL14, MRPL24, RPS, RPS2, SYCE1L, RPL12, CMC1, DOHH, RPLP1, BTBD17TSPAN33, ACAT2, GGCT, FDPS, SNHG19, PRMT1, RPL13A, FBXL12, RPS19, RFXANK, NRN1, DCTPP1, ZNF579, YBX1, MRPL18, NTMT1, RPL14, PEF1, RPS21, PEX7, BTBD17, RPL10, and RPS18.

9. 10. The method of claim 8, further comprising administering to the subject a therapeutically effective amount of a suppressor of a transcriptional repressor selected from the group consisting of E2F6, ZNF274, MAX, and IKZF3.

10. The method of claim 8 or 9, wherein the subject is a human.

11. 11. The method of any one of claims 5 and 8-10, wherein the agent or suppressor comprises a small molecule compound, an oligonucleotide, a nucleic acid, a peptide, a polypeptide, a CRISPR / Cas system, or an antibody or antigen-binding portion thereof.

12. The method of claim 11 , wherein the agent is a nucleic acid encoding the transcription factor or the target.

13. The method of any one of claims 8 to 11, wherein the agent, the suppressor, or the cell is administered by intraparenchymal, intracallosal, intracerebroventricular, intrathecal, intracerebral, intracisternal, or intravenous administration.

14. The method of any one of claims 8 to 13, wherein the cells or the progeny are administered to the forebrain, striatum, and / or cerebellum.

15. 15. The method of any one of claims 8 to 14, wherein the condition is a lysosomal storage disease, an autoimmune demyelinating condition (e.g., multiple sclerosis, neuromyelitis optica, transverse myelitis, and optic neuritis), a vascular leukoencephalopathy (e.g., subcortical stroke, diabetic leukoencephalopathy, hypertensive leukoencephalopathy, age-related white matter disease, and spinal cord injury), a radiation-induced demyelinating condition, a leukodystrophy (e.g., Pelizaeus-Merzbacher disease, Tay-Sachs disease, Sandhoff gangliosidosis, Krabbe disease, metachromatic leukodystrophy, mucopolysaccharidosis, Niemann-Pick disease type A, adrenoleukodystrophy, Canavan disease, vanishing white matter disease, and Alexander disease), or periventricular leukomalacia or cerebral palsy.

16. 16. The method of any one of claims 8 to 15, wherein the condition is Huntington's disease or subcortical dementia.

17. The method of any one of claims 8 to 15, wherein the condition is Parkinson's disease.

18. 10. The method of any one of the preceding claims, wherein the glial progenitor cells are derived from pluripotent stem cells.

19. The method of claim 18, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

20. 20. The method of any one of claims 8 to 19, wherein the cell or the progeny is heterologous, xenogenic, allogeneic, syngeneic, or autologous to the subject.

21. 21. The method of any one of claims 8 to 20, wherein the white matter loss, oligodendrocyte loss, or astrocyte loss is age-related.

22. The method of any one of claims 1 to 6 and 8 to 20, wherein the transcription factor is selected from the group consisting of CTCF, E2F1, and ETV4.

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