Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

• the present application relates to competitive replacement of glial cells and its uses in 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 The central nervous system (CNS) is organized into gray matter, which generally contains the cell bodies and dendrite networks of neurons, and white matter, which consists of axon bundles encased by myelin produced by oligodendrocytes. Loss of white matter, oligodendrocyte, or astrocyte can lead to poor outcomes, including cognitive impairment, dementia, urinary incontinence, gait disturbances, depression, and increased risk of stroke and death.
• This loss involves partial loss of myelin, axons, and oligodendroglial cells; mild reactive astrocytic gliosis; sparsely distributed macrophages as well as stenosis resulting from hyaline fibrosis of arterioles and smaller vessels.
• the present disclosure is directed to overcoming these and other deficiencies in the art. SUMMARY This disclosure addresses the need mentioned above in a number of aspects.
• this disclosure provides a method for rejuvenating, or enhancing the development potential of, a glial progenitor cell or a progeny thereof.
• the method comprises increasing in the glial progenitor cell or the progeny a 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.
• the transcription factor is selected from the group consisting of CTCF, E2F1, and ETV4.
• the target can be selected from the group (as listed Table 1 and Figure 5) consisting of 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, LRRC8A, EXOSC5, RAN, DYNLT1, FDPS, ACTL6A, RPS5, DOLPP1, GGCT, RPS2, S
• the glial progenitor cell is an aged glial progenitor cell.
• the progeny is an oligodendrocyte or an astrocyte.
• the increasing step comprises expressing or introducing in the glial progenitor cell or the progeny the transcription factor or the target.
• the increasing step comprises contacting the glial progenitor cell or the progeny with an agent that increase the level or activity of the transcription factor or the target.
• the method further comprises suppressing in the glial progenitor cell or the progeny a transcription repressor selected from the group consisting of E2F6, ZNF274, MAX, and IKZF3.
• the disclosure provides a cell prepared according to the method described herein or a progeny thereof.
• this disclosure provides a method of 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 an agent that increase 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, or (b) a therapeutically effective amount of the cell prepared according to the method described herein or a progeny thereof.
• the transcription factor is selected from the group consisting of CTCF, E2F1, and ETV4.
• the target is selected from the group mentioned above and listed Table 1 and Figure 5.
• the method further comprises administering to the subject a therapeutically effective amount of a suppressor of a transcription repressor selected from the group consisting of E2F6, ZNF274, MAX, and IKZF3.
• the subject is a human.
• the agent comprises a small molecule compound, an oligonucleotide, a nucleic acid, a peptide, a polypeptide, a CRISPR/Cas system, or an antibody or an antigen-binding portion thereof.
• the nucleic acid encodes the transcription factor or the target mentioned above.
• the suppressor comprises a small molecule compound, an oligonucleotide, a nucleic acid, a peptide, a polypeptide, a CRISPR/Cas system, or an antibody or an antigen-binding portion thereof.
• the agent, suppressor, or cell is administered by intraparenchymal, intracallosal, intraventricular, intrathecal, intracerebral, intracisternal, or intravenous administration.
• the cell or progeny is administered to the forebrain, striatum, and/or cerebellum.
• the condition is a lysosomal storage disease, an autoimmune demyelination 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 demyelination condition, a leukodystrophy (e.g., Pelizaeus-Merzbacher Disease, Tay-Sach Disease, Sandhoff’s gangliosidoses, Krabbe's disease, metachromatic leukodystrophy, mucopolysaccharidoses, Niemann-Pick A disease, adrenoleukodystrophy, Canavan's disease, Vanishing White Matter Disease, and Alexander Disease), or periventricular leukomalacia or cerebral palsy.
• the condition is Huntington’s disease or subcortical dementia. In some embodiments, the condition is Parkinson’s disease.
• the glial progenitor cell is derived from a pluripotent stem cell. In some embodiments, the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell. In some embodiments, the cell or progeny is heterologous, xenogenic, allogeneic, isogenic, or autologous to the subject. In some embodiments, the white matter loss, oligodendrocyte loss, or astrocyte loss is age-related. BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one drawing executed in color.
• FIG. 1A shows an experimental design and analytical endpoints.
• STR striatum (caudate-putamen); LV, lateral ventricle; CTX, cortex. Dashed rectangle (orange) represents inset (B’).
• Scale B, 500 ⁇ m; C’, 100 ⁇ m; D, 50 ⁇ m; E, 10 ⁇ m; I: 100 ⁇ m; I’: 10 ⁇ m.
• FIG. 1B shows that engraftment of WT glia (mCherry + , red) into the striatum of HD chimeras yielded progressive replacement of HD glia (EGFP + , green) creating extensive exclusive domains in their advance. Dashed outlines (white) demarcate the striatal outlines within which human cells were mapped and quantified.
• Figs. 1C-D. show that the border between advancing WT and retreating HD hGPCs was typically well-delineated, such that exclusive domains are formed as WT GPCs (Olig2 + , white) displace their HD counterparts.
• FIG. 1E shows that GPC replacement preceded astrocytic replacement, as within regions colonized by WT hGPCs, stray HD astrocytes (hGFAP + , white) could still be found.
• Fig. 1F shows mapped distributions of human glia in host striata. Human glia were mapped in 15 equidistant sections (5 are shown as example) and reconstructed in 3D. Their distribution was measured radially as a function of distance to the injection site.
• Fig.1G shows rendered examples of mapped striata.
• Fig. 1H shows volumetric quantification indicating that WT gradually replaced their HD counterparts as they expanded from their implantation site; H 1 : WT vs.
• Fig.1I shows that at the boundary between WT and HD glia, a high incidence of Ki67 + (white) cells can be seen exclusively within the WT glial population. I’. Higher magnification of two WT daughter cells at the edge of the competitive boundary.
• Fig.1J shows that quantification of Ki67 + glia within each population as a function of time shows a significant proliferative advantage by WT glia, that is sustained throughout the experiment.
• Figs.2A-2I show that WT glia acquired a dominant competitor transcriptional profile in the face of resident HD glia.
• Fig.2A. shows an experimental design.
• Figs. 2B and C show that uniform manifold approximation projection (UMAP) visualization of the integrated (B) and split by group (C) scRNA-seq data identified six major cell populations.
• Fig.2D shows tacked bar plot proportions of cell types in each group.
• Fig.2E shows cell cycle analysis notched box plots of cycling GPCs and GPCs in the G2/M phase.
• the box indicates the interquartile range
• the notch indicates the 95% confidence interval with the median at the center of the notch
• the error bars represent the minimum and maximum non-outlier values.
• Fig.2F shows a Venn diagram of pairwise differentially expressed GPC genes (Log2 fold change > 0.15, adjusted p-value ⁇ 0.05).
• Fig. 2G shows curated ingenuity pathway analysis of genes differentially expressed between GPC groups. The size of circles represent p-value while the shading indicates activation Z-Score with red being more active in the upper group and green being more active in the lower group.
• Fig.2I shows violin plots of pairwise differentially expressed GPC ribosomal gene log2 fold changes. Comparisons between groups in (E) utilized Dunn tests following a Kruskal- Wallis test with multiple comparisons adjusted via the Benjamini-Hochberg method. Figs. 3A-3I show that differences in cell age were sufficient to drive competitive repopulation of humanized striata. B-C. STR, striatum (caudate-putamen); LV, lateral ventricle; CTX, cortex.).
• Fig.3A shows an experimental design and analytical endpoints.
• Fig.3B shows that engraftment of younger WT glia (EGFP + , green) into the striatum of WT chimeras yielded selective replacement of their aged counterparts (mCherry + , red). Dashed outlines demarcate the striatal regions within which human cells were mapped and quantified.
• Fig.3C shows WT chimeric control, engrafted only at birth.
• Fig.3D shows rendered examples of mapped striata.
• Fig.3G shows that at the interface between young and aged WT glia, a higher incidence of Ki67 + (white) cells can be seen within the younger population.
• Fig.3H shows the inset color split represented in the dashed square in Fig.3G.
• Figs. 4A-4I show that WT glia acquired a dominant transcriptional profile when confronting their aged counterparts.
• Fig.4A shows an experimental design.
• Figs.4B-C show uniform manifold approximation projection (UMAP) visualization of the integrated (B) and split by group (C) scRNA-seq data identifies six major cell populations.
• Fig.4D shows stacked bar plot proportions of cell types in each group.
• Fig.4E shows cell cycle analysis notched box plots of cycling GPCs and GPCs in the G2/M phase. The box indicates the interquartile range, the notch indicates the 95% confidence interval with the median at the center of the notch, and the error bars represent the minimum and maximum non-outlier values.
• Fig.4F shows a Venn diagram of pairwise differentially expressed GPC genes (Log2 fold change > 0.15, adjusted p-value ⁇ 0.05).
• Fig. 4G shows curated Ingenuity Pathway analysis of genes differentially expressed between GPC groups. The size of circles represent p-value while the shading indicates activation Z-Score with red being more active in the upper group and green being more active in the lower group.
• Fig.4H shows heatmap of curated pairwise differentially expressed GPC genes.
• Fig.4I shows violin plots of pairwise differentially expressed GPC ribosomal gene log2 fold changes. Comparisons between groups in E utilized Dunn tests, following a Kruskal- Wallis test with multiple comparisons adjusted via the Benjamini-Hochberg method.
• Figs.5A-5F show transcriptional signature of competitive advantage.
• Fig.5A shows schematic of a protocol for identifying transcription factors (TFs) linked specifically to competitive advantage.
• Fig.5B shows a box plot of identified WGCNA module eigengene of interest (blue) in competing and non-competing cells.
• Fig.5C shows that GSEA highlighted prioritized transcription factors whose regulons were enriched for upregulated genes in dominant young WT cells.
• Fig. 5D shows analysis of the relative contribution of each biological factor (age vs genotype) towards the composition of each module eigengene.
• Fig. 5E shows important transcription factors, as predicted by SCENIC to establish competitive advantage, and their relative activities across groups.
• Fig.5A shows schematic of a protocol for identifying transcription factors (TFs) linked specifically to competitive advantage.
• Fig.5B shows a box plot of identified WGCNA module eigengene of interest (blue) in competing and non-competing cells.
• FIG. 5F shows regulatory network including downstream targets and their functional signaling pathways. Target expressions are controlled by at least one other important transcription factor in (E).
• NES Network Enrichment Score.
• Fig.6A shows an experimental design and analytical endpoints.
• Fig.6B shows that neonatally engrafted HD glia (EGFP + , green) expanded within the murine striatum yielding substantial humanization of the tissue over time. Dashed lines demarcate the striatal borders within which human cells were mapped and quantified.
• Figs.6C-D shows that their expansion was concomitant with an increase in the number of HD glia harbored in the murine striatum over time (C) at the cost of their Ki67 + proliferative cell pool (D).
• Fig. 6E shows a strategy employed to assess the extent of striatal humanization 36 weeks following neonatal implantation of HD GPCs.
• HD cell distribution was mapped in 15 equidistant sagittal sections (5 are shown for example) and reconstructed in 3D for analysis.
• Fig. 6F shows a rendered example of a mapped and reconstructed striatum for volumetric analysis.
• Figs.6H-J show that as they colonized the murine striatum, HD glia either expanded and persisted as Olig2 + GPCs (arrows point to Olig2 + /EGFP + (red/green) cells) or differentiated into hGFAP + (red) astrocytes.
• Figs.7A-7H show replacement of HD by WT glial progenitor cells yielded proportional phenotypic replacement. Scale: D-E, G-H, 50 ⁇ m.
• Fig. 7A shows an experimental design and analytical endpoints for the WT Control group.
• Fig.7B shows stereological estimations demonstrating that the total number of HD glia progressively decreased relatively to HD chimera controls as WT glia expanded within the humanized striatum; 2-way ANOVA with ⁇ idák’s multiple comparisons test.
• Figs.7C–E show that WT glia expanded as Olig2 + (white) GPCs displacing their HD counterparts.
• Figs.7F-H show within areas 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 striatal dominance. Orange arrows point to co-labelled cells.
• Figs.8A-8D show that adult-engrafted hGPCs more rapidly dominated already-resident mouse than human hGPCs.
• Fig. 8A shows an experimental design and analytical endpoints for the WT Control group.
• Fig.8B shows that engraftment of WT glia (mCherry + , red) into the adult striatum of Rag1 (-/-) mice yielded substantial humanization of the murine striatum over time.
• FIGs.8C-D show volumetric quantifications demonstrating that adult-transplanted WT glia infiltrated and dispersed throughout the murine striatum over time (C), and (D), and that they do so significantly more broadly than do those grafted into already HD chimeric mice.
• Figs.9A-9F show eexpression of fluorescent transgenes did not influence competitive dominance.
• Fig.9A shows an experimental design for mice that received a 1:1 mixture of mCherry- tagged (WT-mCherry) and untagged (WT-untagged) WT glia.
• Fig.9B shows iimmunolabeling against human nuclear antigen (hN) demonstrating that both WT-mCherry (mCherry + hN + , red, white) and WT-untagged (mCherry- EGFP- hN + , white) glia expanded within the previously humanized striatum, progressively displacing HD glia (EGFP + hN + , green, white).
• Fig.9C shows that vast homotypic domains were formed as mixed WT glia expanded and displaced resident HD glia.
• Fig. 9D shows that in contrast, isogenic WT-mCherry and WT-untagged were found admixing.
• Fig.9E shows that as previously noted, within WT glia dominated domains, only more complex astrocyte-like HD glia could be found, typically within white matter tracts.
• Figs.10A-10D show human GPC chimeric mice were established by either a neonatal striatal injection of HD EGFP-tagged glial (Fig.
• FIG. 11A-11C show that aged human glia were eliminated by their younger counterparts through induced apoptosis. Scale: A –100 ⁇ m, B – 50 ⁇ m.
• Fig. 11A shows that at the border between young (EGFP + , green) and aged WT glia (mCherry + , red), a higher incidence of apoptotic TUNEL + (white) cells were apparent in the aged population.
• Fig.11B shows higher magnification of a competitive interface between these distinct populations demonstrating resident glia selectively undergoing apoptosis.
• Fig. 11A shows that at the border between young (EGFP + , green) and aged WT glia (mCherry + , red), a higher incidence of apoptotic TUNEL + (white) cells were apparent in the aged population.
• Fig.11B shows higher magnification of a competitive interface between these distinct populations demonstrating resident glia selectively undergoing apoptosis.
• compositions and methods for treating a condition mediated by oligodendrocyte loss, astrocyte loss, or white matter loss including age-related oligodendrocyte loss, age-related astrocyte loss, or age-related white matter loss.
• This disclosure also relates to (a) rejuvenating a glial progenitor cell or a progeny thereof or (b) enhancing the development potential of a glial progenitor cell or a progeny thereof.
• glial progenitor cells refers to cells having the potential to differentiate into cells of the glial lineage such as oligodendrocytes and astrocytes. Glia 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.
• 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 transmission in the nervous system.
• “Glial cells” as used herein encompasses fully differentiated cells of the glial lineage, such as oligodendrocytes or astrocytes, and well as glial progenitor cells, each of which can be referred to as macroglial cells.
• glial progenitor cells are also known as oligodendrocyte progenitor cells or NG2 cells.
• Conditions Mediated by Loss of While Matter/Oligodendrocytes/Astrocytes and Related Disorders Certain aspects of this disclosure relate to compositions and methods for treating a condition or disorder mediated by oligodendrocyte loss, astrocyte loss, or white matter loss. Such a condition often entails a deficiency in myelin in central nerve system (“CNS”). Examples of such conditions or disorders include any diseases or conditions related to demyelination, insufficient myelination and remyelination, or dysmyelination in a subject.
• CNS central nerve system
• Such a condition or disorder can be inherited, acquired, or due to the ageing process, i.e., age- related.
• the condition is that of age-related white matter disease defined as or characterized by oligodendrocyte loss, astrocyte loss, or white matter atrophy in the setting of normal otherwise healthy aging.
• ageing represents the accumulation of changes in a human being over time and can encompass physical, psychological, and social changes. Ageing increases the risk of human diseases such as cancer, diabetes, cardiovascular disease, stroke, and many more, including demyelination in the CNS, which are often seen in various neurodegenerative diseases.
• the condition or disorder is mediated by age-related oligodendrocyte loss, age-related astrocyte loss, or age-related white matter loss.
• Demyelination in the CNS may occur in response to genetic mutation (leukodystrophies), autoimmune disease (e.g., multiple sclerosis), or trauma (e.g., traumatic brain injury, spinal cord injury, or ischemic stroke).
• Perturbation of myelin function may play a critical role in neurologic 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.
• ASSD Autism Spectrum Disorder
• Alzheimer’s disease Huntington’s disease
• Multiple System Atrophy Parkinson’s disease
• Fragile X syndrome schizophrenia
• various leukodystrophies various leukodystrophies.
• Leukodystrophies are a group of rare, primarily inherited neurological disorders that result from the abnormal production, processing, or development of myelin and are the result of genetic defects (mutations). Some forms are present at birth, while others may not produce symptoms until a child becomes older. A few 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 diseases (ADLD), 4H Leukodystrophy (Pol III- related leukodystrophy), Zellweger Spectrum Disorders (ZSD), Childhood ataxia with central nervous system hypomyelination or CACH (also called vanishing white matter disease or VWMD), Cerebrotendinous xanthomatosis (CTX), Alexander disease (AXD), SOX10- associated peripheral demyelinating neuropathy, central dysmyelinating le
• Suitable subjects for treatment in accordance with the methods described herein include any human subject having 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.
• the condition mediated by a deficiency in myelin is selected from the group consisting of pediatric leukodystrophies, the lysosomal storage diseases, congenital dysmyelination, cerebral palsy, inflammatory demyelination, post-infectious and post-vaccinial leukoencephalitis, radiation- or chemotherapy induced demyelination, and vascular demyelination.
• condition mediated by a deficiency in myelin requires myelination.
• condition mediated by a deficiency in myelin requires remyelination.
• condition requiring remyelination is selected from the group consisting of multiple sclerosis, neuromyelitis optica, transverse myelitis, optic neuritis, subcortical stroke, diabetic leukoencephalopathy, hypertensive leukoencephalopathy, age-related white matter disease, white matter dementia, Binswanger's disease, spinal cord injury, radiation- or chemotherapy induced demyelination, post-infectious and post-vaccinial leukoencephalitis, periventricular leukomalacia, and cerebral palsy.
• the condition mediated by a deficiency in myelin is neurodegenerative disease.
• the neurodegenerative disease is Huntington’s disease.
• Huntington’s disease is an autosomal dominant neurodegenerative disease characterized by a relentlessly progressive movement disorder with devastating psychiatric and cognitive deterioration.
• Huntington's disease is associated with a consistent and severe atrophy of the neostriatum which is related to a marked loss of the GABAergic medium-sized spiny projection neurons, the major output neurons of the striatum.
• Huntington's disease is characterized by abnormally long CAG repeat expansions in the first exon of the Huntingtin gene.
• neurodegenerative diseases treatable in accordance with the present application include frontotemporal dementia, Alzheimer’s disease, Parkinson’s disease, multisystem atrophy, and amyotrophic lateral sclerosis.
• the condition mediated by a deficiency in myelin is a neuropsychiatric disease.
• the neuropsychiatric disease is schizophrenia. Schizophrenia is a serious mental illness that affects how a person thinks, feels, and behaves.
• the symptoms of schizophrenia generally fall into the following three categories: (1) psychotic symptoms including altered perceptions, (2) negative symptoms including loss of motivation, disinterest and lack of enjoyment, and (3) cognitive symptoms including problems in attention, concentration, and memory.
• Other neuropsychiatric diseases treatable in accordance with the present application include autism spectrum disorder and bipolar disorder.
• the pathological processes underlying many of these disorders remain poorly understood and few disease-modifying therapies exist. There are unmet needs for therapeutics for treating these disorders.
• This disclosure address these needs in a number of ways, such as competitive replacement of aged or older glial progenitor cells in the brain and rejuvenation of glial progenitor cells or their progeny cells.
• Competitive Replacement of Glial Progenitor Cells in Adult Brain Some aspects of this disclosure relate to competitive replacement of glial progenitor cells. Competition among cell populations in development and oncogenesis is well- established, and yet competition among cells in the adult brain has remained little-studied. In particular, it is unknown whether allografted human glia can outcompete diseased cells to achieve therapeutic replacement in the adult human brain.
• the WT hGPCs outcompeted and ultimately eliminated their human HD counterparts, repopulating the host striata with healthy glia.
• Single-cell RNA-Seq revealed that WT donor hGPCs acquired a YAP1/MYC/E2F- defined dominant competitor phenotype upon interaction with the resident HD-derived glia.
• Astrocytic and oligodendrocytic pathology have been associated with the genesis and progression of a number of both neurodegenerative and neuropsychiatric disorders, including conditions as varied as amyotrophic lateral sclerosis (ALS) 1-4 , Huntington’s disease (HD) 5-10 and Parkinson’s disease 11, 12 , as well as schizophrenia and bipolar disease 13-19 .
• ALS amyotrophic lateral sclerosis
• HD Huntington’s disease
• Parkinson’s disease 11 12 as well as schizophrenia and bipolar disease 13-19 .
• the replacement of diseased glia by healthy glial progenitor cells (hGPCs) might provide real therapeutic benefit 20 , given their ability to disperse and colonize their hosts while giving rise to new astrocytes and oligodendrocytes.
• human GPCs can outcompete and replace their murine counterparts in a variety of experimental therapeutic models 21-23 , it has remained unclear if allografted human GPCs can replace other human cells, diseased or otherwise.
• human glial-chimeric mice 24 were used to model competition between healthy and diseased human glia in vivo, by engrafting healthy hGPCs into the striata of adult mice neonatally chimerized with hGPCs derived from subjects with HD.
• HD is a prototypic monogenic neurodegenerative disease, resulting from the expression of a mutant, CAG-repeat expanded, Huntingtin (mHTT) gene 25 .
• scRNA-seq Single cell RNA sequence analysis
• glial pathology In light of the contribution of glial pathology to a broad variety of neurodegenerative and neuropsychiatric disorders 36, 37 , it was sought here to establish the relative fitness of wild- type and both diseased and aged human GPCs in vivo, so as to assess the potential for allogeneic glial replacement as a therapeutic strategy.
• Some parts of this disclosure focused on Huntington’s disease, given the well-described role of glial pathology in HD 5, 8, 10, 38, 39 . It was found that when WT hGPCs were introduced into brains already chimerized with HD hGPCs, that the WT cells out-competed and ultimately replaced the already-resident HD glial progenitors.
• the selective expansion of the healthy cells was associated with the active elimination of the resident HD glia, and was further supported by the proliferative advantage of the healthy donor cells relative to their already-resident diseased counterparts.
• Single-cell RNA sequencing revealed that the dominance of healthy WT hGPCs encountering HD glia in these adult chimeric mouse brains was linked to their expression of a transcriptional signature characteristic of competitively dominant cells in invertebrate systems.
• transplanted young WT hGPCs acquired the gene expression signature of a dominant competitor phenotype in vivo, whether challenged by already-resident older HD or isogenic WT hGPCs; indeed, the analysis described herein suggested that cellular youth was an even stronger determinant of competitive fitness than was disease genotype.
• cell replacement was driven by a recapitulation of developmental cell competition, an evolutionarily conserved selection process by which less fit clones are sensed and eliminated from a tissue by their fitter neighbors 40-43 , but as manifested here dynamically in the adult brain. This process has been shown in a variety of systems to comprise the active elimination of relatively slowly growing cells by their faster growing, more competitively fit neighbors 44-48 .
• WT hGPCs typically expanded from their implantation sites in an advancing proliferative wave. These younger hGPCs largely eliminated their hitherto stably resident – and hence older - counterparts, whether the latter were mHTT-expressing HD cells, or isogenic WT cells that had been transplanted months earlier. In both cases, the younger cells ultimately recolonized their host brains with healthy new hGPCs (Figs.1 and 3), and in both cases the younger donor cells differentially expressed gene sets associated with competitive dominance (Figs.2, 4 and 5).
• the competitive replacement of resident glia by younger hGPCs observed resembles that of mouse glial replacement by implanted human GPCs, as their expansion within the murine brain is also sustained by a relative proliferative advantage, and progresses with the elimination of their murine counterparts upon contact 22 .
• the winning population of young WT hGPCs appears to trigger the apoptotic death and local elimination of the resident losing population, whether comprised of older isogenic WT or sibling HD cells.
• the relative localization of dying host cells to the advancing wavefronts of younger WT cells suggests that the latter trigger the death of already-resident hGPCs, likely via contact-dependent means.
• glial progenitor cell delivery and glial replacement offers a viable and broadly applicable strategy towards the cell-based treatment of those diseases of the brain in which glial cells are causally involved.
• Rejuvenation of Glial Progenitor Cells or Progenies Thereof Some aspects of this disclosure relate to rejuvenation of glial progenitor cells or their progeny cells.
• Human glial progenitor cells emerge during the 2 nd trimester to colonize the brain, in which a parenchymal pool remains throughout adulthood. While fetal hGPCs are highly migratory and proliferative, their expansion competence diminishes with age, as well as following demyelination-associated turnover.
• the present disclosure provides a method of rejuvenating a glial progenitor cell or a progeny thereof. Also provided is a method of enhancing the development potential of, a glial progenitor cell or a progeny thereof.
• Each of the methods comprises upregulating or increasing in the glial progenitor cell or the progeny a 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. Shown below are some examples of human CEBPZ, CTCF, E2F1, MYC, NFYB, and ETV4 proteins.
• CEBPZ, CTCF, E2F1, MYC, NFYB, or ETV4 of non-human species can also be used in the expression cassette, genetic construct, vector, composition, or method disclosed herein.
• the term CEBPZ, CTCF, E2F1, MYC, NFYB, or ETV4 also encompasses all the alternatively spliced variants, isoforms, functional fragments, or derivatives that substantially retain transcription factor activity of the CEBPZ, CTCF, E2F1, MYC, NFYB, or ETV4 described herein.
• a functional fragment or derivative retains at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of its transcription factor activity.
• a CEBPZ, CTCF, E2F1, MYC, NFYB, or ETV4 transcription factor can include conservative amino acid substitutions that do not substantially alter its activity. Suitable conservative substitutions of amino acids are known to those of skill in this art and may be made generally without altering the biological activity of the resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. Conservative and non-conservative amino acid substitutions have been described herein. As used herein, the term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter the activity of one of the above-described protein.
• Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been 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, e.g., a protein having one or more substitutions, point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof. It retains substantially the activity to of the parent peptide, polypeptide, or protein (such as those disclosed herein).
• a conservative modification or functional equivalent is at least 60% (e.g., any number between 60% and 100%, inclusive, e.g., 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) identical to a parent (e.g., one of the human or non-human CEBPZ, CTCF, E2F1, MYC, NFYB, or ETV4 sequences or the targets disclosed herein).
• a parent e.g., one of the human or non-human CEBPZ, CTCF, E2F1, MYC, NFYB, or ETV4 sequences or the targets disclosed herein.
• Amino acid substitutions can be made, in some cases, by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target sit; or (c) the bulk of the side chain.
• 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 influence chain orientation (glycine and proline); and (6) aromatic amino acids (tryptophan, tyrosine, and phenylalanine). Substitutions made within these groups can be considered conservative substitutions.
• substitutions include, without limitation, substitution of valine for alanine, lysine for arginine, glutamine for asparagine, glutamic acid for aspartic acid, serine for cysteine, asparagine for glutamine, aspartic acid for glutamic acid, proline for glycine, arginine for histidine, leucine for isoleucine, isoleucine for leucine, arginine for lysine, leucine for methionine, leucine for phenylalanine, glycine for proline, threonine for serine, serine for threonine, tyrosine for tryptophan, phenylalanine for tyrosine, and/or leucine for valine.
• substitutions are shown in the table below.
• Amino acid substitutions may be introduced into a human CEBPZ, CTCF, E2F1, MYC, NFYB, or ETV4 and the products screened for retention of the biological activity of the parent protein.
• the methods described herein can also achieved by upregulating or increasing in a glial progenitor cell or the progeny a level or activity of one or more of targets of one or more of the transcription factors described above.
• targets include those listed in Table 1 and Figure 5, including but not limited to: 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, LRRC8A, EXOSC5, RAN, DYNLT1, FDPS, ACTL6A, RPS5, DOLPP1, GGCT, RPS2, SYCE1
• one or more of the targets may also have highly conserved protein domains, conserved in several species including human, mouse, rat, chicken, fish and Drosophila. Accordingly, a homologue of non-human species can also be used in the expression cassette, genetic construct, vector, composition, or method disclosed herein. Accordingly, each of the target also encompasses functional fragments or derivatives that substantially retain the respective activity of the target described herein. Typically, a functional fragment or derivative retains at least 50% of 60%, 70%, 80%, 90%, 95%, 99% or 100% of its parent’s activity. It is also intended that a target may include conservative amino acid substitutions that do not substantially alter its activity.
• a method disclosed herein can further comprise suppressing in the glial progenitor cell or the progeny a transcription repressor selected from the group consisting of E2F6, ZNF274, MAX, and IKZF3.
• nucleic acid sequences 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 references.
• this suppressing can be achieved by administering to a subject in need thereof or a target cell in need thereof a suppressor or inhibitor of one or more of the transcription repressor.
• a suppressor or inhibitor can comprise or be a small molecule compound, an oligonucleotide, a nucleic acid, a peptide, a polypeptide, a CRISPR/Cas system, or an antibody or an antigen-binding portion thereof.
• the suppressor/inhibitor examples include activators, agonists, or potentiators of the related YAP or MYC pathway signaling pathways (e.g., the Hippo signaling pathway). Various activators for this signaling pathway are known in the art.
• the suppressor is an inhibitory nucleic acid or interfering nucleic acid, such as siRNA, shRNA, miRNA, antisense oligonucleotides (ASOs), 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 references.
• suppressing or knocking down of one or more of the repressor genes described herein can also be achieved via a CRISPR-Cas guided nuclease using a CRISPR/Cas system and related methods known in the art. Examples include those described in PCT/US22/78344 and PCT/US22/78356, the contents of which are incorporated by references.
• Expression Cassettes and Expression Vectors The disclosure also provides an expression cassette, comprising or consisting of a recombinant nucleic acid encoding a transcription factor or a target thereof described above. Where such recombinant nucleic acid may not already comprise a promoter, the expression cassette may additionally comprise a promoter.
• an expression cassette according to the present disclosure comprises, in 5' to 3' direction, a promoter, a coding sequence, and optionally a terminator or other elements.
• the expression cassette allows an easy transfer of a nucleic acid sequence of interest into an organism, preferably a cell and preferably a disease cell.
• the expression cassette of the present disclosure is preferably comprised in a vector.
• the vector of the present disclosure allows to transform, transfect, transduce, infect, or introduce into a cell with a nucleic acid sequence of interest.
• the 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 such as to allow for the expression of the nucleic acid sequence of interest.
• Exogenous genetic material e.g., a nucleic acid, an expression cassette, or an expression vector encoding one or more therapeutic or inhibitory proteins or RNAs
• Various expression vectors i.e., vehicles for facilitating delivery of exogenous genetic material into a target cell are known to one of ordinary skill in the art.
• exogenous genetic material refers to a nucleic acid or an oligonucleotide, either natural or synthetic, that is not naturally found in the cells; or if it is naturally found in the cells, it is not transcribed or expressed at biologically significant levels by the cells.
• exogenous genetic material includes, for example, a non-naturally occurring nucleic acid that can be transcribed into an RNA.
• transfection of cells refers to the acquisition by a cell of new genetic material by incorporation of added nucleic acid (DNA, RNA, or a hybrid thereof) without use of a viral delivery vehicle. Thus, transfection refers to the introducing of nucleic acid into a cell using physical or chemical methods.
• transfection techniques are known to those of ordinary skill in the art including: calcium phosphate nucleic acid co-precipitation, strontium phosphate nucleic acid co-precipitation, DEAE-dextran, electroporation, cationic liposome- mediated transfection, and tungsten particle-facilitated microparticle bombardment.
• transduction of cells refers to the process of transferring nucleic acid into a cell using a DNA or RNA virus.
• An RNA virus e.g., a retrovirus
• Exogenous genetic material contained within the virus can be incorporated into the genome of the transduced cell.
• a cell that has been transduced with a chimeric DNA virus may not have the exogenous genetic material incorporated into its genome but may be capable of expressing the exogenous genetic material that is retained extrachromosomally within the cell.
• the exogenous genetic material may include a heterologous gene (coding for a therapeutic RNA or protein) together with a promoter to control transcription of the new gene.
• the promoter characteristically has a specific nucleotide sequence necessary to initiate transcription.
• the exogenous genetic material further includes additional sequences (i.e., enhancers) required to obtain the desired gene transcription activity.
• the exogenous genetic material may introduced into the cell genome immediately downstream from the promoter so that the promoter and coding sequence are operatively linked so as to permit transcription of the coding sequence.
• a retroviral expression vector may include an exogenous promoter element to control transcription of the inserted exogenous gene.
• exogenous promoters include both constitutive and inducible promoters. Naturally-occurring constitutive promoters control the expression of essential cell functions. As a result, a gene under the control of a constitutive promoter is expressed under all conditions of cell growth.
• Exemplary constitutive promoters include the promoters for the following genes that encode certain constitutive or "housekeeping" functions: hypoxanthine phosphoribosyl transferase, dihydrofolate reductase, adenosine deaminase, phosphoglycerol kinase, pyruvate kinase, phosphoglycerol mutase, the actin promoter, ubiquitin, elongation factor-1 and other constitutive promoters known to those of skill in the art.
• many viral promoters function constitutively in eucaryotic cells.
• any of the above-referenced constitutive promoters can be used to control transcription of a heterologous gene insert.
• Genes that are under the control of inducible promoters are expressed only in, or largely controlled by, the presence of an inducing agent, (e.g., transcription under control of the metallothionein promoter is greatly increased in presence of certain metal ions).
• Inducible promoters include responsive elements (REs) which stimulate transcription when their inducing factors are bound.
• REs for serum factors there are REs for serum factors, steroid hormones, retinoic acid and cyclic AMP.
• Promoters containing a particular RE can be chosen in order to obtain an inducible response and in some cases, the RE itself may be attached to a different promoter, thereby conferring inducibility to the recombinant gene.
• the appropriate promoter constitutitutive versus inducible; strong versus weak
• the gene encoding the therapeutic agent is under the control of an inducible promoter
• delivery of the therapeutic agent in situ is triggered by exposing the genetically modified cell in situ to conditions for permitting transcription of the therapeutic agent, e.g., by injection of specific inducers of the inducible promoters which control transcription of the agent.
• in situ expression by genetically modified cells of a therapeutic agent encoded by a gene under the control of the metallothionein promoter is enhanced by contacting the genetically modified cells with a solution containing the appropriate (i.e., inducing) metal ions in situ.
• the amount of therapeutic agent that is delivered in situ is regulated by controlling such factors 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 that are inserted into the cell; (3) the number of transduced/transfected cells that are administered (e.g., implanted) to 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 are left in place; and (7) the production rate of the therapeutic agent by the genetically modified cell.
• factors 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 that are inserted into the cell; (3) the number of transduced/transfected cells that are administered
• the expression vector may include a selection gene, for example, a neomycin resistance gene or a fluorescent protein gene, for facilitating selection of cells that have been transfected or transduced with the expression vector.
• the cells are transfected with two or more expression vectors, at least one vector containing the gene(s) encoding the therapeutic agent(s), the other vector containing a selection gene.
• a coding sequence of the present disclosure can be inserted into any type of target or host cell.
• the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
• the expression vector can be transferred into a host cell by physical, chemical, or biological means.
• Carrier/Delivery of polynucleotides As disclosed herein, the polynucleotides or nucleic acid molecules described above can be used for treating a disorder in a subject.
• this disclosure provides systems and methods for delivery of the polynucleotides to a target cell or a subject.
• Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like.
• Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).
• Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
• Viral vectors and especially 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 lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
• Chemical means for introducing a polynucleotide into a host cell 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).
• a liposome e.g., an artificial membrane vesicle.
• the polynucleotides or nucleic acids described herein e.g., protein-coding nucleic acids, inhibitory nucleic acids, those encoding a CRISPR-Cas system, expression cassettes, and expression vectors
• Methods for the delivery of nucleic acid molecules are known in the art. See, e.g., U.S. Pat. No.
• Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see for example 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 for example U.S. Pat. No.
• the present application provides carrier systems containing the nucleic acid molecules described herein.
• the carrier system is a lipid-based carrier system, cationic lipid, or liposome nucleic acid complexes, a liposome, a micelle, a virosome, a lipid nanoparticle or a mixture thereof.
• the carrier system is a polymer-based carrier system such as a cationic polymer-nucleic acid complex.
• the carrier system is a cyclodextrin-based carrier system such as a cyclodextrin polymer-nucleic acid complex.
• the carrier system is a protein-based carrier system such as a cationic peptide-nucleic acid complex.
• the carrier system in a lipid nanoparticle formulation.
• Lipid nanoparticle (“LNP”) formulations described herein can be applied to any nucleic acid molecules (e.g., an RNA molecule) or combination of nucleic acid molecules described herein.
• the nucleic acid molecules described herein are formulated as a lipid nanoparticle composition such as is described in U.S. Patent Nos.7514099 and 7404969.
• this application features a composition comprising a nucleic acid molecule formulated as any of formulation as described in US 20120029054, such as 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.
• this disclosure features conjugates and/or complexes of nucleic acid molecules described herein.
• conjugates and/or complexes can be used to facilitate delivery of nucleic acid molecules into a biological system, such as a cell.
• the conjugates and complexes provided by hereon can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the disclosure.
• Non-limiting, examples of such conjugates are described in e.g., U.S. Pat. Nos.7,833,992; 6,528,631; 6,335,434; 6, 235,886; 6,153,737; 5,214,136; 5,138,045.
• PEG polyethylene glycol
• the attached PEG can be any molecular weight, preferably from about 100 to about 50,000 daltons (Da). Accordingly, the disclosure features compositions or formulations comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes) and nucleic acid molecules described herein. See, e.g., WO 96/10391, WO 96/10390, and WO 96/10392).
• the nucleic acid molecules can also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneimine- polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine- polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives.
• polyethyleneimine and derivatives thereof such as polyethyleneimine- polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine- polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives.
• the nucleic acid molecules can be formulated in the manner described in U.S. 20030077829.
• nucleic acid molecules described herein can be complexed with membrane disruptive agents such as those described in U.S. 20010007666.
• the membrane disruptive agent or agents and the molecule can be complexed with a cationic lipid or helper lipid molecule, such as those lipids described in U.S. Pat. No. 6,235,310.
• nucleic acid molecules described herein can be complexed with delivery systems as described in U.S. Patent Application Publication Nos.2003077829; 20050287551; 20050164220; 20050191627; 20050118594; 20050153919; 20050085486; and 20030158133; and IWO 00/03683 and WO 02/087541.
• a liposomal formulation described herein can comprise a nucleic acid molecule described herein formulated or complexed with compounds and compositions described in U.S. Pat. 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.
• nucleic acid molecules described above can be used for treating a disorder in a subject.
• Vectors such as recombinant plasmids and viral vectors as discussed above can be used to deliver a therapeutical agent described herein.
• Delivery of the vectors can be systemic, such as by intravenous or intra-muscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell.
• recombinant vectors can also be administered directly or in conjunction with a suitable delivery reagents, including, for example, the Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes lipid-based carrier system, cationic lipid, or liposome nucleic acid complexes, a micelle, a virosome, a lipid nanoparticle.
• a suitable delivery reagents including, for example, the Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes lipid-based carrier
• a polynucleotide encoding the RNA molecule or protein can be inserted into, or encoded by, vectors such as plasmids or viral vectors.
• the polynucleotide is inserted into, or encoded by, viral vectors.
• Viral vectors may be Herpesvirus (HSV) vectors, retroviral vectors, adenoviral vectors, AAV vectors, lentiviral vectors, and the like.
• the viral vectors are AAV vectors.
• the RNA may be encoded by a retroviral vector (See, e.g., U.S. Pat.
• Lentiviral vectors Lentiviruses, such as HIV, are “slow viruses.” Vectors derived from lentiviruses can be expressed long-term in the host cells after a few administrations to the patients, e.g., via ex vivo transduced stem cells or progenitor cells. For most diseases and disorders, including genetic diseases, cancer, and neurological disease, long-term expression is crucial to successful treatment. Regarding safety with lentiviral vectors, a number of strategies for eliminating the ability of lentiviral vectors to replicate have now been known in the art.
• Lentiviral vectors are particularly suitable to achieving long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as CNS cells.
• a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO01/96584 and WO01/29058; and U.S. Pat. No. 6,326,193).
• a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
• CMV immediate early cytomegalovirus
• Another example of a suitable promoter is EF1a.
• 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, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.
• SV40 simian virus 40
• MMTV mouse mammary tumor virus
• HSV human immunodeficiency virus
• LTR long terminal repeat
• MoMuLV promoter MoMuLV promoter
• an avian leukemia virus promoter an Epstein-Barr virus immediate early promoter
• Rous sarcoma virus promoter as well as human gene promoters such as
• Inducible promoters include, but are not limited to a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
• the present disclosure provides a recombinant lentivirus capable of infecting dividing and non-dividing cells, such oligodendrocytes, astrocytes, or glial progenitor cells.
• the virus is useful for the in vivo and ex vivo transfer and expression of nucleic acid sequences.
• Lentiviral vectors of the present disclosure may be lentiviral transfer plasmids or infectious lentiviral particles.
• Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid to a variety of cell types in vivo, and have been used extensively in gene therapy protocols, including for targeting genes to neural cells and glial cells.
• Various replication defective adenovirus and minimum adenovirus vectors have been described for nucleic acid therapeutics (See, e.g., PCT Patent Publication Nos.
• adenoviral vectors may also be used to deliver therapeutic molecules of the present disclosure to cells.
• Adeno-Associated Virus The adeno-associated virus is a widely used gene therapy vector due to its clinical safety record, non-pathogenic nature, ability to infect non-dividing cells (like 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 of hemophilia B, spinal muscular atrophy, alpha 1 antitrypson, and Leber congenital amaurosis. Because of their safety, nonpathogenic 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 infusion, these serotypes exhibit a dominant neuronal tropism and expression in oligodendrocytes is low, especially when gene expression is driven by a constitutive promoter, which restricts their potential for use in treating white matter diseases.
• AAV1/2, AAV2, and AAV8 have been shown transduce oligodendrocytes. Reliance on cell-specific promoters for expression specificity allows for the possibility of nonselective cellular uptake and leaky transgene expression through cryptic promoter activity in non–oligodendrocyte lineage cells.
• the approach described herein to alleviate these issues includes using AAV serotypes with high tropism for oligodendrocytes or astrocytes or glial progenitor cells.
• AAV/Olig001 a chimeric AAV capsid with strong selectivity for oligodendrocytes
• Adeno-associated virus and/or “AAV” refer to parvoviruses with a linear single-stranded DNA genome and variants thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. Parvoviruses, including AAV, are useful as gene therapy vectors as they can penetrate a cell and introduce a nucleic acid (e.g., transgene) into the nucleus.
• the introduced nucleic acid forms circular concatemers that persist as episomes in the nucleus of transduced cells.
• a transgene is inserted in specific sites in the host cell genome. Site-specific integration, as opposed to random integration, is believed to likely result in a predictable long-term expression profile.
• the insertion site of AAV into the human genome is referred to as AAVS1.
• AAV is not associated with any pathogenic disease in humans
• a nucleic acid delivered by AAV can be used to express a therapeutic RNA or polypeptide for the treatment of a disease, disorder and/or condition in a human subject.
• Multiple serotypes of AAV exist in nature with at least fifteen wild type serotypes having been identified from humans thus far (i.e., AAV1-AAV15).
• Naturally occurring and variant serotypes are distinguished by having a protein capsid that is serologically distinct from other AAV serotypes.
• Examples include AAV1, AAV2, AAV, AAV3 (including AAV3A and AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV12, AAVrh10, AAVrh74 (see WO 2016/210170), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, and recombinantly produced variants (e.g., capsid variants with insertions, deletions and substitutions, etc.), such as variants referred to as AAV2i8, NP4, NP22, NP66, DJ, DJ/8, DJ/9, LK3, RHM4-1, among many others.
• Prime AAV refers to AAV that infect primates
• non-primate AAV refers to AAV that infect non- primate mammals
• bovine AAV refers to AAV that infect bovine mammals, and so on.
• Serotype distinctiveness is determined on the basis of the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences and antigenic determinants (e.g., due to VP1, VP2, and/or VP3 sequence differences of AAV serotypes).
• serotype refers to both serologically distinct viruses, as well as viruses that are not serologically distinct but that may be within a subgroup or a variant of a given serotype.
• Genomic sequences of various serotypes of AAV, as well as sequences of the native ITRs, rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Numbers 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 by reference herein.
• a “recombinant adeno-associated virus” or “rAAV” is distinguished from a wild-type AAV by replacement of all or part of the endogenous viral genome with a non-native sequence.
• rAAV vector can include a heterologous polynucleotide encoding a desired RNA or protein or polypeptide (e.g., an RNA molecule disclosed herein).
• a recombinant vector sequence may be encapsidated or packaged into an AAV capsid and referred to as an “rAAV vector,” an “rAAV vector particle,” “rAAV viral particle” or simply a “rAAV.”
• the present disclosure provides for an rAAV vector comprising a polynucleotide sequence not of AAV origin (e.g., a polynucleotide heterologous to AAV).
• the heterologous polynucleotide may be flanked by at least one, and sometimes by two, AAV terminal repeat sequences (e.g., inverted terminal repeats).
• the heterologous polynucleotide flanked by ITRs also referred to herein as a “vector genome,” typically encodes an RNA or a polypeptide of interest, or a gene of interest, such as a target for therapeutic treatment.
• Delivery or administration of an rAAV vector to a subject provides encoded RNAs/proteins/peptides to the subject.
• an rAAV vector can be used to transfer/deliver a heterologous polynucleotide for expression for, e.g., treating a variety of diseases, disorders and conditions.
• rAAV vector genomes generally retain 145 base ITRs in cis to the heterologous nucleic acid sequence that replaced the viral rep and cap genes.
• ITRs are useful to produce a recombinant AAV vector; however, modified AAV ITRs and non-AAV terminal repeats including partially or completely synthetic sequences can also serve this purpose.
• ITRs form hairpin structures and function to, for example, serve as primers for host-cell-mediated synthesis of the complementary DNA strand after infection.
• ITRs also play a role in viral packaging, integration, etc. ITRs are the only AAV viral elements which are required in cis for AAV genome replication and packaging into rAAV vectors.
• An rAAV vector genome optionally comprises two ITRs which are generally at the 5’ and 3’ ends of the vector genome comprising a heterologous sequence (e.g., a transgene encoding a gene of interest, or a nucleic acid sequence of interest including, but not limited to, an antisense, and siRNA, a CRISPR molecule, among many others).
• a 5’ and a 3’ ITR may both comprise the same sequence, or each may comprise a different sequence.
• An AAV ITR may be from any AAV including by not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or any other AAV.
• An rAAV vector of the disclosure may comprise an ITR from an AAV serotype (e.g., wild-type AAV2, a fragment or variant thereof) that differs from the serotype of the capsid (e.g., AAV8, Olig001).
• an rAAV vector comprising at least one ITR from one serotype, but comprising a capsid from a different serotype may be referred to as a hybrid viral vector (see U.S. Patent No.7,172,893).
• An AAV ITR may include the entire wild type ITR sequence, or be a variant, fragment, or modification thereof, but will retain functionality.
• an rAAV vector genome is linear, single-stranded and flanked by AAV ITRs.
• a single stranded DNA genome of approximately 4700 nucleotides Prior to transcription and translation of the heterologous gene, a single stranded DNA genome of approximately 4700 nucleotides must be converted to a double-stranded form by DNA polymerases (e.g., DNA polymerases within the transduced cell) using the free 3’-OH of one of the self-priming ITRs to initiate second-strand synthesis.
• DNA polymerases e.g., DNA polymerases within the transduced cell
• full length-single stranded vector genomes i.e., sense and anti-sense
• anneal to generate a full length-double stranded vector genome This may occur when multiple rAAV vectors carrying genomes of opposite polarity (i.e., sense or anti-sense) simultaneously transduce the same cell.
• the cell can transcribe and translate the double-stranded DNA and express the heterologous gene.
• the efficiency of transgene expression from an rAAV vector can be hindered by the need to convert a single stranded rAAV genome (ssAAV) into double-stranded DNA prior to expression.
• This step can be circumvented by using a self-complementary AAV genome (scAAV) that can package an inverted repeat genome that can fold into double-stranded DNA without the need for DNA synthesis or base-pairing between multiple vector genomes. See, e.g., U.S. Patent No. 8,784,799; McCarty, (2008) Molec.
• a viral capsid of an rAAV vector may be from a wild type AAV or a variant 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 (WO 2015/013313), RHM15-1, RHM15-2, RHM15- 3/RHM15-5, RHM15-4, RHM15-6, AAV hu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9,45, AAV2i8, AAV
• Capsids may be derived from a number of AAV serotypes disclosed in U.S. Patent 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; and include true type AAV (AAV-TT) variants disclosed in WO 2015/121501, and RHM4-1, RHM15-1 through RHM15-6, and variants thereof, disclosed in WO 2015/013313.
• a full complement of AAV cap proteins includes VP1, VP2, and VP3.
• the ORF comprising nucleotide sequences encoding AAV VP capsid proteins may comprise less than a full complement AAV Cap proteins or the full complement of AAV cap proteins may be provided.
• an rAAV vector comprising a capsid protein encoded by a nucleotide sequence derived from more than one AAV serotype e.g., wild type AAV serotypes, variant AAV serotypes
• a chimeric vector or “chimeric capsid” (See U.S. Patent No. 6,491,907, the entire disclosure of which is incorporated herein by reference).
• a chimeric capsid protein is encoded by a nucleic acid sequence derived from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more AAV serotypes.
• a recombinant AAV vector includes a capsid sequence derived from e.g., AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh74, AAVrh10, AAV2i8, or variant thereof, resulting in a chimeric capsid protein comprising a combination of amino acids from any of the foregoing AAV serotypes (see, Rabinowitz et al.
• a chimeric capsid can comprise a mixture of a VP1 from one serotype, a VP2 from a different serotype, a VP3 from yet a different serotype, and a combination thereof.
• a chimeric virus capsid may include an AAV1 cap protein or subunit and at least one AAV2 cap protein or subunit.
• a chimeric capsid can, for example include an AAV capsid with one or more B19 cap subunits, e.g., an AAV cap protein or subunit can be replaced by a B19 cap protein or subunit.
• a VP3 subunit of an AAV capsid can be replaced by a VP2 subunit of B19.
• a chimeric capsid is an Olig001 capsid as described in WO2021221995 and WO2014052789, which are incorporated herein by reference.
• chimeric vectors have been engineered to exhibit altered tropism or tropism for a particular tissue or cell type.
• the term “tropism” refers to preferential entry of the virus into certain cell (e.g., oligodendrocytes) or tissue types and/or preferential interaction with the cell surface that facilitates entry into certain cell or tissue types.
• AAV tropism is generally determined by the specific interaction between distinct viral capsid proteins and their cognate cellular receptors (Lykken et al. (2016) J. Neurodev. Disord.10:16).
• sequences e.g., heterologous sequences such as a transgene
• the vector genome e.g., an rAAV vector genome
• a “tropism profile” refers to a pattern of transduction of one or more target cells in various tissues and/or organs.
• a chimeric AAV capsid may have a tropism profile characterized by efficient transduction of oligodendrocytes or astrocytes or oligodendrocyte progenitor cells with only low transduction of neurons and other CNS cells. See WO2014/052789, incorporated herein by reference.
• Such a chimeric capsid may be considered specific for oligodendrocytes or astrocytes or glial progenitor cells exhibiting tropism for oligodendrocytes or astrocytes or glial progenitor cells, and referred to herein as “glialtropism,” if when administered directly into the CNS, preferentially transduces oligodendrocytes or astrocytes or oligodendrocyte progenitor cells over neurons and other CNS cell types.
• At least about 80% of cells that are transduced by a capsid specific for oligodendrocytes or oligodendrocyte progenitor cells are oligodendrocytes or oligodendrocyte progenitor cells, e.g., at least about 85%, 90%, 95%, 96%, 97%, 98% 99% or more of the transduced cells are oligodendrocytes or oligodendrocyte progenitor cells.
• Gene and Cell Therapies The nucleic acids, genetic constructs, expression cassettes, expression vectors, and cells described herein may be used for gene therapy treatment and/or prevention of a disease, disorder or condition.
• oligodendrocyte or myelin can be used for treating or preventing a disease, disorder or condition associated with deficiency or dysfunction of oligodendrocyte or myelin by increasing the expression of a transcription factor or a target thereof, and of any other condition and or illness in which increasing the expression of the protein may produce a therapeutic benefit or improvement, e.g., a disease, disorder or condition mediated by, or associated with, a decrease in the level or function of the protein compared with the level or function of the protein in an otherwise healthy individual.
• a disorder of myelin As used herein a disorder of myelin, a disease of myelin, a myelin-related disorder, a myelin-related disease, a myelin disorder, a disorder mediated by a deficiency in myelin, and a myelin disease are used interchangeably. They include a condition mediated by loss of while matter/oligodendrocytes/astrocytes and any disease, condition (e.g., those occurring from traumatic spinal cord injury and cerebral infarction), or disorder related to demyelination, insufficient myelination and remyelination, or dysmyelination in a subject. Such a disorder can be inherited or acquired or both.
• myelination refers to the act of demyelinating, or the loss of the myelin sheath insulating the nerves, and is the hallmark of some neurodegenerative autoimmune diseases, including multiple sclerosis, transverse myelitis, chronic inflammatory demyelinating polyneuropathy, and Guillain-Barre Syndrome.
• Leukodystrophies are caused by inherited enzyme deficiencies, which cause abnormal formation, destruction, and/or abnormal turnover of myelin sheaths within the CNS white matter. Both acquired and inherited myelin disorders share a poor prognosis leading to major disability.
• some embodiments of the present disclosure can include methods for the treatment of neurodegenerative autoimmune diseases in a subject.
• Remyelination of neurons requires oligodendrocytes.
• the term "remyelination”, as used herein, refers to the re- generation of the nerve's myelin sheath by replacing myelin producing cells or restoring their function.
• Myelin related diseases or disorders which may be treated or ameliorated by the methods of the present disclosure include diseases, disorders or injuries which relate to dysmyelination or demyelination in a subject's brain cells, e.g., CNS neurons.
• Such diseases include, but are not limited to, diseases and disorders in which the myelin which surrounds the neuron is either absent, incomplete, not formed properly, or is deteriorating.
• diseases include, but are not limited to, multiple sclerosis (MS), neuromyelitis optica (NMO), progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelolysis (CPM), adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher disease (PMD), Wallerian Degeneration, optic neuritis, transverse myelitis, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, post radiation injury, neurologic complications of chemotherapy, stroke, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, Bassen-Kornzweig syndrome, Marchiafava-Bignami syndrome, metachromatic le
• Myelin related diseases or disorders which may be treated or ameliorated by the methods of the present disclosure include a disease or disorder characterized by a myelin deficiency. Insufficient myelination in the central nervous system has been implicated in a wide array of neurological disorders. Among these are forms of cerebral palsy in which a congenital deficit in forebrain myelination in children with periventricular leukomalacia, contributes to neurological morbidity (Goldman et al., 2008) Goldman, S. A., Schanz, S., and Windrem, M. S. (2008). Stem cell-based strategies for treating pediatric disorders of myelin. Hum Mol Genet.17, R76-83.
• myelin loss and ineffective repair may contribute to the decline in cognitive function associated with senescence (Kohama et al., 2011) Kohama, S. G., Rosene, D. L., and Sherman, L. S. (2011) Age (Dordr). Age- related changes in human and non-human primate white matter: from myelination disturbances to cognitive decline. Therefore, it is contemplated that effective compositions and methods of enhancing myelination and/or remyelination may have substantial therapeutic benefits in halting disease progression and restoring function in a wide array of myelin-related disorders.
• one aspect of this disclosure provides a method of 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 an agent that increase 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, or (b) a therapeutically effective amount of the cell prepared according to the method described herein or a progeny thereof.
• the target is selected from the group mentioned above and listed Table 1 and Figure 5.
• Such an agent may comprise or be a small molecule compound, an oligonucleotide, a nucleic acid, a genetic construct, a peptide, a polypeptide, a CRISPR/Cas system, or an antibody or an antigen-binding portion thereof.
• the agent include activators, agonists, or potentiators of the related CEBPZ, CTCF, E2F1, MYC, NFYB, and ETV4 signaling pathways.
• activators for this signaling pathway are known in the art.
• the agent comprises or is (i) the polypeptide of CEBPZ, CTCF, E2F1, MYC, NFYB, or ETV4, or target thereof (e.g., those listed Table 1 and Figure 5) or (ii) a nucleic acid, a genetic construct, or vector encoding the polypeptide.
• the agent may be or comprise (i) a suppressor of a transcription repressor selected from the group consisting of E2F6, ZNF274, MAX, and IKZF3, or (ii) a nucleic acid, a genetic construct, or vector encoding the suppressor.
• the one or more genetic constructs may activate transcription of one or more of the genes described herein via a CRISPR-Cas9 guided nuclease (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 hereby incorporated by reference in their entirety).
• CRISPR-Cas9 guided nuclease Gaj et al., “ZFN, TALEN, and CRISPR/Cas-based
• CRISPR-Cas9 is a genetic technique which allows for sequence- specific control of gene expression in prokaryotic and eukaryotic cells by guided nuclease double-stranded DNA cleavage. It is based on the bacterial immune system-derived CRISPR (clustered regularly interspaced palindromic repeats) pathway.
• the one or more genetic constructs may be packaged in a suitable delivery vehicle or carrier for delivery to the 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.
• the genetic construct is packaged into a therapeutic expression vector to facilitate delivery.
• Suitable expression vectors are well known in the art and include, without limitation, viral vectors such as adenovirus vectors, adeno- associated virus vectors, retrovirus vectors, lentivirus vectors, or herpes virus vectors.
• the viral vectors or other suitable expression vectors comprise sequences encoding the genetic constructs of the present application and any suitable promoter and/or enhancer for expressing the genetic construct.
• Suitable promoters include, for example, and without limitation, the U6 or HI RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art.
• the expression vectors may also comprise inducible or regulatable promoters for expression of the inhibitory nucleic acid molecules in a tissue or cell-specific manner. Gene therapy vectors carrying the therapeutic genetic construct or nucleic acid molecule are administered to a subject by, for example, intravenous injection, local administration (U.S. Patent No.
• the pharmaceutical preparation of the therapeutic vector can include the therapeutic vector in an acceptable diluent, or can comprise a slow release matrix in which the therapeutic delivery vehicle is imbedded.
• the pharmaceutical preparation can include one or more cells which produce the therapeutic delivery system.
• Gene therapy vectors typically utilize constitutive regulatory elements which are responsive to endogenous transcriptions factors.
• Another suitable approach for the delivery of the genetic construct of the present disclosure involves the use of liposome delivery vehicles or nanoparticle delivery vehicles.
• the delivery vehicle is a nanoparticle.
• nanoparticle delivery vehicles are known in the art and are suitable for delivery of the genetic constructs of the present application (see, e.g., van Vlerken et al., “Multi-functional Polymeric Nanoparticles for Tumour-Targeted Drug Delivery,” Expert Opin.
• Suitable nanoparticles include, without limitation, poly(beta-amino esters) (Sawicki et al., “Nanoparticle Delivery of Suicide DNA for Epithelial Ovarian Cancer Cell Therapy,” Adv. Exp. Med. Biol.622:209–219 (2008), which is hereby incorporated by reference in its entirety), polyethylenimine-alt-poly(ethylene glycol) copolymers (Park et al., “Degradable Polyethylenimine-alt-Poly(ethylene glycol) Copolymers As Novel Gene Carriers,” J.
• nanoparticle vehicles suitable for use in the present application include microcapsule nanotube devices disclosed in U.S. Patent Publication No. 2010/0215724 to Prakash et al., which is hereby incorporated by reference in its entirety.
• the genetic construct is contained in a liposome delivery vehicle.
• liposome means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.
• liposomes include: their biocompatibility and biodegradability, incorporation of a wide range of water and lipid soluble drugs; and they afford protection to encapsulated molecules from metabolism and degradation. Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.
• 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 hereby incorporated by reference in their entirety.
• the genetic construct, expression cassette, or expression vector can be administered in association with a glial progenitor cell-targeted fusogen or a glial progenitor cell-selective surface-binding moiety.
• the genetic construct, expression cassette, or expression vector can be in or associated with a fusosome.
• fusogen refers to an agent or molecule that creates an interaction between two membrane enclosed lumens. In embodiments, the fusogen facilitates fusion of the membranes.
• the fusogen creates a connection, e.g., a pore, between two lumens (e.g., a lumen of a liposome and a cytoplasm of a target cell, or a lumen of a viral vector and a cytoplasm of a target cell).
• the fusogen comprises a protein or a complex of two or more proteins having a targeting domain or binding moiety.
• the targeting domain or binding moiety specifically targets or binds to a molecule on glial progenitor cell or a glial progenitor cell.
• a targeting domain or binding moiety can be a receptor ligand, a peptide/polypeptide, an antibody, or an 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 in, e.g., US 20210198698 and US 20210137839, which are incorporated by reference in their entireties.
• fusosome refers to a bilayer of amphipathic lipids enclosing a lumen or cavity and a fusogen that interacts with the amphipathic lipid bilayer.
• the fusosome comprises a nucleic acid.
• the fusosome is a membrane enclosed preparation.
• the fusosome is derived from a source cell. Fusosomes can take various forms. For example, in some embodiments, a fusosome described herein is derived from a source cell.
• a fusosome may be or comprise, e.g., an extracellular vesicle, a microvesicle, a nanovesicle, an exosome, a microparticle, or any combination thereof.
• a fusosome is released naturally from a source cell, and in some embodiments, the source cell is treated to enhance formation of fusosomes.
• the fusosome is between about 10-10,000 nm in diameter, e.g., about 30-100 nm in diameter.
• the fusosome comprises one or more synthetic lipids.
• the fusosome is or comprises a virus, e.g., a retrovirus, e.g., a lentivirus.
• the fusosome's bilayer of amphipathic lipids is or comprises the viral envelope.
• the viral envelope may comprise a fusogen, e.g., a fusogen that is endogenous to the virus or a pseudotyped fusogen.
• the fusosome's lumen or cavity comprises a viral nucleic acid, e.g., a retroviral nucleic acid, e.g., a lentiviral nucleic acid.
• the viral nucleic acid may be a viral genome.
• the fusosome further comprises one or more viral non-structural proteins, e.g., in its cavity or lumen.
• Fusosomes may have various structures or properties that facilitate delivery of a payload to a target cell.
• the fusosome and the source cell together comprise nucleic acid(s) sufficient to make a particle that can fuse with a target cell.
• these nucleic acid(s) encode proteins having one or more of (e.g., all of) the following activities: gag polyprotein activity, polymerase activity, integrase activity, protease activity, and fusogen activity.
• compositions of the present disclosure can be administered to a subject that does not have, and/or is not suspected of having, a myelin related disorder in order to enhance or promote a myelin dependent process.
• compositions described herein can be administered to a subject to promote myelination of CNS neurons in order to enhance cognition, which is known to be a myelin dependent process, in cognitive healthy subjects.
• compositions described herein can be administered in combination with cognitive enhancing (nootropic) agents.
• Exemplary agents include any drugs, supplements, or other substances that improve cognitive function, particularly executive functions, memory, creativity, or motivation, in healthy individuals.
• Non limiting examples include racetams (e.g., piracetam, oxiracetam, and aniracetam), nutraceuticals (e.g., bacopa monnieri, panax ginseng, ginko biloba, and GABA), stimulants (e.g., amphetamine pharmaceuticals, methylphenidate, eugeroics, xanthines, and nicotine), L-Theanine, Tolcapone, Levodopa, Atomoxetine, and Desipramine.
• racetams e.g., piracetam, oxiracetam, and aniracetam
• nutraceuticals e.g., bacopa monnieri, panax ginseng, ginko biloba, and GABA
• stimulants e.g., amphetamine pharmaceuticals, methylphenidate, eugeroics, xanthines, and nicotine
• L-Theanine Tolcapone
• the overall dosage of a therapeutic agent (e.g., a protein, a polynucleotide encoding the protein, or a vector, such as an rAAV vector, or a cell) will be a therapeutically effective amount depending on several factors including the overall health of a subject, the subject's disease state, severity of the condition, the observation of improvements and the formulation and route of administration of the selected agent(s). Determination of a therapeutically effective amount is within the capability of those skilled in the art. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the subject's condition.
• Cell Replacement Therapy Also within scope of this disclosure is a host cell comprising the genetic construct, cassette, or expression vector described above, or a progeny cell of the host cell.
• the host cell can be a stem cell or a progenitor cell.
• Example of the stem cell 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 and others.
• the host cell is a glial progenitor cell, such as an oligodendrocyte progenitor cell.
• the glial progenitor cells described herein may be derived from any suitable source of pluripotent stem cells, such as, for example and without limitation, human induced pluripotent stem cells (iPSCs) and embryonic stem cells, as described in more detail below.
• iPSCs human induced pluripotent stem cells
• glial progenitor cells can be cells rejuvenated from glial progenitor cells or progenies thereof as described herein.
• the host cell or a progeny thereof can be used as a therapeutic cell or agent for treating the disorders or conditions described herein.
• One aspect of the present application relates to a method of alleviating 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 an age-related loss.
• This method includes identifying a subject, e.g., an adult subject, undergoing 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 population of isolated glial progenitor cells is then introduced into CNS (such as the brain and/or brain stem) of the selected subject to at least partially replace cells in the subject’s brain in the location undergoing the adverse effects of oligodendrocyte loss, astrocyte loss, or white matter loss.
• 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 signal transmission in the nervous system.
• “Glial cells” as used herein encompasses fully differentiated cells of the glial lineage, such as oligodendrocytes or astrocytes, and as well as glial progenitor cells.
• Glial progenitor cells are cells having the potential to differentiate into cells of the glial lineage such as oligodendrocytes and astrocytes.
• glial progenitor cells or rejuvenated cells are young glial or glial progenitor cells, or are younger than the counterparts in the subject to be treated.
• the term “young” glial or glial progenitor cells refers to cells that are induced to start differentiation into glial progenitor cell in an in vitro setting (about 105 days from cell isolation from fetal donor tissue).
• the term “young glial cells” refers to differentiated glial progenitor cells that are ready for transplantation into an animal (about 160 days from cell isolation from fetal donor tissue).
• the term “young glial cells” refers to glial progenitor cells or their progeny that are within 1-20 weeks of transplantation.
• the term “older glial cells” is used in relative to the term “young glial cells”.
• young glial cells may have one or more of the following characteristics: (i) growing or proliferating or dividing faster, (ii) having lower levels than old of senescence-associated transcripts encoding CDKN1A (p21Cip1) and CDKN2/p16(INK4) and p14(ARF), and (iii) longer telomeres or higher telomerase activity or both.
• older glial cells are glial cells that are derived from glial progenitor cells that have been transplanted into a host for 5, 10, 20, 30 or 40 weeks. In some embodiments, the older glial cells are glial cells that have been cultured for an additional 5, 10, 20, 30 or 40 weeks from differentiated glial progenitor cells (e.g., about 160 days from the initial tissue harvest). In some embodiments, the older glial cells are glial cells that have been cultured for an additional 5, 10, 20, 30 or 40 weeks from the introduction of differentiation (e.g., about 105 days from the initial tissue harvest).
• the glial progenitor cells described herein may be derived from any suitable source of pluripotent stem cells, including iPSCs.
• iPSCs are pluripotent cells that are derived from non- pluripotent cells, such as somatic cells.
• 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.
• somatic cells can be reprogrammed to an embryonic stem cell-like state using genetic manipulation.
• exemplary somatic cells suitable for the formation of iPSCs include fibroblasts (see e.g., Streckfuss-Bomeke et al., Eur. Heart J.
• fibroblasts obtained by a skin sample or biopsy
• synoviocytes from synovial tissue
• keratinocytes mature B cells
• mature T cells pancreatic ⁇ cells
• melanocytes melanocytes
• hepatocytes foreskin cells
• cheek cells or lung fibroblasts.
• Methods of producing induced pluripotent stem cells are known in the art and typically involve expressing a combination of reprogramming factors in a somatic cell.
• Suitable reprogramming factors that promote and induce iPSC generation include one or more of Oct4, Klf4, Sox2, c-Myc, Nanog, C/EBP ⁇ , Esrrb, Lin28, and Nr5a2.
• at least two reprogramming factors are expressed in a somatic cell to successfully reprogram the somatic cell.
• at least three reprogramming factors are expressed in a somatic cell to successfully reprogram the somatic cell.
• iPSCs may be derived by methods known in the art, including the use integrating viral vectors (e.g., lentiviral vectors, inducible lentiviral vectors, and retroviral vectors), excisable vectors (e.g., transposon and floxed lentiviral vectors), and non-integrating vectors (e.g., adenoviral and plasmid vectors) to deliver the genes that promote cell reprogramming (see e.g., Takahashi and Yamanaka, Cell 126:663-676 (2006); Okita. et al., Nature 448:313-317 (2007); Nakagawa et al., Nat.
• viral vectors e.g., lentiviral vectors, inducible lentiviral vectors, and retroviral vectors
• excisable vectors e.g., transposon and floxed lentiviral vectors
• non-integrating vectors e.g., adenoviral
• the methods of iPSC generation described above can be modified to include small molecules that enhance reprogramming efficiency or even substitute for a reprogramming factor.
• small molecules include, without limitation, epigenetic modulators such as, the DNA methyltransferase inhibitor 5’-azacytidine, the histone deacetylase inhibitor VPA, and the G9a histone methyltransferase inhibitor BIX-01294 together with BayK8644, an L-type calcium channel agonist.
• TGF- ⁇ inhibitors e.g., kenpaullone
• kinase inhibitors e.g., kenpaullone
• Methods of obtaining highly enriched preparations of glial progenitor cells from the iPSCs that are suitable for making the non-human mammal models described herein are disclosed in WO2014/124087 to Goldman and Wang, and Wang et al., Cell Stem Cell 12(2):252-264 (2013), which are hereby incorporated by reference in their entirety.
• the glial progenitor cells are derived from embryonic stem cells.
• Embryonic stem cells are derived from totipotent cells of the early mammalian embryo and are capable of unlimited, undifferentiated proliferation in vitro.
• the term “embryonic stem cells” refer to a cells isolated from an embryo, placenta, or umbilical cord, or an immortalized version of such a cells, i.e., an embryonic stem cell line. Suitable embryonic stem cell lines include, without limitation, lines WA-01 (H1), WA-07, WA-09 (H9), WA-13, and WA-14 (H14) (Thomson et al., Science 282 (5391): 1145-47 (1998) and U.S.
• Patent No.7,029,913 to Thomson et al. which are hereby incorporated by reference in their entirety.
• Other suitable embryonic stem cell lines includes the HAD-C100 cell line (Tannenbaum et al., PLoS One 7(6):e35325 (2012), which is hereby incorporated by reference in its entirety, the WIBR4, WIBR5, WIBR6 cel lines (Lengner et al., Cell 141(5):872-83 (2010), which is hereby incorporated by reference in its entirety), and the human embryonic stem cell lines (HUES) lines 1-17 (Cowan et al., N. Engl. J. Med.350:1353-56 (2004), which is hereby incorporated by reference in its entirety).
• glial progenitor cells provide a virtually unlimited source of clonal/genetically modified cells potentially useful for tissue replacement therapies.
• Methods of obtaining highly enriched preparations of glial progenitor cells from embryonic cells that are suitable for making the non-human mammal model of the present disclosure are described herein as disclosed in Wang et al., Cell Stem Cell 12:252-264 (2013), which is hereby incorporated by reference in its entirety.
• glial progenitor cells are derived from a pluripotent population of cells, i.e., iPSCs or embryonic stem cells, using a protocol that directs the pluripotent cells through serial stages of neural and glial progenitor cell differentiation.
• Stage 1 of this process involves culturing the pluripotent cell population under conditions effective to induce embryoid body formation.
• the pluripotent cell population may be maintained in co-culture with other cells, such as embryonic fibroblasts, in an embryonic stem cell (ESC) media (e.g., DMEM/F12 containing a suitable serum replacement and bFGF).
• ESC embryonic stem cell
• the pluripotent cells are passaged before reaching 100% confluence, e.g., 80% confluence, when colonies are approximately 250-300 ⁇ m in diameter.
• the pluripotential state of the cells is readily assessed using markers to SSEA4, TRA-1-60, OCT-4, NANOG, and/or SOX2.
• EBs embryoid bodies
• Stage 2 embryoid bodies
• EBs embryoid bodies
• EBs embryoid bodies
• Stage 3 EBs are plated and cultured in neural induction medium supplemented with bFGF, heparin, laminin, then switched to neural induction media supplemented with retinoic acid.
• Neuroepithelial differentiation is assessed by the co-expression of PAX6 and SOX1, which characterize central neural stem and progenitor cells.
• pre-OPCs pre-oligodendrocyte progenitor cell
• neuroepithelial cell colonies can be cultured in the presence of additional factors including retinoic acid, B27 supplement, and a sonic hedgehog (shh) agonist (e.g., purmophamine).
• shh sonic hedgehog
• the appearance 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 progenitor cells, NKX2.2 is a more specific indicator of oligodendroglial differentiation.
• an early pre-oligodendrocyte progenitor cell stage is marked by OLIG + /NKX2.2- cell colonies.
• OLIG + /NKX2.2- early pre-OPCs are differentiated into later-stage OLIG + /NKX2.2 + pre-OPCs by replacing retinoic acid with bFGF.
• Stage 5 a significant percentage of the cells are pre-OPCs as indicated by OLIG2 + /NKX2.2 + expression profile.
• Pre-OPCs can be further differentiated into bipotential glial progenitor cells by culture in glial induction media supplemented with growth factors such as triiodothyronine (T3), neurotrophin 3 (NT3), insulin 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 to maximize the production of myelinogenic glial progenitor cells when desired. Cell preparations suitable for transplantation into an appropriate subject are identified as containing PDGFR ⁇ + glial progenitor cells.
• T3 triiodothyronine
• NT3 neurotrophin 3
• IGF-1 insulin growth factor
• PDGF-AA platelet-derived growth factor-AA
• the population of glial progenitor cells used in carrying out the method of the present application may comprise at least about 80% glial progenitor cells, including, for example, about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% glial cells.
• the selected preparation of glial progenitor cells can be relatively devoid (e.g., containing less than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of other cells types such as neurons and neuronal progenitor cells.
• the cell population can be a substantially pure populations of glial progenitor cells.
• the subject being treated in accordance with the method of the present application can be an adult afflicted with age-related white matter/oligodendrocyte/astrocyte loss in the brain.
• This method alleviates the adverse effects of this condition which can arise as part of the normal aging process.
• “treating” or “treatment” refers to any indication of success in amelioration of an injury, pathology, or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology, or condition more tolerable to the patient; slowing the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject’s physical or mental well-being.
• Treating can include the administration of glial progenitor cells or/and other agent(s) to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with the disease, condition or disorder.
• Treateutic effect refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of a disease, condition or disorder in the subject.
• Treatment may be prophylactic (to prevent or delay the onset or worsening of the 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 the disease, condition or disorder.
• white matter relates to a component of the central nervous system, in the brain and superficial spinal cord, which consists mostly 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.
• the glial progenitor cells may be introduced into the subject needing alleviation of the adverse effects by a variety of know techniques. These include, but are not limited to, injection, deposition, and grafting as described herein.
• the glial progenitor cells can be transplanted bilaterally into multiple sites of the subject, as described 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 hereby incorporated by reference in their entirety).
• Typical procedures include intraparenchymal, intracallosal, intraventricular, intrathecal, and intravenous transplantation.
• Intraparenchymal transplantation can be achieved by injection or deposition of tissue within the host brain so as to be apposed to the brain parenchyma at the time of transplantation.
• the two main procedures for intraparenchymal transplantation are: (1) injecting the donor cells within 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), which is hereby incorporated by reference in its entirety).
• Both methods provide parenchymal apposition between the donor cells and host brain tissue at the time of grafting, and both facilitate anatomical integration between the graft and host brain tissue. This is of importance if it is required that the donor cells become an integral part of the host brain and survive for the life of the host.
• Glial progenitor cells can also be delivered intracallosally as described in U.S. Patent Application Publication No. 20030223972 to Goldman, which is hereby incorporated by reference in its entirety.
• the glial progenitor cells can also be delivered directly to the forebrain subcortex, specifically into the anterior and posterior anlagen of the corpus callosum.
• Glial progenitor cells can also be delivered to the cerebellar peduncle white matter to gain access to the major cerebellar and brainstem tracts.
• Glial progenitor cells can also be delivered to the spinal cord. Alternatively, the cells may be placed in a ventricle, e.g., a cerebral ventricle.
• Grafting cells in the ventricle may be accomplished by injection of the donor cells or by growing the cells in a substrate such as 30% collagen to form a plug of solid tissue which may then be implanted into the ventricle to prevent dislocation of the graft cells.
• the cells may be injected around the surface of the brain after making a slit in the dura.. Suitable techniques for cell delivery are described supra.
• said preparation of glial progenitor cells is administered to the striatum, forebrain, brain stem, and/or cerebellum of the subject. Delivery of the cells to the subject can include either a single step or a multiple step injection directly into the nervous system.
• a single injection can be used for localized disorders such as demyelination of the optic nerve.
• adult and fetal oligodendrocyte precursor cells disperse widely within a transplant recipient’s brain, for widespread disorders, multiple injections sites can be performed to optimize treatment.
• Injection is optionally directed into areas of the central nervous system such as white matter tracts like the corpus callosum (e.g., into the anterior and posterior anlagen), dorsal columns, cerebellar peduncles, cerebral peduncles.
• Such injections can be made unilaterally or bilaterally using precise localization methods such as stereotaxic surgery, optionally with accompanying imaging methods (e.g., high resolution MRI imaging).
• the cellular transplants can be optionally injected as dissociated cells but can also be provided by local placement of non-dissociated cells. In either case, the cellular transplants optionally comprise an acceptable solution.
• acceptable solutions include solutions that avoid undesirable biological activities and contamination.
• Suitable solutions include an appropriate amount of a pharmaceutically-acceptable salt to render the formulation isotonic.
• the pharmaceutically-acceptable solutions include, but are not limited to, saline, Ringer’s solution, dextrose solution, and culture media.
• the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.
• the injection of the dissociated cellular transplant can be a streaming injection made across the entry path, the exit path, or both the entry and exit paths of the injection device (e.g., a cannula, a needle, or a tube). Automation can be used to provide a uniform entry and exit speed and an injection speed and volume.
• the number of glial progenitor cells administered to the subject can range from about 10 2 -10 8 at each administration (e.g., injection site), depending on the size and species of the recipient, and the volume of tissue requiring cell replacement.
• Single administration (e.g., injection) doses can span ranges of 10 3 -10 5 , 10 4 -10 7 , and 10 5 -10 8 cells, or any amount in total for a transplant recipient patient.
• immunosuppressant agents and their dosing regimens are known to one of skill in the art and include such agents as Azathioprine, Azathioprine Sodium, Cyclosporine, Daltroban, Gusperimus Trihydrochloride, Sirolimus, and Tacrolimus.
• Dosages ranges and duration of the regimen can be varied with the disorder being treated; the extent of rejection; the activity of the specific immunosuppressant employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the specific immunosuppressant employed; the duration and frequency of the treatment; and drugs used in combination.
• One of skill in the art can determine acceptable dosages for and duration of immunosuppression.
• the dosage regimen can be adjusted by the individual physician in the event of any contraindications or change in the subject’s status.
• one or more immunosuppressant agents can be administered to the subject starting at 10 weeks prior to cell administration.
• the one or more immunosuppressant agents are administered to the subject starting at 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, ⁇ 24 hours prior to cell administration.
• one or more immunosuppressant agents are administered to the subject starting on the day of cell administration and continuing for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months post administration.
• the one or more immunosuppressant agents are administered to the subject for > 1 year following administration.
• Suitable subjects for treatment in accordance with the methods described herein include any mammalian subject afflicted with age-related white matter loss.
• Exemplary mammalian subjects include humans, mice, rats, guinea pigs, and other small rodents, dogs, cats, sheep, goats, and monkeys.
• the subject is human.
• the above-described rejuvenation therapy and cell therapy can be used together.
• the nucleic acid molecules, the inhibitory molecules, CRISPR/Cas systems, expression cassettes, or expression vectors described above can be used as therapeutic reagents in ex vivo applications.
• the reagents can be introduced into tissue or cells that are transplanted into a subject for therapeutic effect.
• the cells and/or tissue can be derived from an organism or subject that later receives the explant (e.g., isogenic or autologous), or can be derived from another organism or subject (e.g., a relative, a sibling, or a HLA matching donor) prior to transplantation (e.g., heterologous, xenogenic, allogeneic, or isogenic).
• the reagents can be used to modulate the expression of one or more genes in the cells or tissue, such that the cells or tissue obtain a desired phenotype or are able to perform a function when transplanted in vivo.
• certain target cells from a patient are extracted or isolated.
• a pharmaceutically effective dose of the therapeutic reagent or pharmaceutical composition can be administered to the subject.
• a pharmaceutically effective dose is a dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state.
• a therapeutically effective dose of the reagent to be administer to a given subject by taking into account factors, such as the size and weight of the subject, the extent of the disease progression or penetration, the age, health, and sex of the subject, the route of administration m and whether the administration is regional or systemic. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.
• the therapeutic reagent or pharmaceutical composition can be administered in a single dose or in multiple doses.
• the cell, protein, or nucleotide compositions described herein may be administered in an amount effective to enhance myelin production in the CNS of a subject by an increase in 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) of 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% as compared to the level of myelin proteins of an untreated subject.
• myelin proteins e.g
• compositions may be administered in an amount effective to promote survival of CNS neurons in a subject by an increase in the number of surviving neurons of 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% as compared to the number of surviving neurons in an untreated CNS neurons or subject.
• Another strategy for treating a subject suffering from myelin-related disorder is to administer a therapeutically effective amount of a cell or nucleotide composition described herein along with a therapeutically effective amount of an oligodendrocyte differentiation and/or proliferation inducing agent(s) and/or anti-neurodegenerative disease agent.
• anti-neurodegenerative disease agents include L-dopa, cholinesterase inhibitors, anticholinergics, dopamine agonists, steroids, and immunomodulators including interferons, monoclonal antibodies, and glatiramer acetate. Therefore, in a further aspect of the disclosure, the compositions described herein can be administered as part of a combination therapy with adjunctive therapies for treating neurodegenerative and myelin related disorders.
• oligodendrocyte precursor differentiation inducing compositions described herein and a therapeutic agent as part of a specific treatment regimen intended to provide a beneficial effect from the co-action of these therapeutic agents.
• the oligodendrocyte precursor differentiation inducing compound and a therapeutic agent can be formulated as separate compositions.
• Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually minutes, hours, days, or weeks depending upon the combination selected).
• 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 infusion into the brain ventricles.
• compositions for preventing or treating an inherited or acquired disorder of myelin.
• a pharmaceutical composition comprises one or more of the above-described protein molecule, polynucleotide, expression cassette, expression vector (e.g., viral vector genome, expression vector, rAAV vector), system (e.g., a CRISPR/Cas system or nucleic acid(s) encoding components of the system), and host cell or a progeny thereof.
• the pharmaceutical composition further comprises a pharmaceutically-acceptable carrier, adjuvant, diluent, excipient and/or other medicinal agents.
• a pharmaceutically acceptable carrier, adjuvant, diluent, excipient or other medicinal agent is one that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject without causing undesirable biological effects which outweigh the advantageous biological effects of the material.
• Any suitable pharmaceutically acceptable carrier or excipient can be used in the preparation of a pharmaceutical composition according to the disclosure (See e.g., Remington The Science and Practice of Pharmacy, Adeboye Adejare (Editor) Academic Press, November 2020).
• a pharmaceutical composition is typically sterile, pyrogen-free and stable under the conditions of manufacture and storage.
• a pharmaceutical composition may be formulated as a solution (e.g., water, saline, dextrose solution, buffered solution, or other pharmaceutically sterile fluid), microemulsion, liposome, or other ordered structure suitable to accommodate a high product (e.g., viral vector particles, microparticles or nanoparticles) concentration.
• a pharmaceutical composition comprising the above-described protein, polynucleotide, expression cassette, expression vector, vector genome, cell, or rAAV vector of the disclosure is formulated in water or a buffered saline solution.
• a carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
• Proper fluidity can be maintained, for example, by use of a coating such as lecithin, by maintenance of a required particle size, in the case of dispersion, and by the use of surfactants.
• Prolonged adsorption of an injectable composition can be brought about by including, in the composition, an agent which delays absorption, e.g., a monostearate salt and gelatin.
• a nucleic acid, vector and/or host cell of the disclosure may be administered in a controlled release formulation, for example, in a composition which includes a slow-release polymer or other carrier that protects the product against rapid release, including an implant and microencapsulated delivery system.
• a pharmaceutical composition of the disclosure is a parenteral pharmaceutical composition, including a composition suitable for intravenous, intraarterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intraarticular, intraparenchymal (IP), intrathecal (IT), intracerebroventricular (ICV) and/or intracisternal magna (ICM) administration.
• a pharmaceutical composition of this disclosure is formulated for administration by ICV injection.
• a vector e.g., a viral vector such as AAV
• molecule or polynucleotide, or vector (e.g., vector genome, rAAV vector), or system (e.g., a CRISPR/Cas systems or nucleic acid(s) encoding components of the system), or a cell may be administered to a subject (e.g., a patient) or a target cell in order to treat the subject.
• Administration of a vector to a human subject, or an animal in need thereof can be by any means known in the art for administering a vector.
• a target cell include cells of the CNS, preferably oligodendrocytes, astrocytes, or the progenitor cells thereof.
• a vector can be administered in addition to, and as an adjunct to, the standard of care treatment. That is, the vector can be co-administered with another agent, compound, drug, treatment or therapeutic regimen, either simultaneously, contemporaneously, or at a determined dosing interval as would be determined by one skilled in the art using routine methods. Uses disclosed herein include administration of an rAAV vector of the disclosure at the same time, in addition to and/or on a dosing schedule concurrent with, the standard of care for the disease as known in the art.
• a combination composition includes one or more immunosuppressive agents.
• a combination composition includes an rAAV vector comprising a transgene (e.g., a polynucleotide encoding an RNA molecule disclosed herein) and one or more immunosuppressive agents.
• a method includes administering or delivering an rAAV vector comprising the transgene to a subject and administering an immunosuppressive agent to the subject either prophylactically prior to administration of the vector, or after administration of the vector (i.e., either before or after symptoms of a response against the vector and/or the protein provided thereby are evident).
• a vector of the disclosure e.g., an rAAV vector
• Exemplary methods of systemic administration include, but are not limited to, intravenous (e.g., portal vein), intraarterial (e.g., femoral artery, hepatic artery), intravascular, subcutaneous, intradermal, intraperitoneal, transmucosal, intrapulmonary, intralymphatic and intramuscular administration, and the like, as well as direct tissue or organ injection.
• intravenous e.g., portal vein
• intraarterial e.g., femoral artery, hepatic artery
• intravascular subcutaneous, intradermal, intraperitoneal, transmucosal, intrapulmonary, intralymphatic and intramuscular administration, and the like
• direct tissue or organ injection includes administration to areas directly affected by oligodendrocyte deficiency (e.g., brain and/or central nervous system).
• vectors of the disclosure, and pharmaceutical compositions thereof are administered to the brain parenchyma (i.e., by intraparenchymal administration), to the spinal canal or the subarachnoid space so that it reaches the cerebrospinal fluid (CSF) (i.e., by intrathecal administration), to a ventricle of the brain (i.e., by intracerebroventricular administration) and/or to the cisterna magna of the brain (i.e., by intracisternal magna administration).
• CSF cerebrospinal fluid
• a vector of the present disclosure is administered by direct injection into the brain (e.g., into the parenchyma, ventricle, cisterna magna, etc.) and/or into the CSF (e.g., into the spinal canal or subarachnoid space) to treat a disorder of myelin.
• a target cell of a vector of the present disclosure includes a cell located in the cortex, subcortical white matter of the corpus callosum, striatum and/or cerebellum.
• a target cell of a vector of the present disclosure is an oligodendrocyte or a progenitor cell thereof.
• Additional routes of administration may also comprise local application of a vector under direct visualization, e.g., superficial cortical application, or other stereotaxic application.
• a vector of the disclosure is administered by at least two routes.
• a vector is administered systemically and also directly into the brain. If administered via at least two routes, the administration of a vector can be, but need not be, simultaneous or contemporaneous. Instead, administration via different routes can be performed separately with an interval of time between each administration.
• the above-described protein, or polynucleotide encoding the protein, or a vector genome, or a vector (e.g., an rAAV vector) comprising the polynucleotide may be used for transduction of a cell ex vivo or for administration directly to a subject (e.g., directly to the CNS of a patient with a disease).
• a transduced cell e.g., a host cell
• a disease, disorder or condition e.g., cell therapy for the disease.
• an rAAV vector comprising a therapeutic nucleic acid can be preferably administered to an oligodendrocyte, an astrocyte, or a progenitor cell thereof in a biologically-effective amount.
• the dosage amount of a vector depends upon, e.g., the mode of administration, disease or condition to be treated, the stage and/or aggressiveness of the disease, individual subject's condition (age, sex, weight, etc.), particular viral vector, stability of protein to be expressed, host immune response to the vector, and/or gene to be delivered.
• doses range from at least 1 x 10 8 , or more, e.g., 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 , 1 x 10 15 or more vector genomes (vg) per kilogram (kg) of body weight of the subject to achieve a therapeutic effect.
• a polynucleotide encoding a protein described herein may be administered as a component of a DNA molecule (e.g., a recombinant nucleic acid) having a regulatory element (e.g., a promoter) appropriate for expression in a target cell (e.g., an oligodendrocyte, an astrocyte, or a progenitor cell thereof).
• a target cell e.g., an oligodendrocyte, an astrocyte, or a progenitor cell thereof.
• the polynucleotide may be administered as a component of a plasmid or a viral vector, such as an rAAV vector.
• An rAAV vector may be administered in vivo by direct delivery of the vector (e.g., directly to the CNS) to a patient in need of treatment.
• kits may be administered to a patient ex vivo by administration of the vector in vitro to a cell from a donor patient in need of treatment, followed by introduction of the transduced cell back into the donor (e.g., cell therapy).
• Kit The present disclosure provides a kit with packaging material and one or more components described therein.
• a kit typically includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo or ex vivo, of the components therein.
• a kit can contain a collection of such components, e.g., the above- described polynucleotide, nucleic acid, expression cassette, expression vector (e.g., viral vector genome, expression vector, rAAV vector), and cell, and optionally a second active agent such as a compound, therapeutic agent, drug or composition.
• a kit refers to a physical structure that contains one or more components of the kit.
• Packaging material can maintain the components in a sterile manner and can be made of material commonly used for such purposes (e.g., paper, glass, plastic, foil, ampules, vials, tubes, etc).
• a label or insert can include identifying information of one or more components therein, dose amounts, clinical pharmacology of the active ingredients(s) including mechanism of action, pharmacokinetics and pharmacodynamics.
• a label or insert can include information identifying manufacture, lot numbers, manufacture location and date, expiration dates.
• a label or insert can include information on a disease (e.g., an inherited or acquired or age-related disorder of myelin such as HD) for which a kit component may be used.
• a label or insert can include instructions for a clinician or subject for using one or more of the kit components in a method, use or treatment protocol or therapeutic regimen. Instructions can include dosage amounts, frequency of duration and instructions for practicing any of the methods, uses, treatment protocols or prophylactic or therapeutic regimens described herein.
• a label or insert can include information on potential adverse side effects, complications or reaction, such as a warning to a subject or clinician regarding situations where it would not be appropriate to use a particular composition.
• the term “about,” or “approximately” refers to a measurable value such as an amount of the biological activity, homology or length of a polynucleotide or polypeptide sequence, dose, time, temperature, and the like, and is meant to encompass variations 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 (greater than or less than) of the specified amount unless otherwise stated, otherwise evident from the context, or except where such number would exceed 100% of a possible value.
• transgene refers to a heterologous polynucleotide that is introduced into a cell and is capable of being transcribed into RNA and optionally, translated and/or expressed under appropriate conditions. In aspects, it confers a desired property to a cell into which it was introduced, or otherwise leads to a desired therapeutic or diagnostic outcome. In another aspect, it may be transcribed into a molecule that mediates RNA interference, such as miRNA, siRNA, or shRNA.
• homologous or “homology,” refers to two or more reference entities (e.g., a nucleic acid or polypeptide sequence) that share at least partial identity over a given region or portion.
• a homologous peptide will retain activity or function associated with the unmodified or reference peptide and the modified peptide will generally have an amino acid sequence “substantially homologous” with the amino acid sequence of the unmodified sequence.
• substantially homology or “substantial similarity,” means that when optimally aligned with appropriate insertions or deletions with another polypeptide, nucleic acid (or its complementary strand) or fragment thereof, there is sequence identity in at least about 70% to 99% of the sequence.
• a nucleic acid or polynucleotide refers to a DNA molecule (e.g., a cDNA or genomic DNA), an RNA molecule (e.g., an mRNA), or a DNA or RNA analog.
• a DNA or RNA analog can be synthesized from nucleotide analogs.
• the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
• An isolated or recombinant nucleic acid refers to a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid.
• the term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene
• nucleic acid described above can be used to express the protein of this disclosure.
• a "recombinant nucleic acid” is a combination of nucleic acid sequences that are joined together using recombinant technology and procedures used to join together nucleic acid sequences.
• a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of shuffling or recombination.
• the terms mean that the two nucleic acid segments are not from the same gene or, if form the same gene, one or both of them are modified from the original forms.
• the terms also include non-naturally occurring multiple copies of a naturally occurring DNA molecule.
• the terms refer to a nucleic acid segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found.
• Exogenous DNA segments are expressed to yield exogenous RNAs or polypeptides.
• a "homologous DNA molecule” is a DNA molecule that is naturally associated with a host cell into which it is introduced.
• a “regulatory sequence” includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences.
• the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein or RNA desired, and the like.
• the expression vector can be introduced into host cells to produce an RNA or a polypeptide of interest.
• 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 which causes RNAs to be initiated at high frequency.
• a "promoter” is a nucleotide sequence which initiates and regulates transcription of a polynucleotide. Promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters.
• promoter or "control element” includes full-length promoter regions and functional (e.g., controls transcription or translation) segments of these regions.
• “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
• a given promoter operably linked to a nucleic acid sequence is capable of effecting 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 the expression thereof.
• intervening untranslated yet 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.
• operably linked is intended to encompass any spacing or orientation of the promoter element and the DNA sequence of interest which allows for initiation of transcription of the DNA sequence of interest upon recognition of the promoter element by a transcription complex.
• the term “genetic construct” or “nucleic acid construct,” refers to a non- naturally occurring nucleic acid molecule resulting from the use of recombinant DNA technology (e.g., a recombinant nucleic acid).
• a genetic or nucleic acid construct is a nucleic acid molecule, either single or double stranded, which has been modified to contain segments of nucleic acid sequences, which are combined and arranged in a manner not found in nature.
• a nucleic acid construct may be a “cassette” or a “vector” (e.g., a plasmid, an rAAV vector genome, an expression vector, etc.), that is, a nucleic acid molecule designed to deliver exogenously created DNA into a host cell.
• "Expression cassette” as used herein means a nucleic acid sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, which may include a promoter operably linked to the nucleotide sequence of interest that may be operably linked to termination signals. It also may include sequences required for proper translation of the nucleotide sequence.
• the coding region usually codes for an RNA or protein of interest.
• the expression cassette including the nucleotide sequence of interest may be chimeric.
• the expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
• the expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of a regulatable promoter that initiates transcription only when the host cell is exposed to some particular stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development.
• a vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
• the vector may or may not be capable of autonomous replication or integrate into a host DNA.
• Examples include a plasmid, virus (e.g., an rAAV), cosmid, or other vehicle that can be manipulated by insertion or incorporation of a nucleic acid (e.g., a recombinant nucleic acid).
• a vector can be used for various purposes including, e.g., genetic manipulation (e.g., cloning vector), to introduce/transfer a nucleic acid into a cell, to transcribe or translate an inserted nucleic acid in a cell.
• a vector nucleic acid sequence contains at least an origin of replication for propagation in a cell.
• a vector nucleic acid includes a heterologous nucleic acid sequence, an expression control element(s) (e.g., promoter, enhancer), a selectable marker (e.g., antibiotic resistance), a poly-adenosine (polyA) sequence and/or an ITR.
• an expression control element(s) e.g., promoter, enhancer
• a selectable marker e.g., antibiotic resistance
• polyA poly-adenosine
• ITR an ITR.
• the nucleic acid sequence when delivered to a host cell, the nucleic acid sequence is propagated.
• the cell when delivered to a host cell, either in vitro or in vivo, the cell expresses the polypeptide encoded by the heterologous nucleic acid sequence.
• the nucleic acid sequence, or a portion of the nucleic acid sequence is packaged into a capsid.
• a host cell may be an isolated cell or a cell within a host organism.
• a nucleic acid sequence e.g., transgene
• additional sequences e.g., regulatory sequences
• regulatory sequences may be present within the same vector (i.e., in cis to the gene) and flank the gene.
• regulatory sequences may be present on a separate (e.g., a second) vector which acts in trans to regulate the expression of the gene.
• Plasmid vectors may be referred to herein as “expression vectors.”
• the term “vector genome” refers to a recombinant nucleic acid sequence that is packaged or encapsidated to form an rAAV vector.
• a vector genome includes a heterologous polynucleotide sequence, e.g., a transgene, regulatory elements, ITRs not originally present in the capsid.
• a recombinant plasmid is used to construct or manufacture a recombinant vector (e.g., rAAV vector)
• the vector genome does not include the entire plasmid but rather only the sequence intended for delivery by the viral vector.
• This non- vector genome portion of the recombinant plasmid is typically referred to as the “plasmid backbone,” which is important for cloning. selection and amplification of the plasmid, a process that is needed for propagation of recombinant viral vector production, but which is not itself packaged or encapsidated into an rAAV vector.
• viral vector generally refers to a viral particle that functions as a nucleic acid delivery vehicle and which comprises a vector genome (e.g., comprising a transgene instead of a nucleic acid encoding an AAV rep and cap) packaged within the viral particle (i.e., capsid) and includes, for example, lenti- and parvo- viruses, including AAV serotypes and variants (e.g., rAAV vectors).
• a recombinant viral vector does not comprise a vector genome comprising a rep and/or a cap gene.
• the term “overexpressing,” “overexpress,” “overexpressed,” or “overexpression,” when referring to the production of a nucleic acid or a protein in a host cell means that the nucleic acid or protein is produced in greater amounts than it is produced in its naturally occurring environment. It is intended that the term encompass overexpression of endogenous, as well as exogenous or heterologous nucleic acids and proteins. As such, the terms and the like are intended to encompass increasing the expression of a nucleic acid or a protein in a cell to a level greater than that the cell naturally contains.
• the expression level or amount of the 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% as compared to the level or amount that the cell naturally contains.
• the terms “overexpressing,” “overexpress,” “overexpressed,” and “overexpression,” and the like are intended to encompass increasing the expression of a nucleic acid or a protein to a level greater than that a mutant cell, a diseased cell, a wildtype cell, or a non-diseased cell contains.
• the expression level or amount of the 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% as compared to the level or amount that a mutant cell, a diseased cell, a wildtype cell, or a non- diseased cell contains.
• Anti-sense refers to a nucleic acid sequence, regardless of length, that is complementary to the coding strand or mRNA of a nucleic acid sequence. Antisense RNA can be introduced to an individual cell, tissue or organanoid. An anti-sense nucleic acid can contain a modified backbone, for example, phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural internucleoside linkages. As referred to herein, a "complementary nucleic acid sequence” is a nucleic acid sequence capable of hybridizing with another nucleic acid sequence comprised of complementary nucleotide base pairs.
• hybridize pair to form a double-stranded molecule between complementary nucleotide bases (e.g., adenine (A) forms a base pair with thymine (T), as does guanine (G) with cytosine (C) in DNA) under suitable conditions of stringency.
• a “suppressor” or an “inhibitor” refers to an agent that causes a decrease in the expression or activity of a target gene or protein, respectively.
• inhibitor refers to the reduction in the expression of a gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, below that observed in the absence of an inhibitor, suppressor or repressor, such as the inhibitory nucleic acid molecules (e.g., siRNA) described herein.
• Down-regulation can be associated with post-transcriptional silencing, such as, RNAi mediated cleavage or by alteration in DNA methylation patterns or DNA chromatin structure.
• 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 decrease in the expression of a target gene.
• a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule.
• expression of a target gene is reduced by 10%, 25%, 50%, 75%, or even 90-100%.
• siRNA intends a double-stranded RNA molecule that interferes with the expression of a specific gene or genes post-transcription.
• the siRNA functions to interfere with or inhibit gene expression using the RNA interference pathway. Similar interfering or inhibiting effects may be achieved with one or more of short hairpin RNA (shRNA), microRNA (mRNA) and/or nucleic acids (such as siRNA, shRNA, or miRNA) comprising one or more modified nucleic acid residue--e.g. peptide nucleic acids (PNA), locked nucleic acids (LNA), unlocked nucleic acids (UNA), or triazole-linked DNA.
• shRNA short hairpin RNA
• mRNA microRNA
• nucleic acids such as siRNA, shRNA, or miRNA
• PNA peptide nucleic acids
• LNA locked nucleic acids
• UNA unlocked nucleic acids
• a siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2-base overhang at its 3′ end.
• These dsRNAs can be introduced to an individual cell or culture system.
• Such siRNAs are used to downregulate mRNA levels or promoter activity.
• the terms “treat,” “treating” or “treatment” refer to administration of a therapy that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition.
• the term “ameliorate” means a detectable or measurable improvement in a subject’s disease, disorder or condition, or symptom thereof, or an underlying cellular response.
• a detectable or measurable improvement includes a subjective or objective decrease, reduction, inhibition, suppression, limit or control in the occurrence, frequency, severity, progression or duration of, complication cause by or associated with, improvement in a symptom of, or a reversal of a disease, disorder or condition.
• the term “associated with” refers to with one another, if the presence, level and/or form of one is correlated with that of the other.
• a particular entity e.g., polypeptide, genetic signature, metabolite, microbe, etc.
• a particular entity e.g., polypeptide, genetic signature, metabolite, microbe, etc.
• a particular disease, disorder, or condition if its presence, level and/or form correlates with incidence of and/or susceptibility to the disease, disorder, or condition (e.g., across a relevant population).
• the term “prevent” or “prevention” refers to delay of onset, and/or reduction in frequency and/or severity of one or more sign or symptom of a particular disease, disorder or condition (e.g., a myelin disease).
• 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 decrease in the development, frequency and/or intensity of one or more sign or symptom of the disease, disorder or condition is observed in a population susceptible to the disease, disorder or condition. Prevention may be considered complete when onset of disease, disorder or condition has been delayed for a predefined period of time.
• 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 that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder or condition.
• a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition.
• a therapeutically effective amount does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment.
• “Population” of cells refers to any number of cells greater than 1, but is at least 1 ⁇ 10 3 cells, at least 1 ⁇ 10 4 cells, at least at least 1 ⁇ 10 5 cells, at least 1 ⁇ 10 6 cells, at least 1 ⁇ 10 7 cells, at least 1 ⁇ 10 8 cells, at least 1 ⁇ 10 9 cells, or at least 1 ⁇ 10 10 cells.
• stem cells refers to cells with the ability to both replace themselves and to differentiate into more specialized cells. Their self-renewal capacity generally endures for the lifespan of the organism.
• a pluripotent stem cell can give rise to all the various cell types of the body.
• a multipotent stem cell can give rise to a limited subset of cell types.
• a hematopoietic stem cell can give rise to the various types of cells found in blood, but not to other types of cells.
• Multipotent stem cells can also be referred to as somatic stem cells, tissue stem cells, lineage-specific stem cells, and adult stem cells.
• the non- stem cell progeny of multipotent stem cells are progenitor cells (also referred to as restricted- progenitor cells).
• Progenitor cells give rise to fully differentiated cells, but a more restricted set of cell types than stem cells.
• Progenitor cells also have comparatively limited self-renewal capacity; as they divide and differentiate they are eventually exhausted and replaced by new progenitor cells derived from their upstream multipotent stem cell.
• iPS cells commonly abbreviated as iPS cells or iPSCs, refer to a type of pluripotent stem cell artificially prepared from a non-pluripotent cell, typically an adult somatic cell, or terminally differentiated cell, such as fibroblast, a hematopoietic cell, a myocyte, a neuron, an epidermal cell, or the like, by introducing certain factors, referred to as reprogramming factors.
• Pluripotency refers to a stem cell that has the potential to differentiate into all cells constituting one or more tissues or organs, or particularly, any of the three germ layers: endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm (epidermal tissues and nervous system).
• endoderm internal stomach lining, gastrointestinal tract, the lungs
• mesoderm muscle, bone, blood, urogenital
• ectoderm epidermal tissues and nervous system.
• “Pluripotent stem cells” used herein refer to cells that can differentiate into cells derived from any of the three germ layers, for example, direct descendants of totipotent cells or induced pluripotent cells.
• therapeutic cells refers to a cell population that ameliorates a condition, disease, and/or injury in a patient.
• Therapeutic cells may be autologous (i.e., derived from the patient), allogeneic (i.e., derived from an individual of the same species that is different from the patient) or xenogeneic (i.e., derived from a different species than the patient). Therapeutic cells may be homogenous (i.e., consisting of a single cell type) or heterogeneous (i.e., consisting of multiple cell types).
• therapeutic cell includes both therapeutically active cells as well as progenitor cells capable of differentiating into a therapeutically active cell.
• autologous refers to any material derived from the same subject or individual to which it is later to be re-introduced.
• the autologous cell therapy method described herein involves collection of glial cells, or progenitors thereof from a donor, e.g., a patient, which are then engineered to express, e.g., a transgene, and then administered back to the same donor, e.g., patient.
• a donor e.g., a patient
• transgene e.g., a transgene
• heterologous refers to any material (e.g., cells or tissue scaffold) derived from a different subject or individual.
• heterologous or non-endogenous or “exogenous” also refers to any material (e.g., gene, protein, compound, molecule, cell, or tissue or tissue component) or activity that is not native to a host cell or a host subject, or is any gene, protein, compound, molecule, cell, tissue or tissue component, or activity native to a host or host cell but has been altered or mutated such that the structure, activity or both is different as between the native and mutated versions.
• allogeneic refers to any material (e.g., cells or tissue) derived from one individual which is then introduced to another individual of the same species, e.g., allogeneic cell transplantation.
• cells may be obtained from a first subject, modified ex vivo according to the methods described herein and then administered to a second subject in order to treat a disease.
• the cells administered to the subject are allogeneic and heterologous cells.
• xenogenic refers to any material (e.g., cells or tissue) derived from an individual of a different species.
• isogenic refers to any materials (e.g., cells or tissue) characterized by essentially identical genes.
• the term “subject” refers to an organism, for example, a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, a dog).
• a subject is a non-human disease model.
• a human subject is an adult, adolescent, or pediatric subject.
• a subject is suffering from a disease, disorder or condition, e.g., a disease, disorder or condition that can be treated as provided herein.
• a subject is suffering from a disease, disorder or condition associated with deficient or dysfunctional myelin.
• a subject is susceptible to a disease, disorder, or condition.
• a susceptible subject is predisposed to and/or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing a disease, disorder or condition.
• a subject displays one or more symptoms of a disease, disorder or condition.
• a subject does not display a particular symptom (e.g., clinical manifestation of disease) or characteristic of a disease, disorder, or condition.
• a subject does not display any symptom or characteristic of a disease, disorder, or condition.
• a subject is a human patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
• 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 that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition.
• a therapeutically effective amount does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment.
• the examples below are intended to exemplify the practice of embodiments of the disclosure but are by no means intended to limit the scope thereof.
• hESCs Human embryonic stem cell lines and culture conditions Sibling human embryonic stem cells (hESCs) lines GENEA019 (WT: 18;15 CAG) and GENEA020 (HD: 48;17 CAG), both female, were obtained from GENEA, Inc. (Sydney, Australia). hESCs were regularly cultured under feeder-free conditions on 0.55 ug/cm 2 human recombinant laminin 521 (Biolamina, cat. no. LN521) coated cell culture flasks with mTeSR1 medium (StemCell Technologies, cat. no. 85850). Daily medium changes were performed. hESCs were routinely passaged at 80% confluency onto freshly coated flasks.
• Electroporation was performed using an Amaxa 4D-Nucleofector (Lonza) with the P3 primary cell kit (Lonza, cat. no. V4XP-3024) according to manufacturer’s guidelines. After nucleofection, the electroporated hESC suspensions were transferred to 10 cm cell culture dishes and cultured with mTeSR1 supplemented with 10 ⁇ M Y-27632 (Tocris, cat. no.1254) for the first 24h.
• Electroporated hESCs were grown for 48-72h, then treated with 0,5 ⁇ g/ ⁇ L puromycin (ThermoFisher, cat. A1113803). Electroporated hESC cultures were kept under puromycin until individual colonies were large enough to be picked manually. Colonies were assessed by fluorescence microscopy and transferred to a 96-well plate based on uniformity of reporter expression. Following expansion, each clone was split for further expansion and genotyping. For genotyping, DNA was extracted using the prepGEM Tissue DNA extraction kit (Zygem).
• BEBP12-769E then expanded for karyotype and array comparative genomic hybridization (aCGH) prior to hGPC production.
• aCGH karyotype and array comparative genomic hybridization
• Human GPCs were derived from both reporter WT and HD hESCs using the protocol described in Wang et al., Cell Stem Cell 12, 252--264 (2013) with minor modifications to the embryoid body (EB) generation step.
• Cells were collected for transplant between 150 and 200 DIV, at which time the cultures derived from both WT-mCherry/EGFP and HD-EGFP hESCs were comprised predominantly of PDGFR ⁇ + /CD44 + bipotential GPCs.
• Xenotransplantation Cell preparation To prepare cells for transplant, glial cultures were collected in Ca 2+ /Mg 2+ -free Hanks’ balanced salt solution (HBSS (-/-) ; ThermoFisher, cat. no. 14170112), then mechanically dissociated to small clusters by gentle , and counted with a hemocytometer. The cell suspension was then spun and resuspended in cold HBSS (-/-) at 10 5 cells/ ⁇ l, and kept on ice until transplanted.
• HBSS Ca 2+ /Mg 2+ -free Hanks’ balanced salt solution
• ThermoFisher cat. no. 14170112
• Neonatal grafts To generate human-mouse chimeras harboring mHTT-expressing human glia (HD chimeras), newborn immunocompromised Rag1 (-/-) pups 66 were cryoanesthetized, secured in a custom baked clay stage, and injected bilaterally with 100,000 HD glia (50,000 per hemisphere) into the presumptive striatum within 48h of birth. Cells were delivered using a 10 ⁇ l syringe (Hamilton, cat. no. 7653-01) with pulled glass pipettes at a depth of 1.2-1.4 mm. The pups were then returned to their mother until weaned.
• mice were compared to HD chimeric littermates that did not receive WT glia and to na ⁇ ve rag1 (-/-) mice that received WT glia at 36 weeks of age following this exact procedure.
• Human glial striatal isografts To evaluate the effects of cell age as a determinant of competitive dominance between human glia, newborn Rag1 (-/-) mice were injected following the same perinatal transplant protocol described above, but instead glia derived from WT- mCherry was delivered to generate human-mouse chimeras harboring WT human glia (WT chimeras).
• WT chimeras were then injected following the same adult transplant above described, but instead isogenic WT-EGFP glia was delivered.
• experimental animals were compared to WT chimeric littermates that did not receive WT- EGFP glia and to na ⁇ ve rag1 (-/-) mice that received WT-EGFP glia at 40 weeks of age following this exact procedure.
• Aseptic technique was used for all xenotransplants. All mice were housed in a pathogen-free environment, with ad libitum access to food and water, and all procedures were performed in agreement with protocols approved by the University of Rochester Committee on Animal Resources.
• the blocks were then cut as 20 ⁇ m sections on a CM1950 cryostat (Leica), serially collected on adhesion slides and stored at -20oC until further use.
• Identification and phenotyping of human cells was accomplished by immunostaining for their respective fluorescent reporter, together with a phenotypic marker, including Olig2 (GPCs and oligodendroglia), GFAP (astrocytes), or Ki67 (proliferating cells).
• Genetically- expressed fluorescent reporters were used as markers for human cells, as their expression remained stable throughout the animal’s life. In mice that received a 1:1 mixture of WT- mCherry and WT-untagged human glia, the latter were identified by the expression of human nuclear antigen (hN), and the lack of fluorescent reporter expression.
• Immunolabeled sections were rehydrated with PBS, then incubated in permeabilization/blocking buffer (PBS + 0.1% Triton-X (Sigma-Aldrich cat. no. T8787) + 10% Normal Goat Serum (ThermoFisher, cat. no.16210072)) for 2h. Sections were then incubated overnight with primary antibodies at 4oC. The following day, the sections were rinsed with PBS, and secondary antibodies applied for 1h. After again rinsing with PBS, a second round of primary antibodies, this time against fluorescent reporters, were applied to the sections overnight at 4oC. These were rinsed with PBS the following day and the sections incubated with secondary antibodies for 1h.
• permeabilization/blocking buffer PBS + 0.1% Triton-X (Sigma-Aldrich cat. no. T8787) + 10% Normal Goat Serum (ThermoFisher, cat. no.16210072)
• TUNEL terminal deoxynucleotidyl transferase-dUTP nick end labeling
• Quantitative histology Transplant mapping and 3D reconstruction To map human cell distribution, whole brain montages of 15 equidistantly spaced, 160 ⁇ m apart, sagittal sections spanning the entire striatum were captured using a Nikon Ni-E Eclipse microscope equipped with a DS-Fi3 camera at 10x magnification and stitched in the 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). When applicable, the site of adult injection was mapped as a reference point for volumetric quantification of human cell distribution. Mapped sections were then aligned using the lateral ventricle as a reference to produce a 3D reconstructed model of the humanized murine striatum. After 3D reconstruction, the cartesian coordinates for each human cell marker, injection site and striatal outlines were exported for further analysis.
• each quantified section was given an upper and lower boundary ⁇ ⁇ , ⁇ ⁇ , by representing the striatal outline as two identical polygons separated from each other by the section thickness (20 ⁇ m). Then, since the depth-wise location of each cell marker within each individual section is unknown, mapped cells within each section were represented as uniform point probability functions with constant probability across the section.
• each cell marker in a section from ⁇ ⁇ to ⁇ ⁇ has a probability function:
• 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 glia delivery site in radial increments of 125 ⁇ m (For control HD or WT chimeras, an average of the coordinates of the adult WT glia delivery site was used).
• Mapped cells were counted as 1 if their respective representative line segments were fully inside, 0 if fully outside, and partially if intersecting the spherical shell at either the upper or lower boundary of its corresponding section.
• the montages were then loaded onto Stereo Investigator and outlines of the striatum were defined.
• a set of 200 ⁇ 200 ⁇ m counting frames was placed by the software in a systematic random fashion within a 400 ⁇ 400 ⁇ m grid covering the outlined striatum of each section. Counting was performed in the entire section height (without guard zones), and cells were counted based on their immunolabelling in the optical section in which they first came into focus. Representative images showing whole striata were generated from whole brain montages using the ‘crop’ function, and by adjusting the ‘min/max’ levels in NIS-Elements imaging software.
• TUNEL + human cells To assess the distribution and proportion of apoptotic cells within each human cell pool, whole striatal montages of 5 equidistantly spaced, 480 ⁇ m apart, sagittal sections spanning the entire striatum were captured using a Nikon Ni-E Eclipse microscope equipped with a DS-Fi3 camera, at 10x magnification and stitched in the NIS-Elements imaging software. The striatum was outlined within each section, and immunolabeled human cells identified and mapped based on their TUNEL labelling within the outlined striatum using Stereo Investigator.
• Representative images showing whole humanized striata were generated from previously acquired whole brain montages using the ‘crop’ function and adjusting the ‘min/max’ levels in NIS-Elements imaging software.
• Representative images of human glial competitive interfaces were then captured as large field z-stacked montages, using a Nikon Ti- E C2+ confocal microscope equipped with 488nm, 561nm and 640nm laser lines, and a standard PMT detector. Images were captured at 40x or 60x magnification with oil-immersion objectives and stitched in NIS-Elements. Maximum intensity projections were then generated, and the ‘min/max’ levels adjusted in NIS-Elements.
• FACS Fluorescence activated cell sorting
• the brains were immersed in ice-cold sterile HBSS for about 5 minutes to facilitate the microdissection. Under a dissecting microscope, the striata from each mouse was dissected and placed in sterile HBSS on ice. The striatal tissues were transferred to a Petri dish containing sterile HBSS without magnesium chloride and calcium chloride, chopped into small pieces using sterile disposable scalpels, transferred into a sterile tube, and then incubated in a 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 in order to achieve a single cell suspension.
• the cells were then pelleted, resuspended into MEM, and filtered for flow cytometry.
• Single cell preparations were isolated based on their expression of mCherry, EGFP, or their absence, using a BD FACSAria Fusion (BD Biosciences).
• DAPI 4′,6-diamidino-2-phenylindole
• Single-cell RNA sequencing analysis Primary data acquisition Isolated cells were captured for scRNA-seq on a 10X Genomics chromium controller (v3.1 chemistry).
• TwopassMode basic
• limitSjdbInsertNsj 2000000
• soloUMIfiltering MultiGeneUMI.
• Differential expression analysis Human data were imported into R 71 using Seurat 72 . Cells were filtered (Unique genes >250 and percent mitochondrial genes ⁇ 15). 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 the mouse sample and cell line in addition to the number of unique genes and percent mitochondrial gene expression.
• 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. Data were then re-imported into Seurat, and differential expression was carried out using MAST 74 . Genes were considered for differential expression if their expression was detected in at least 3% of all GPCs.
• the model design for differential expression utilized the number of unique genes in a cell and the experimental group (cell line/age of the cell, and if the cell was in the presence or absence of an opposing clone).
• regulons are referred to as regulons and are assigned “Area Under the Curve” (AUC) values to represent their activities in each cell, with higher values indicating a stronger enrichment of such regulon.
• AUC Average Under the Curve
• the resulting AUC matrix was then used to look for important transcription factors.
• 1 was assigned to cells from the young WT samples, and 0 was assigned to cells from the aged WT or aged HD samples.
• Lasso logistic regression was then performed on predetermined 0/1 outcome with all TF’s AUCs as predictor using glmnet. Lambda for logistic regression was automatically defined with cv.glmnet.
• Inventors isolated TFs with positive coefficients, and further filtered based on their mean activity per group, such that TF mean activity in the young WT should be higher than that in the aged counterpart.
• the final step was to perform gene set enrichment analysis (GSEA) 76 on regulons identified thus far, to determine if they were enriched for differentially upregulated genes in young WT cells compared to aged HD and WT cells (adjusted p ⁇ x10 -3 , NES > 0).
• GSEA gene set enrichment analysis
• WGCNA Weighted gene co-expression network analysis
• Data are represented as the mean ⁇ standard error of mean (SEM).
• Data Availability The sequencing datasets reported in this paper can be accessed at GEO, via accession number GSE206322.
• Code Availability The program for the quantification of mapped cell populations in 3D-reconstructed tissues is publicly available through the following link: QIM / Tools / Thick Section Point Density GitLab (dtu.dk). All codes used to analyze and generate Figures for the genomics dataset are accessible on Github, at: https://github.com/CTNGoldmanLab/HD_Competition_2022.
• Example 2 Generation of distinctly color-tagged human glia from WT and HD hESCs
• fluorophore-tagged reporter lines of WT and HD human embryonic stem cells were first generated so as to enable the production of spectrally-distinct GPCs of each genotype, whose growth in vivo could then be independently monitored.
• 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) and mHtt-expressing (HD, GENEA020) hESCs 27, 28 .
• Both WT-mCherry and HD-EGFP hESCs were then differentiated using a protocol for generating hGPCs 21 and assays were carried out to assess both their capacity to differentiate into glia and the stability of their reporter expression upon acquisition of glial fate.
• mice whose striata were substantially chimerized by tagged mHTT- expressing glia were generated by neonatally injecting hGPCs derived from EGFP-tagged HD hESCs into the neostriata of immunodeficient Rag1 (-/-) mice (Figs. 6A).
• the HD glia rapidly infiltrated the striata of these mice, migrating and expanding first within the striatal white matter tracts, and then progressively displacing their murine counterparts from the striatal neuropil (Figs.6B).
• the murine striatum was substantially humanized by HD glia ( Figs.6B, 6F, and 6G).
• hGPCs derived from WT hESCs engineered to express mCherry were engrafted into the striata of 36 week-old HD chimeras, and monitored their expansion histologically as they competed with the already-resident HD glia (Fig.1A and Figs.7A-B).
• the WT glia pervaded the previously humanized striatum, gradually displacing their HD counterparts as they expanded from their implantation site (Fig. 1B). This process was slow but sustained, over time yielding substantial repopulation of the HD striatum with WT glia (Fig. 1B, G and H 1 ; 54 weeks: P ⁇ 0.0001; 72 weeks, P ⁇ 0.0001).
• Example 5 Human WT glia enjoy a proliferative advantage relative to resident HD glia Since striatal humanization by HD glia decelerated with time as the fraction of proliferative HD hGPCs fell (Fig. 6D), assays were carried out to examine if the selective expansion of younger WT glia within the HD striatum was sustained by a difference in proliferative capacity between the two populations. To do so, the expression of Ki67 in both WT and HD glial populations was assessed as competitive striatal repopulation unfolded.
• Leiden community detection revealed six major populations of human glia; these included hGPCs, cycling hGPCs, immature oligodendrocytes (iOL), neural progenitor cells (NPCs), astrocytes, and their intermediate progenitors (astrocyte progenitor cells, APCs) (Figs. 2B-D).
• hGPCs high-mobility progenitor cells
• NPCs neural progenitor cells
• astrocyte progenitor cells astrocyte progenitor cells
• Figs. 2B-D intermediate progenitors
• cell cycle analysis predicted higher G2/M scores in competing WT hGPCs compared to their HD counterparts (Fig.2E), aligning with histological observations (Fig.1J).
• inventors focused on hGPCs as the primary competing population in the model.
• Pairwise differential expression revealed discrete sets of differentially expressed genes across groups (Fig. 2F), and subsequent functional analysis with Ingenuity pathway analysis (IPA) within the hGPC population revealed numerous salient terms pertaining to their competition (Fig.2G). It was found that during competition, WT GPCs activate pathways driving protein synthesis, whereas HD GPCs were predicted to downregulate them. Predicted upstream transcription factor activation identified YAP1, MYC, and MYCN – conserved master regulators of cell growth and proliferation 32-34 – as significantly modulated across experimental groups. Importantly, it was found YAP1 and MYC targets to be selectively down-regulated in competing HD GPCs relatively to their controls (Fig.2G).
• Example 7 Age differences drive competitive human glial repopulation Since WT cells transplanted into adult hosts were fundamentally younger than the resident host cells that they displaced and replaced, assays were carried out to examine if differences in cell age, besides disease status, might have contributed to the competitive success of the late donor cells. To that end, hGPCs newly produced from WT hESCs engineered to express EGFP were engrafted into the striata of 40 week-old adult glial chimeras, which had been perinatally engrafted with hGPCs derived from mCherry-tagged, otherwise isogenic WT hESCs (Fig. 3A).
• Example 8 Young cells replace their older counterparts via the induction of apoptosis Since younger glia appeared to exert clear competitive dominance over their older counterparts, assays were carried out to examine whether the elimination of the older glia by younger cells occurred passively, as a result of the higher proliferation rate of the younger cells leading to the relative attrition of the older residents during normal turnover, or whether replacement was actively driven by the induction of programmed cell death in the older cells by the more fit younger cells. To address this question, the TUNEL assay was used to compare the rates of apoptosis in aged and young WT glial populations as they competed in the host striatum, as well as at their respective baselines in singly-transplanted controls.
• genes functionally associated with protein synthesis including ribosomal genes and E2F family members, as well as upstream MYC and MYCN signaling, were all activated in competing young WT GPCs relative to their aged counterparts (Fig.4G). Yet despite these similarities, in other respects aged WT GPCs responded differently than did HD GPCs to newly implanted WT GPCs. In contrast to HD GPCs, aged WT cells confronted with younger isogenic competitors upregulated both MYC and MYCN targets relative to their non-competing controls (Fig.4G) with a concomitant upregulation of ribosomal genes (Fig.4I).
• Example 10 Competitive advantage is linked to a discrete set of transcription factors Assays were carried out to examine what gene signatures would define the competitive advantage of newly-transplanted human GPCs over resident cells. To that end, a multi-stepped analysis was applied using lasso-regulated logistic regression (Fig.5A), that pinpointed 6 TFs (CEBPZ, CTCF, E2F1, MYC, NFYB, ETV4) whose activities could significantly explain the dominance of young WT GPCs over both aged HD and aged WT GPCs (Fig.5E).
• Fig.5A lasso-regulated logistic regression
• WGCNA Weighted gene co- expression network analysis
• module blue was primarily influenced by age (Fig. 5D), in contrast to other modules identified from WGCNA.
• MYC whose regulated pathway activation had already been inferred as conferring competitive advantage (Figs. 2 and 4), was also one of the six prioritized TFs.
• Fig. 5F MYC was part of module blue and regulated these blue modular genes, whose expression levels were higher in the competing versus non-competing paradigms.
• the targets in this network were enriched for pathways regulating cell proliferation (TP53, YAP1, RICTOR), transcription (MYCN, MLXIPL), and protein synthesis (LARP1), each of which had been previously noted as differentially-expressed in each competitive scenario (Figs. 2 and 4).
• the output of this competition-triggered regulatory network thus appeared to confer competitive advantage upon young WT hGPCs when introduced into the adult brain, whether confronted by older, HD-derived or isogenic hGPCs.
• RAG-1-deficient mice have no mature B and T lymphocytes.
• STARsolo accurate, fast and versatile mapping/quantification of single-cell and single-nucleus RNA-seq data. bioRxiv, 2021.2005.2005.442755 (2021). 70. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21 (2013). 71. R Core Team R: A language and environment for statistical computing. (2013). 72. Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nature Biotechnology 36, 411--420 (2016). 73.

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