Patent Application
20240167030
Oligodendrocytes are cells in the central nervous system that are critical for brain health and function by producing myelin, which insulates and protects neuronal axons. Oligodendrocytes arise from a progenitor population in the brain called oligodendrocyte progenitor cells (OPCs), which are present in both the adult and developing brain and are responsible for maintaining oligodendrocyte formation in development and disease.
Oligodendrocyte formation is a multi-step process proceeding from OPC, to intermediate pre-myelinating or newly formed oligodendrocytes, to fully mature myelinating oligodendrocytes. Oligodendrocyte formation has been shown to be stalled at the intermediate premyelinating stage in numerous demyelinating diseases, such as multiple sclerosis.
There is a need for novel therapeutics to accelerate maturation of OPCs to myelin-forming oligodendrocyte to treat numerous demyelinating diseases.
Embodiments described herein relate to methods and compositions to accelerate formation of fully mature myelinating oligodendrocytes from precursor stages for the treatment of myelin-related disorders using gene therapy or genome engineering.
Thus, in one aspect, the invention provides a method for accelerating cellular maturation of a cell, the method comprising impairing the activity of developmental transcriptional condensates at an intermediate stage of a cell lineage for the cell.
In some embodiments, the cell is an oligodendrocyte.
In some embodiments, the developmental transcriptional condensates are regulated by Sox6.
In some embodiments, the method comprises delivering to the cell an agent to decrease expression of endogenous Sox6.
In some embodiments, the agent is an antisense oligonucleotide (ASO), an siRNA, a CRISPR interference agent (Cas effector enzyme and guide RNA), TALE/zinc finger protein, NgAO, micro RNA, or a coding sequence therefor.
In some embodiments, the method comprises contacting the cell with a delivery vehicle comprising the agent or the coding sequence therefor.
In some embodiments, the delivery vehicle is an AAV vector, an adenoviral vector, or a lentivirus vector.
In some embodiments, the cell is contacted in vitro, in vivo, or ex vivo.
In some embodiments, the method accelerates oligodendrocyte maturation.
In some embodiments, the method enhances myelination.
In another aspect, the invention provides a modified cell generated by any one of the methods of the invention.
In certain embodiments, the cell is accelerated to maturation by impairing the activity of developmental transcriptional condensates at an intermediate stage.
In some embodiments, the cell is an oligodendrocyte.
In some embodiments, the developmental condensates are regulated by Sox6.
In some embodiments, Sox6 expression is inhibited. In some embodiments, Sox6 expression is inhibited by an antisense oligonucleotide (ASO), siRNA, CRISPR interference, or micro RNA targeting Sox6
In some embodiments, the cell is selected from a neural stem cell (NSC), oligodendrocyte progenitor cell (OPC), or oligodendrocyte cell.
In some embodiments, the modified cell descended from NSC, OPC or oligodendrocyte cell.
In another aspect, the invention provides a composition comprising a modified cell of the invention.
In certain embodiments, the cell is accelerated to maturation by impairing the activity of developmental transcriptional condensates at an intermediate stage.
In some embodiments, the cell is an oligodendrocyte.
In some embodiments, the developmental condensates are regulated by Sox6.
In some embodiments, Sox6 expression is inhibited. In some embodiments, Sox6 expression is inhibited by an antisense oligonucleotide (ASO), siRNA, CRISPR interference, or micro RNA targeting Sox6.
In another aspect, the invention provides a method of treating a myelin related disorder in a subject, the method comprising administering to the subject an agent that impairs the activity of Sox6 to arrest developmental transcriptional condensates at an intermediate stage of the oligodendrocyte precursor—oligodendrocyte lineage, thereby accelerating or promoting maturation of said oligodendrocyte precursor to myelin-producing oligodendrocytes.
In another aspect, the invention provides a method of treating a myelin related disorder in a subject, the method comprising administering to the subject an oligodendrocyte cell accelerated to maturation by impairing the activity of developmental transcriptional condensates at an intermediate stage, thereby introducing mature and myelin-producing oligodendrocyte cell in the subject.
In some embodiments, the myelin-related disorder is selected from multiple sclerosis (MS), neuromyelitis optica (NMO), transverse myelitis, chronic inflammatory demyelinating polyneuropathy, Guillain-Barre Syndrome, progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelolysis (CPM), adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher disease (PMD), Wallerian Degeneration, optic neuritis, amylotrophic 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, AR, Bassen-Kornzweig syndrome, Marchiafava-Bignami syndrome, metachromatic leukodystrophy, trigeminal neuralgia, acute dissmeminated encephalitis, Marie-Charcot-Tooth disease and Bell's palsy.
In some embodiments, the method alleviates at least one symptom(s) of the subject associated with said myelin related disorder.
In some embodiments, the method restores a function of the subject to at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100% of a control subject without the myelin related disorder, preferably, the function is motor coordination, locomotion, or axon conduction velocity.
In some embodiments, the cell is selected from the group consisting of a modified NSC, OPC, and oligodendrocyte.
In some embodiments, the subject is a mammal, such as a human.
It should be understood that any one embodiment of the invention, including those described only in the examples or claims, can be combined with one or more additional embodiment(s) of the invention, unless explicitly/expressly disclaimed or otherwise improper.
Understanding the developmental regulators that ultimately produce functional cell types is a central goal of biology and medicine. While the mechanisms controlling early differentiation have been well-studied for many important cell lineages, the regulation of subsequent maturation timing remains unknown.
The invention described herein is partly based on the discovery that the timing of cellular maturation is governed by a transient form of transcriptional condensates. During oligodendrocyte development, the transcription factor SOX6 dramatically re-localizes from nearly all super enhancers in oligodendrocyte progenitor cells (OPCs) to form developmental condensates across a small set of gene bodies in immature oligodendrocytes. Genes in condensate loci are highly expressed in immature cells but turn off upon maturation.
Consistent with this, CRISPR-, RNAi, or miRNA-mediated suppression of SOX6 reduced developmental condensate gene activation, and accelerated maturation directly to mature myelinating oligodendrocytes.
The data presented herein collectively demonstrates that Sox6-regulated developmental condensates govern processivity along the continuum of oligodendrocyte formation from OPCs. Thus the invention described herein provides, among other things, methods for modulating biomolecular condensates to control maturation rate during development. The methods of the invention can be used, for example, to accelerate the regeneration of mature cell types in numerous diseases.
For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
The terms “comprise,” “comprising,” “include,” “including,” “have,” and “having” are used in the inclusive, open sense, meaning that additional elements may be included. The terms “such as,” “e.g.,” as used herein are non-limiting and are for illustrative purposes only. “Including” and “including but not limited to” are used interchangeably.
The term “or” as used herein should be understood to mean “and/or,” unless the context clearly indicates otherwise.
As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
The phrases “parenteral administration” and “administered parenterally” are
The term “treating” is art-recognized and includes inhibiting a disease, disorder or condition in a subject, e.g., impeding its progress; and relieving the disease, disorder or condition, e.g., causing regression of the disease, disorder and/or condition. Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected.
The term “preventing” is art-recognized and includes stopping a disease, disorder or condition from occurring in a subject, which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it. Preventing a condition related to a disease includes stopping the condition from occurring after the disease has been diagnosed but before the condition has been diagnosed.
The term “pharmaceutical composition” refers to a formulation containing the disclosed compounds in a form suitable for administration to a subject. In a preferred embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or a vial. The quantity of active ingredient (e.g., a formulation of the disclosed compound or salts thereof) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved. One skilled in the art will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, inhalational, and the like. Dosage forms for the topical or transdermal administration of a compound described herein includes powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, nebulized compounds, and inhalants. In a preferred embodiment, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.
The phrase “pharmaceutically acceptable” is art-recognized. In certain embodiments, the term includes compositions, polymers and other materials and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The phrase “pharmaceutically acceptable carrier” is art-recognized, and includes, for example, pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of a subject composition and not injurious to the patient. In certain embodiments, a pharmaceutically acceptable carrier is non-pyrogenic. Some examples of materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.
The terms “prophylactic” or “therapeutic” treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal such as, but not limited to, myelination disturbances, myelin deficiencies, myelin loss and ineffective myelin repair) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).
The terms “therapeutic agent,” “drug,” “medicament” and “bioactive substance” are art-recognized and include molecules and other agents that are biologically, physiologically, or pharmacologically active substances that act locally or systemically in a patient or subject to treat a disease or condition. The terms include without limitation pharmaceutically acceptable salts thereof and prodrugs. Such agents may be acidic, basic, or salts; they may be neutral molecules, polar molecules, or molecular complexes capable of hydrogen bonding; they may be prodrugs in the form of ethers, esters, amides and the like that are biologically activated when administered into a patient or subject.
The phrase “therapeutically effective amount” or “pharmaceutically effective amount” is an art-recognized term. In certain embodiments, the term refers to an amount of a therapeutic agent that produces some desired effect at a reasonable benefit/risk ratio applicable to any medical treatment. In certain embodiments, the term refers to that amount necessary or sufficient to eliminate, reduce or maintain a target of a particular therapeutic regimen. The effective amount may vary depending on such factors as the disease or condition being treated, the particular targeted constructs being administered, the size of the subject or the severity of the disease or condition. One of ordinary skill in the art may empirically determine the effective amount of a particular compound without necessitating undue experimentation. In certain embodiments, a therapeutically effective amount of an agent (e.g., a composition or genetically modified cells described herein) for in vivo use will likely depend on a number of factors, including: the rate of release of an agent from a polymer matrix, which will depend in part on the chemical and physical characteristics of the polymer; the identity of the agent; the mode and method of administration; and any other materials incorporated in the polymer matrix in addition to the agent.
The terms “nucleic acid,” “nucleotide,” “polynucleotide,” and “oligonucleotide” are used interchangeably and refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form. For the purposes of the present disclosure, these terms are not to be construed as limiting with respect to the length of a polymer. The terms can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones). In general, an analogue of a particular nucleotide has the same base-pairing specificity; i.e., an analogue of A will base-pair with T.
The terms “polypeptide,” “peptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of a corresponding naturally-occurring amino acids.
A “functional domain” is a domain of a polypeptide comprising a specific activity. Non-limiting examples of activities that a functional domain may possess are nuclease activity, transcriptional regulatory activity, viral capsid recognition activity and the like.
“Binding” refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific. Such interactions are generally characterized by a dissociation constant (Kd) of 10−6 M−1 or lower. “Affinity” refers to the strength of binding: increased binding affinity being correlated with a lower Kd.
A “binding protein” is a protein that is able to bind to another molecule. A binding protein can bind to, for example, a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA-binding protein) and/or a protein molecule (a protein-binding protein). In the case of a protein-binding protein, it can bind to itself (to form homodimers, homotrimers, etc.) and/or it can bind to one or more molecules of a different protein or proteins. A binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA-binding, RNA-binding and protein-binding activity.
In general, “CRISPRs” (Clustered Regularly Interspaced Short Palindromic Repeats), also known as SPIDRs (SPacer Interspersed Direct Repeats), refer a family of DNA loci that are usually specific to a particular bacterial species. The CRISPR locus comprises a distinct class of interspersed short sequence repeats (SSRs) that were recognized in E. coli (Ishino et al. (1987) J. Bacteriol., 169:5429-5433; and Nakata et al., J. Bacteriol. (1989) 171:3553-3556), and associated genes. Similar interspersed SSRs have been identified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena, and Mycobacterium tuberculosis (See, Groenen et al. (1993) Mol. Microbiol., 10:1057-1065; Hoe et al. (1999) Emerg. Infect. Dis., 5:254-263; Masepohl et al. (1996) Biochim. Biophys. Acta 1307:26-30; and Mojica et al. (1995) Mol. Microbiol., 17:85-93). The CRISPR loci typically differ from other SSRs by the structure of the repeats, which have been termed short regularly spaced repeats (SRSRs) (Janssen et al. (2002) OMICS J. Integ. Biol., 6:23-33; and Mojica et al. (2000) Mol. Microbiol., 36:244-246). In general, the repeats are short elements that occur in clusters that are regularly spaced by unique intervening sequences with a substantially constant length (Mojica et al. (2000), supra). Although the repeat sequences are highly conserved between strains, the number of interspersed repeats and the sequences of the spacer regions typically differ from strain to strain (van Embden et al., J. Bacteriol. (2002) 182:2393-2401). CRISPR loci have been identified in more than 40 prokaryotes including, but not limited to Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacteriumn, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thernioplasnia, Corynebacterium, Mycobacterium, Streptomyces, Aquifrx, Porphvromonas, Chlorobium, Therms, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myrococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga.
“CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. In some embodiments, one or more elements of a CRISPR system is derived from a class 1 type I or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a class 2 type II, or type V CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system). In the context of formation of a CRISPR complex, “target sequence” refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. A sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an “editing template” or “editing polynucleotide” or “editing sequence.” In aspects of the invention, an exogenous template polynucleotide may be referred to as an editing template. In an aspect of the invention the recombination is homologous recombination.
The invention is further illustrated by the following examples, which are not intended to limit the scope of the claims.
Cells are equipped with transcriptional blueprints that provide unique instructions to establish cellular identity, maintain homeostasis, and shape how cells respond to extracellular cues from the environment (Enver et al., 2009; Mullen et al., 2011). Delineating these transcriptional networks offers insight to the regulation of pathways and processes crucial to cellular function in normal development and how these go awry in disease (Lee and Young, 2013; Ott et al., 2018). Progenitor populations in particular are equipped with transcriptional networks that have the added responsibility to stabilize or direct cell state during differentiation (Adam et al., 2015; Boyer et al., 2005; Tsankov et al., 2015). While differentiation was previously assumed to be a straight line, it is now appreciated that this complex process (Enver et al., 2009; Iacono et al., 2019; MacLean et al., 2018). In particular, interrogating the transcriptional circuitry governing the rate of maturation along a differentiation trajectory provides novel insight to the lineage of interest, and more broadly gives clues to how cellular differentiation is controlled in development and disease.
In the central nervous system (CNS), the oligodendrocyte lineage undergoes a multi-step differentiation process and the transcriptional mechanisms governing the rate of transitioning between states remain poorly understood (Elbaz and Popko, 2019; Emery, 2010; Emery and Lu, 2015; Monje, 2018). Mature myelinating oligodendrocytes are responsible for wrapping neuronal axons in myelin, which is critical for proper action potential propagation and providing neurons with trophic support (Nave, 2010). These mature oligodendrocytes arise from a progenitor population in the brain called oligodendrocyte progenitor cells (OPCs), but first go through an intermediate pre-myelinating oligodendrocyte state (Emery and Lu, 2015; Howng et al., 2010; Zhang et al., 2014). The importance of understanding this differentiation trajectory is highlighted by the numerous diseases of oligodendrocytes, some of which have been shown to impair the lineage at specific states (Allan et al., 2020; Elitt et al., 2018; Gibson et al., 2019; Mathys et al., 2019; Yeung et al., 2019). Knowledge of this differentiation process also provides targets for rational drug design to drive myelin formation in a disease context specific manner (Allan et al., 2020; Elitt et al., 2018; Wang et al., 2020).
Most knowledge of the transcriptional programs regulating oligodendrocyte formation is derived from gene expression data or known disease-causing mutations that lead to hypo-myelinating phenotypes (Emery and Lu, 2015; He et al., 2016; Howng et al., 2010; Sun et al., 2018; Zhou et al., 2001). While effective, an alternative approach to investigate the core circuitry of cell states is through elucidation of super-enhancers, which are chromatin regions enriched for master lineage transcription factors and harbor high levels of transcription of genes critical for cellular identity (Adam et al., 2015; Gryder et al., 2017; Hnisz et al., 2013; Ott et al., 2018; Whyte et al., 2013). In this study, super-enhancer profiling was performed with extended transcriptional network analysis to elucidate the transcriptional mechanisms governing the processivity of oligodendrocyte formation from their progenitors.
Mouse epiblast derived OPC preparation and culture: Data were generated using OPCs generated from epiblast stem cell (EpiSC) lines EpiSC5 (rep 1 OPCs) and 12901 (rep 2 OPCs), which were derived from mouse strain 129SvEv and 129S1/SvImJ respectively and both have been used and validated in multiple publications (Allan et al., 2020; Hubler et al., 2018; Najm et al., 2015; Najm et al., 2011). These EpiSC derived OPCs were purified by fluorescence activated cell sorting using conjugated CD140a (eBioscience, 17-1401; 1:80) and NG2-AF488 (Millipore, AB5320A4; 1:100) antibodies and purity was verified by staining for markers of OPCs, astrocytes, and oligodendrocytes (
Mouse OPC differentiation to oligodendrocytes: OPC differentiation to oligodendrocytes follows the same protocol used previously (Allan et al., 2020; Hubler et al., 2018). In brief, 96 well and 384 well plates were coated with PO (or pre-coated with poly-D lysine) and laminin. OPCs were then seeded at either 40,000 cells per well (96-well plate) or 15,000 cells per well (384-well plate) in oligodendrocyte differentiation media consisting of DMEM/F12 supplemented with N2 Max (R&D systems), B27 (ThermoFisher), 100 ng/mL noggin, 100 ng/mL IGF-1, 10 uM cyclic AMP, 10 ng/mL NT3 and 40 ng/mL T3. Cultures typically were allowed to proceed for 3 days unless otherwise noted.
Mouse oligodendrocyte myelination assay using microfibers: Parallel-aligned 2-4 μm electrospun fibers composed of poly-L-lactic acid were synthesized and suspended by fitting into 12-well plate inserts by The Electrospinning Company. Inserts were placed in 12 well plates. Prior to use, inserts were incubated in 70% ethanol. Next, fibers were coated with 100 μg/mL poly(L-ornithine) followed by 10 μg/ml laminin. EpiSC5 OPCs seeded at 250,000 OPCs per well in oligodendrocyte differentiation medium. Cells were allowed to attach for 2 hours and then were transfected with 25 nM microRNA mimics, inhibitors, or siRNAs. T3 positive control cells were not transfected. Media was replaced after 16 hours. No other transfection was performed. For T3 positive controls, medium was supplemented with 40 ng/mL thyroid hormone from days 0-3. On day 3 thyroid hormone was removed, and medium was subsequently changed every third day. At day 10 plates were fixed. All plates were immunostained with rat anti-MBP, Olig2 and counterstained with DAPI. Plates were imaged on the Operetta® High Content Imaging and Analysis system. A total of 30 fields were captured at 20× using Acapella® software, and images were analyzed using Harmony® software and Columbus™ software. Using this analysis software we developed an Acapella® script to quantify the total fiber area surrounded by MBP+ oligodendrocytes. All staining, imaging, and analysis steps were performed simultaneously for each microfiber plate using identical procedures to reduce plate-to-plate variability.
Immunohistochemistry: For antigens requiring live staining (01), antibodies were diluted in N2B27 media supplemented with 10% Dokey Serum (017-000-121, Jackson ImmunoResearch) and added to cells in a tissue culture incubator for 18 minutes maintained at 5% CO2 and 37° C. Cells were then fixed by removing either primary live stain solution or cell culture media followed by addition of cold 4% paraformaldehyde (PFA, Electron microscopy sciences). The plates were then incubated at room temperature for 18 minutes, washed with PBS and permeabilized and blocked in blocking solution, which consisted of 0.1% Triton X-100 in PBS supplemented with 10% normal donkey serum (017-000-121, Jackson ImmunoResearch), for at least 30 minutes at room temperature. Primary antibodies (see chart above) were then diluted in blocking solution and incubated on cells overnight at 4° C. The following day, cells were rinsed with PBS and incubated in blocking solution containing appropriate secondary antibodies conjugated to an Alexa-Fluor (ThermoFisher). Secondary antibodies were added at a dilution of 1:500 along with the nuclear stain DAPI (Sigma) at a dilution of 1:10,000.
High content imaging and unbiased quantification using Columbus analysis software: Immunocytochemistry performed on cells in 96-well and 384-well plates were imaged using the Operetta High Content imaging and analysis system (PerkinElmer). Fields were captured at 20× magnification and 8-10 fields (96-well plate) or 5 fields (384-well plate) were captured per well. Images were then uploaded and analyzed with PerkinElmer Harmony and Columbus software as described previously (Allan et al., 2020; Hubler et al., 2018). In brief, total cell number was identified using a threshold for area of DAPI staining of nuclei to exclude pyknotic nuclei and debris. To identify oligodendrocytes, each DAPI nucleus was expanded by 50% and then the intensity of staining of an oligodendrocyte marker (MBP/O1) in a separate channel was calculated. Expanded nuclei that intersected O1/MBP above a threshold were called as oligodendrocytes in that image. The number of oligodendrocytes were then divided by the total number of cells as indicated by DAPI staining to get the percentage of oligodendrocytes per field. All fields were combined to give statistics on a per well basis. Thresholding to call a DAPI positive cell an oligodendrocyte varied on a per-plate basis based on different levels of background, staining intensity, etc. However, the same script is applied to the entire plate to ensure that the same threshold is applied across conditions.
Generation of mouse CRISPR KO OPCs: CRISPR knockout (KO) OPCs were created following a similar protocol used previously (Allan et al., 2020; Tripathi et al., 2019). In brief, guides were selected from the Brie library (Doench et al., 2016), cloned into the linearized CRISPRv2 backbone (Addgene 52961) (Sanjana et al., 2014), and sequence verified by sanger sequencing. HEK293T cells (Clonetech) were than transfected using lenti-X shots (manufacturer's protocol, Clonetech). Transfection media was replaced with N2B27 base media the following day. Lentivirus containing N2B27 was then collected after an additional 2 days, filtered, and supplemented with OPC growth factors PDGFA and FGF2 and added to OPCs at a ratio of 1:2 viral media to OPC growth media. The viral media was switched out for virus-free OPC growth the next day and allowed to recover for an additional 2 days. At this point, infected OPCs were selected for 96 hours in OPC growth media supplemented with a lethal dose of puromcyin (500 ng/mL). OPCs were allowed to recover in selection free media for at least 24 hours prior to being aliquoted and frozen down. For all experiments, infected CRISPR KO and non-targeting control OPCs were derived from the same original batch of EpiSC-derived mouse OPCs. qPCR was performed to validate a reduction of gene targets for each batch of CRISPR KO OPCs generated.
Compound screening and validation dose curves: The EpiSC-derived OPC high-throughput screen was performed previously (Factor et al., 2020). In brief, Poly-D lysine coated 384-well CellCarrier ultra plates (PerkinElmer) were coated with laminin diluted in N2B27 base media using a BioTek EL406 Microplate Washer Dispenser (BioTek) equipped with a 5 μl dispense cassette (BioTek). These plates incubated at 37° C. in 5% CO2 for at least an hour. The Selleck bioactives library (X compounds) diluted in dimethylsulfoxide (DMSO) was then dispensed using a 50 nL solid pin tool attached to a Janus automated workstation (Perkin Elmer) at a 1:1000 dilution giving a final concentration of 3 μM per well. OPCs were then plated in OPC differentiation media using the BioTek EL406 Microplate Washer Dispenser. Control columns for this screen included DMSO only as well as a separate column containing FGF2 to impair differentiation and ensure proper response of OPCs to inhibition of differentiation for each plate of the screen. OPCs were then differentiated at 37° C. in 5% CO2 for 3 days. At the end of the assay, cells were fixed, washed, and stained using the BioTek EL406 Microplate Washer Dispenser. Primary antibody used was MBP (Abcam, ab7349; 1:100) along with Olig2 and DAPI (Sigma; 1 μg/ml) in order to give total cell counts. The plates were imaged using the Operetta High Content Imaging and Analysis system (PerkinElmer). Oligodendrocyte percentage was calculated by taking the number of MBP+ cells and dividing by the number of Olig2+ cells. The oligodendrocyte percentage was normalized between plates. Differentiation inhibitors were called.
Dose curve plates (
Magnetic sorting to enrich for intermediate oligodendrocytes (O1+): In order to purify pre-myelinating oligodendrocytes for downstream applications such as RNA-seq and ChIP-seq, we performed magnetic sorting using an antibody against O1 (Zhang et al., 2014) (Bob Miller, 1:100) and followed the manufacturer's protocol for the magnetic anti-mouse IgG secondary beads (Miltenyi, 130-048-402). In brief, OPCs were differentiated in Oligodendrocyte differentiation media for 3 days and OPCs were cultured concurrently in growth media. At the end of 3 days, oligodendrocytes and OPCs were harvested using accutase and live stained in suspension with O1 (1:100 with 10 million cells/mL in N2B27 supplemented with 1:15 BSA fraction V and 1:250 EDTA) for 20 minutes on a rocker at 4° C. Cells were then washed with MACs solution (1:20 BSA Miltenyi, 130-091-376 in autoMACs buffer Miltenyi, 130-091-222), pelleted and resuspended in MACs solution (80 ul per 10 million cells) with 20 ul of magnetic anti-mouse IgG beads per 10 million cells. Cells were then rocked at 4° C. for 20 minutes. Cells were then washed, pelleted and resuspended with MACs solution and then strained using 0.22 um flow tube filter caps to remove any clumps. Cells were then processed by the autoMACs cell sorter using the “Posseld2” sorting option to obtain a positive and flow through population. Oligodendrocytes were harvested from the positively selected cells. OPCs grown in growth media were also put through the autoMACs and the negative flow through was used as the OPC population. Enrichment was verified by RNA-seq, demonstrating an enrichment for oligodendrocyte and myelination pathways in the O1 positive population and enrichment for proliferation and stem cell pathways in the OPC population.
ChIP-seq and alignment to genome: OPCs and purified intermediate O1+ oligodendrocytes were harvested using the autoMACs automated cell sorter as described above. Fixation, nuclei isolation and chromatin shearing were performed as previously described using the Covaris TruChIP protocol following the manufacturer's instructions for the “high-cell” format (Allan et al., 2020). In brief, 5 million OPCs and sorted 01+ oligodendrocytes were used for H3K27Ac ChIP-seq whereas 60-90 million OPCs and sorted 01+ oligodendrocytes were used for Sox6 ChIP-seq. Cells were fixed at room temperature for 10 minutes and then immediately proceeded for nuclei extraction. Isolated nuclei were then sonicated using the Covaris S2 with 5% Duty factor, 4 intensity and 4 60-second cycles. Sheared chromatin was then incubated with protein G magnetic DynaBeads (Thermofisher) that were pre-incubated with primary ChIP-grade antibodies overnight at 4° C. Beads were then washed and ChIP DNA was eluted, purified and used to construct illumina sequencing libraries that were submitted to UChicago's genomics core and sequenced on the HiSeq2500 with single-end 50 bp reads with at least 20 million reads per sample. For aligning reads to the genome (mouse, mm10), reads were quality and adapter trimmed using Trim Galore! Version 0.3.1. Trimmed reads were aligned to the mouse genome (mm10) with Bowtie2 version 2.3.2 and duplicate reads were removed using Picard MarkDuplicates. Peaks were called with MACSv2.1.1 with an FDR<0.001 to define broad peaks for histone marks (H3K27Ac) and narrow peaks for transcription factors (Sox6) and normalized to background input genomic DNA. Peaks were visualized using the Interactive Genomics Viewer (IGV, Broad Institute).
Super enhancer analysis: Super enhancers (SE) for OPCs and O1+ sorted oligodendrocytes were called using the ROSE algorithm on H3K27Ac ChIP-Seq data. Super enhancers were called separately for each biological replicate. Gene targets of these SEs were called. miRNA targets of these SEs were called. Super enhancer controlled genes or miRNAs were overlapped between biological replicates 1 and 2 for both OPCs and O1+ sorted oligodendrocytes in order to obtain common SE controlled genes and miRNAs between both replicates.
Omni ATAC-seq: Omni ATAC-Seq was performed on 50,000 OPCs and sorted O1+ oligodendrocytes based on available protocols (Corces et al., 2017). In brief, nuclei were extracted from cells and treated with transposition mixture containing Nextera Tn5 Transposase for (Illumina, FC-121-1030). Transposed fragments were then purified using Qiagen MinElute columns (Qiagen, 28004), PCR amplified, and libraries were purified with Agencourt AMPure XP magnetic beads (Beckman Coulter) with a sample to bead ratio of 1:1.2. Samples were submitted to UChicago's genomics core (https://fgf.uchicago.edu/) and sequenced on the HiSeq2500 with single-end 50 bp reads with nearly 100 million reads per sample. Reads were aligned to the mm10 mouse genome following the same pipeline that was used for aligning ChIP-seq data and peaks were called using the same pipeline for H3K27Ac ChIP-seq.
Motif enrichment analysis: Motifs were called under significant Sox6 peaks (FDR<0.001) or ATAC-Seq peaks (FDR<0.001) within super enhancers using HOMERv4.11.1 (Adam et al., 2015; Heinz et al., 2010). The FindMotifsGenome.pl tool was used with 200 bp windows for ATAC-Seq regions and Sox6 peaks using mm10 as the reference genome.
Western blot: For cell culture derived protein samples, at least 1 million OPCs were collected and lysed in RIPA buffer (Sigma) supplemented with protease and phosphatase inhibitor (78441, Thermo Fisher) for at least 15 minutes and cleared by centrifugation at 13,000 g at 4° C. for 15 minutes. Protein concentrations were determined using the Bradford assay (Bio-Rad Laboratories). Protein was diluted, boiled at 95° C. for 5 minutes, run using NuPAGE Bis-Tris gels (NP0335BOX, Thermo Fisher), and then transferred to PVDF membranes (LC2002, Thermo Fisher). Membranes incubated with primary antibodies at 4° C. overnight in blocking solution consisting of 5% nonfat drug milk (Nestle Carnation) in TBS plus 0.1% Tween 20 (TBST). Primary antibodies used included Sox6 (1 μg/mL, Abcam, ab30455) and B-Actin peroxidase (1:50,000, Sigma, A3854). Membranes were then imaged and analyzed using Image Studio™ software. Westerns were normalized to the Beta-Actin loading control unless otherwise noted.
qPCR: OPCs were lysed in TRIzol (Ambion) followed by purification and elution of RNA using phenol-chloroform extraction and the RNeasy Mini Kit (74104, Qiagen). RNA quality and quantity were determined using a NanoDrop spectrophotometer. cDNA was generated using the iSCRIPT kit following the manufacturer's instructions (1708891, Biorad). qRT-PCR was performed using pre-designed TaqMan gene expression assays (Thermo Fisher) as detailed in the chart below. qPCR was performed using the Applied Biosystems 7300 real-time PCR system and probes were normalized to Rp113a endogenous control.
Bulk RNA-seq sample preparation and alignment: OPCs were lysed in TRIzol and RNA was isolated as described for qPCR. Purified RNA was then submitted to Novogene for library preparation and sequencing on the Illumina NovaSeq (150 bp paired end). For gene expression analysis, reads were aligned to the mm10 genome and quantified in transcripts per million (TPM) values using salmon 0.14.1 (github dot com slash COMBINE-lab slash salmon). Transcripts were summarized as gene-level TPM abundances with tximport. A gene with TPM>1 was considered expressed. Differential expression analysis was performed using DESEQ2 (bioconductor.org slash packages slash release slash bioc slash html slash DESeq2 dot html). Significant genes were called based on p-adj and fold change values as described in the results section.
Gene ontology analysis: Metascape (Zhou et al., 2019) was used to identify significant pathways from gene lists called using fold change and significance (p-adj) cut-offs described in the results section.
Statistics and replicates: GraphPad Prism was used to perform statistical analyses unless otherwise noted. Statistical tests and replicate descriptions are detailed in each figure legend. Black filled-in circles for bar graphs indicate biological replicates (independent experiments) whereas open circles represent technical replicates. Statistics were only performed on samples with biological replicates. Data is typically graphed as mean±standard deviation (SD) or ±standard error of the mean (SEM) as detailed in the figure legend. A p-value less than 0.05 was considered significant unless otherwise noted.
The core transcriptional networks regulating cell state can be defined by super-enhancers, which are large regions of active chromatin densely packed with transcriptional machinery to ultimately regulate genes and pathways critical for cellular identity and function (Hnisz et al., 2013; Whyte et al., 2013). Utilizing scalable two independent batches of scalable and pure populations of mouse pluripotent stem cell-derived OPCs, RNA sequencing (RNA-seq), H3K27Ac chromatin immunoprecipitation coupled with sequencing (ChIP-seq), and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) were performed on OPCs and magnetically sorted O1 positive oligodendrocytes (Najm et al., 2011; Corces et al., 2017; Creyghton et al., 2010) (
Comparing the transcriptional states of in vitro OPCs and oligodendrocytes with in vivo isolated OPCs, intermediate oligodendrocytes, and fully mature myelinating oligodendrocytes revealed that in vitro sorted oligodendrocytes most closely align with intermediate in vivo oligodendrocytes (Zhang et al., 2014) (
The Ranking of Super Enhancer (ROSE) algorithm was then performed using H3K27Ac ChIP-seq to call super enhancers in both OPCs and intermediate oligodendrocytes (Adam et al., 2015; Hnisz et al., 2013; Loven et al., 2013) (
Performing motif analysis under open chromatin regions defined by ATAC-seq within all super-enhancers of both states revealed an enrichment for OPC-enriched, shared, and oligodendrocyte-enriched transcription factor motifs (
Super-enhancer associated transcription factors play crucial roles regulating the transcriptional network governing cell state (Adam et al., 2015; Mercado et al., 2019). To further parse dominant transcriptional regulators of processivity through the progenitor state, a transcriptional regulatory network analysis approach were utilized to interrogate master regulators of OPC state (Bleu et al., 2019; Federation, 2018; Mercado et al., 2019; Ott et al., 2018) (
Together, these analyses demonstrated that Sox6 is the most connected transcription factor and highly revalent within auto-regulatory cliques in both states (
Alongside transcription factors, super-enhancer controlled micro RNAs (miRNAs) provide an additional layer of feedback to regulate the core circuitry governing cell state and critical cellular processes (Marson et al., 2008; Suzuki et al., 2017; Tiscornia and Izpisua Belmonte, 2010). This is underscored by the fact that miRNA processing machinery and multiple miRNAs have been shown to be critical for oligodendrocyte formation from OPCs (Dugas et al., 2010; Emery and Lu, 2015; Zhao et al., 2010). Performing small RNA sequencing coupled with H3K27Ac ChIP-seq, expressed super-enhancer associated miRNAs in both OPCs and oligodendrocytes were called (
Using an established miRNA target prediction algorithm, how many of these miRNAs predicted to regulate transcription factors in the core transcriptional network (Vlachos et al., 2015) were determined. Combining the connectedness, prevalence in auto-regulatory cliques, and number of predicted targeting super-enhancer associated miRNAs for each transcription factor further highlighted Sox6 as a master regulator of this early state change (
To further unbiasedly uncover miRNAs that interact with the core transcriptional network, miRNAs that are capable of driving the formation of oligodendrocytes from OPCs were screened. Specifically, OPCs were transfected with 1,309 individual miRNA mimics in duplicate followed by high throughput imaging and quantification (
Taken together, Sox6 is identified as a highly connected transcription factor node in the transcriptional and post-transcriptional networks governing the progenitor state such that loss of Sox6 increases oligodendrocyte formation.
Sox6 expression decreases as OPCs differentiate and is thought to primarily maintain OPCs in the progenitor state by both inhibiting oligodendrocyte genes and driving the expression of the OPC signature gene Pdgfra (Baroti et al., 2016; Emery and Lu, 2015; Stolt et al., 2006). However, Sox6 expression does not completely disappear in intermediate pre-myelinating oligodendrocytes and Sox6 was called as a dominant transcription factor in intermediate oligodendrocytes (
Performing ChIP-seq for SOX6 in OPCs and intermediate oligodendrocytes demonstrated a greater number of peaks in OPCs, which agrees with SOX6 expression levels, and an enrichment for SOX6 at super-enhancers in both states (
GO analysis of genes harboring SOX6 aggregation in intermediate oligodendrocytes, demonstrated a clear enrichment for oligodendrocyte differentiation pathways and were expressed most highly in intermediate oligodendrocytes compared to OPCs (
Taken together, this data demonstrates that Sox6 forms nuclear condensates at genes transiently expressed along the trajectory of forming a fully mature myelinating oligodendrocyte.
To test whether Sox6 plays a functional role at these condensates, SOX6 knockout OPCs were generated using CRISPR-Cas9 using two independent single guide RNAs (sgSox6 g1 and sgSox6 g2), which led to a strong reduction in SOX6 expression compared to non-targeting control (sgNTC) (
This data suggests that loss of SOX6 impairs the intermediate pre-myelinating oligodendrocyte state to skip directly to mature myelinating oligodendrocytes.
Given the pronounced acceleration of Sox6 knockout oligodendrocyte gene signatures, whether loss of SOX6 was driving a noticeable change in cell shape of oligodendrocytes formed in 2D culture was tested. As observed earlier, knockdown of Sox6 led to a slight increase in formation of O1 and MBP positive oligodendrocytes at day 3 of differentiation; however, the more dramatic finding was a stark appearance of oligodendrocytes harboring a complex and matted membrane with Sox6 knockdown, which agrees with their precocious maturity (
Performing a differentiation time course following Sox6 knockdown at days 1, 2, and 3 of differentiation demonstrated that this effect on membrane matting occurs concurrently with the appearance of O1 positive oligodendrocytes at day 2 of differentiation and once again is more pronounced than the increase in percentage of oligodendrocytes (
To further test this rapid induction of a myelinating oligodendrocyte signature, OPCs were transfected with miRNAs predicted to target Sox6, namely miR-666-5p and miR-365-3p, and siRNA targeting Sox6, which all led to a reduction in Sox6 expression and increased the formation of wrapping oligodendrocytes in a microfiber differentiation assay relative to control (Bechler et al., 2015) (
Collectively, this data highlights a novel mechanism by which Sox6 governs the rate of oligodendrocyte maturation such that loss of Sox6 dramatically accelerates the formation of myelinating oligodendrocytes by destabilizing the intermediate oligodendrocyte state.
Elucidating transcriptional regulatory networks provides clues as to how cells balance decisions from self-renewal to differentiation to responding to an environmental lesion and identification of dominant transcription factor nodes provides a roadmap that can be used to parse disease pathology (Lee and Young, 2013). For the oligodendrocyte lineage, a majority of what is known regarding core regulators of oligodendrocyte formation has relied on gene expression data and most studies are limited to studying the formation of fully mature myelinating oligodendrocytes and overlook the dominant regulators of intermediate pre-myelinating oligodendrocytes (Elbaz and Popko, 2019; Emery and Lu, 2015; He et al., 2016; Stolt et al., 2002; Sun et al., 2018).
Here, the unique access to scalable and pure populations of OPCs and pre-myelinating oligodendrocytes to rigorously interrogate core transcriptional networks utilizing diverse strategies was capitalized. Coupling super-enhancer profiling with functional miRNA screening pinpointed Sox6 as a highly connected and regulated node governing pre-myelinating oligodendrocyte formation (
The current understanding of Sox6 function in the oligodendrocyte lineage would lead us to predict that Sox6 should avoid oligodendrocyte super-enhancers during differentiation and that loss of Sox6 would increase the pre-myelinating oligodendrocyte gene signature (Baroti et al., 2016; Elbaz and Popko, 2019; Emery and Lu, 2015; Stolt et al., 2006). However, interrogation of the whole-genome binding profile of Sox6 in pre-myelinating oligodendrocytes revealed that Sox6 spread across the gene body at genes associated with super-enhancers (
This work highlights Sox6 as a master regulator of pre-myelinating oligodendrocytes such that loss of Sox6 leads to precocious activation of a myelinating gene signature and skipping of the intermediate pre-myelinating state (
Collectively, this work dissects the transcriptional networks governing pre-myelinating oligodendrocyte formation and establishes a novel role for Sox6 maintaining this intermediate cell state through stabilization of transcriptional condensates. More broadly, this work establishes a novel mechanism by which transcription factors stabilize distinct intermediate stages along the continuum of differentiation.
The data presented herein demonstrates that inhibition of Sox6 function promotes maturation of OPCs to ODC. This example demonstrates that post-natal inactivation of Sox6 can be used to effectively treat or at least reduce the severity of diseases associated with loss or reduction of oligodendrocyte (ODC) function, by enhancing oligodendrocyte maturation and/or functional myelin production, n order to treat patients (including children and adults) having myelin diseases, such as those characterized by myelin damage or loss (e.g., Pelizaeus Merzbacher Disease (PMD)).
To facilitate the delivery of the CRISPR/Cas9 system with sgRNA targeting the Sox6 locus in a patient, several CNS-targeted AAV serotypes, including PHP.B, are generated, and AAV tropism for OPCs is validated. The AAV constructs (AAV9 or AAV-PHP.B) contain a SaCRISPR-Cas9 nuclease (CMV-SaCas9, SaCas9 coding sequence under the control of a CMV promoter) with a site directed guide RNA (sgRNA) against Sox6 (U6-sgRNA, the sgRNA is under the control of the U6 promotor), which is designed to generate indels in the Sox6 gene and thus prevent expression of a functional Sox6 protein.
Mice are maintained in accordance with approved protocols reviewed by Case Western Reserve University's Institutional Animal Care and Use Committee. Mice are housed in a temperature and humidity controlled housing unit under a 12 hour day/light cycle and are allowed ad libitum access to food.
Experimental animals (e.g., mice, such as Jimpy—a severe mouse model of Pelizaeus Merzbacher Disease) are then treated via stereotaxic intraventricular injection with the CRISPR-containing AAVs or controls encoding GFP. Specifically, male postnatal day 0 pups were obtained from (jimpy) mice breeding pairs and rapidly anesthetized using cryoanesthesia.
A 10 μL Hamilton syringe with a 32 gauge needle is loaded with AAV (AAV9 or AAV-PHP.B) packaging CMV-SaCas9 and U6-sgRNA targeting Sox 6. The needle is lowered through the skull to a depth of about 2 mm at a position 2/5 from the intersection of the sagittal suture and lambdoid to the eye. 2 μL of viral solution was injected into the lateral ventricle. The injection is then repeated in the contralateral lateral ventricle using the same coordinates, and injection volume for a total delivery of about 1×1010-1×1011 vector genomes to the ventricular system. The same is repeated for control mice.
Pups are allowed to recover on a heating pad and then reintroduced to their mother. Pups are monitored daily for phenotype improvement as compared to untreated or vehicle-treated animals, which develop severe (motor) phenotype (e.g., intention tremor and seizures) by 2 weeks of age and death by 3 weeks of age.
Treated animals surviving beyond 3 weeks are analyzed using behavioral (e.g., rotarod and open field testing for motor performance, histology (immuno staining of the CNS for myelin proteins and electron microscopy for myelin ultrastructure), or daily monitoring for lifespan extension statistical analysis.
Further, the spatial requirements for the number of cells required to be edited in the CNS (“dose response” of gene edited cells) are determined to generate a functional response with immunohistochemistry.
Finally, the temporal relationship is explored by introducing an optimized construct at post-natal days P1, P7 and P14. An optimized CRISPR/Cas9 approach within a defined therapeutic window reduces expression of the Sox6 protein, increases lifespan of treated individual, and restores myelination of axons.
This example is carried out to demonstrate that post-natal down-regulation or knockdown of Sox6 gene activity using ASO can be used to effectively treat or reduce severity of a myelin disease, such as PMD.
Mice are maintained in accordance with approved protocols reviewed by Case Western Reserve University's Institutional Animal Care and Use Committee. Mice are housed in a temperature and humidity controlled housing unit under a 12 hour day/light cycle and are allowed ad libitum access to food.
Male postnatal day 0 pups are obtained from jimpy (a severe mouse model of Pelizaeus Merzbacher Disease) breeding pairs and rapidly anesthetized using cryoanesthesia. A 10 pL Hamilton syringe with a 32 gauge needle is loaded with antisense oligonucleotides targeting Sox6. The needle is lowered through the skull to a depth of about 2 mm at a position 2/5 from the intersection of the sagittal suture and lambdoid to the eye. 2 pL of ASO solution is injected into the lateral ventricle. The injection is then repeated in the contralateral lateral ventricle using the same coordinates and injection volume for a total delivery of about 10-75 pg of ASO to the ventricular system.
Pups are allowed to recover on a heating pad and then reintroduced to their mother. Pups are monitored daily for phenotype improvement as compared to untreated or vehicle-treated jimpy animals, which develop severe motor phenotype (e.g., intention tremor and seizures) by 2 weeks of age and death by 3 weeks of age.
Treated animals surviving beyond 3 weeks are analyzed using behavioral (e.g., rotarod and open field testing for motor performance), histology (immuno staining of the CNS for myelin proteins and electron microscopy for myelin ultrastructure), or daily monitoring for lifespan extension statistical analysis.
This example is carried out to demonstrate that post-natal down-regulation or knockdown of Sox6 gene activity using RNAi can be used to effectively treat or reduce severity of a myelin disease, such as PMD.
Mice are maintained in accordance with approved protocols reviewed by Case Western Reserve University's Institutional Animal Care and Use Committee. Mice are housed in a temperature and humidity controlled housing unit under a 12 hour day/light cycle and are allowed ad libitum access to food.
Male postnatal day 0 pups are obtained from jimpy (a severe mouse model of Pelizaeus Merzbacher Disease) breeding pairs and rapidly anesthetized using cryoanesthesia. A 10 pL Hamilton syringe with a 32 gauge needle is loaded with AAV (AAV9 or AAV-PHP.B) packaging CMV-RNAi targeting Sox6 (an RNAi construct that can be transcribed inside the cell under the control of a CMV promotor to generate a functional RNAi molecules, which can then be processed to siRNA/shRNA/miRNA targeting Sox6).
Alternatively, the needle is loaded with a non-viral formulation of the RNAi construct targeting Sox6, using non-viral siRNA carriers such as cell-penetrating peptides, polymers, dendrimers, siRNA bioconjugates, and lipid-based siRNA carriers, etc. The needle is lowered through the skull to a depth of about 2 mm at a position 2/5 from the intersection of the sagittal suture and lambdoid to the eye. 2 pL of RNAi solution is injected into the lateral ventricle. The injection is then repeated in the contralateral lateral ventricle using the same coordinates and injection volume.
Pups are allowed to recover on a heating pad and then reintroduced to their mother. Pups are monitored daily for phenotype improvement as compared to untreated or vehicle-treated jimpy animals, which develop severe motor phenotype (e.g, intention tremor and seizures) by 2 weeks of age and death by 3 weeks of age.
Treated animals surviving beyond 3 weeks are analyzed using behavioral (e.g., rotarod and open field testing for motor performance), histology (immuno staining of the CNS for myelin proteins and electron microscopy for myelin ultrastructure), or daily monitoring for lifespan extension statistical analysis.
This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 63/170,152, filed on Apr. 2, 2021, the entire contents of which are incorporated herein by reference.
This invention was made with Government support under Grant Nos. F30HD096784, T32NS077888, and T32GM007250, awarded by the National Institute of Health (NIH). The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/023099 | 4/1/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63170152 | Apr 2021 | US |
