WO2024036106A1 - Complémentation de microglie embryonnaire pour manipulation in vivo de microglie et production d'un modèle animal non humain pour la validation de la fonction génique et le criblage thérapeutique - Google Patents

Complémentation de microglie embryonnaire pour manipulation in vivo de microglie et production d'un modèle animal non humain pour la validation de la fonction génique et le criblage thérapeutique Download PDF

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WO2024036106A1
WO2024036106A1 PCT/US2023/071778 US2023071778W WO2024036106A1 WO 2024036106 A1 WO2024036106 A1 WO 2024036106A1 US 2023071778 W US2023071778 W US 2023071778W WO 2024036106 A1 WO2024036106 A1 WO 2024036106A1
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microglia
human animal
gene
chimeric
cells
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PCT/US2023/071778
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Hiromitsu Nakauchi
Haojun XU
Kota NIIZUMA
Sicong WANG
Seki SHINSUKE
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The Board Of Trustees Of The Leland Stanford Junior University
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    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7153—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for colony-stimulating factors [CSF]
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells

Definitions

  • a non-human animal comprising chimeric microglia and methods of performing blastocyst microglia complementation to produce such a non-human chimeric animal are disclosed.
  • the disclosed methods can be used to produce a non-human animal model carrying microglia mutations of interest for gene validation and therapeutic screening.
  • a method of producing a non-human animal comprising chimeric microglia comprising: a) genetically modifying a non-human animal host embryo by deleting or inactivating a CSF1 R gene; b) transplanting a stem cell comprising a functional CSF1 R gene into the non-human animal host embryo to produce a chimeric non-human animal host embryo; and c) producing a non-human animal from the chimeric non-human animal host embryo, wherein differentiation of the stem cell generates the chimeric microglia in the nervous system of the non-human animal.
  • the non-human animal host embryo is genetically modified to delete or inactivate the CSF1 R gene at the single-celled zygote stage.
  • the stem cell is transplanted into the non-human host animal embryo at the blastocyst stage.
  • the stem cell comprises a mutation of interest.
  • the mutation of interest is linked to a microglia disorder.
  • the non-human animal is a mammal. In some embodiments, the mammal is a rodent.
  • the stem cells are embryonic stem cells, adult stem cells, or induced pluripotent stem cells.
  • the method further comprises performing transcriptomic profiling of the microglia.
  • a non-human animal comprising chimeric microglia produced by a method, described herein, is provided.
  • a non-human animal host embryo comprising: a genetically modified genome comprising a knockout of a CSF1 R gene; and transplanted stem cells having a wild-type CSF1 R gene, wherein a non-human animal can be produced from the chimeric non-human animal host embryo, wherein differentiation of the transplanted stem cells generates chimeric microglia in the nervous system of the non-human animal during development.
  • a method of screening a candidate agent comprising: administering the candidate agent to a non-human animal comprising chimeric microglia, produced by a method, described herein; and detecting an effect of the candidate agent on morphology, gene expression, or activity of the chimeric microglia.
  • detecting morphology comprises detecting ramified, reactive, activated, amoeboid, or rod-like morphology.
  • measuring activity of the chimeric microglia comprises measuring secretion of cytokines, chemokines, NO, glucocorticoids, glutamate, or proteases, phagocytosis, synaptic pruning, or production of reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • measuring secretion comprises detecting cytokines such as interleukin (IL)-4, IL-5, IL-10, IL-13, IL-10, IL-6, IL-12, IL-17, IL-18, IL-23, transforming growth factor (TGF)-0, tumor necrosis factor (TNF)-a, or interferon (IFN)-y, or chemokines such as CCL2, CX3CL1 , or CXCL10, or any combination thereof.
  • cytokines such as interleukin (IL)-4, IL-5, IL-10, IL-13, IL-10, IL-6, IL-12, IL-17, IL-18, IL-23, transforming growth factor (TGF)-0, tumor necrosis factor (TNF)-a, or interferon (IFN)-y
  • TGF tumor necrosis factor
  • IFN interferon
  • measuring gene expression comprises performing microarray analysis, RNA sequencing, or quantitative polymerase chain reaction.
  • measuring gene expression comprises detecting expression of a microglia activation marker.
  • the activation marker is IBA-1 , TLR4, CD14, CD16 CD32, CD86, macrophage receptor with collagenous structure (MARCO), or major histocompatibility complex II.
  • the method further comprises detecting neuroinflammation in the non-human animal.
  • method further comprises measuring the ability of the chimeric microglia to remove foreign material, toxins, pathogens, damaged cells, apoptotic cells, synaptic remnants, myelin debris, DNA fragments, neurofibrillary tangles, or plaques in the central nervous system of the non-human animal.
  • the candidate agent is administered locally to the chimeric microglia.
  • the genome of the non-human animal comprises a mutation linked to a microglia disorder.
  • the mutation is in a gene selected from the group consisting of TREM2, TYROBP, CR1, SPI1, MS4A4A, MS4A4E, MS4A6A, MS4A6E, ABCA7, CD33, INPP5D, CD2AP, SOD1, GRN, PAX2, LRRK2, RIPK1, FMR1, DNMT3A, BIN1, and TET2.
  • a method of transplanting microglia into a mammalian recipient subject comprising transplanting the chimeric microglia from a non-human animal, produced by a method, described herein to the mammalian recipient subject.
  • the stem cells are human stem cells.
  • the mammalian recipient subject is human.
  • a non-human animal host embryo, described herein, in the manufacture of chimeric microglia is provided.
  • at least 90% of the cells in the chimeric microglia are produced from the stem cells.
  • the stem cells are mammalian stem cells.
  • the mammalian stem cells are human stem cells.
  • FIG. 1 Current research limitations for studying microglia.
  • FIG. 2 Microglia replacement models (Xu et al., 2020, Cell Reports 32, 108041 ).
  • FIG. 3 Current microglia replacement model limitations.
  • FIG. 4 Blastocyst complementation of microglia.
  • FIG. 5 Flow cytometry analysis shows adult chimera have high microglia chimerism in the spinal cord.
  • a non-human animal comprising chimeric microglia and methods of performing blastocyst microglia complementation to produce such a non-human chimeric animal are disclosed.
  • the disclosed methods can be used to produce a non-human animal model carrying microglia mutations of interest for gene validation and therapeutic screening.
  • a cell includes a plurality of such cells and reference to “the embryo” includes reference to one or more embryos and equivalents thereof, e.g., blastocysts or morulas, known to those skilled in the art, and so forth.
  • stem cell refers to a cell that retains the ability to renew itself through mitotic cell division and that can differentiate into a diverse range of specialized cell types.
  • Mammalian stem cells can be divided into three broad categories: embryonic stem cells, which are derived from blastocysts, adult stem cells, which are found in adult tissues, and cord blood stem cells, which are found in the umbilical cord. In a developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body by replenishing specialized cells. Totipotent stem cells are produced from the fusion of an egg and sperm cell.
  • Cells produced by the first few divisions of the fertilized egg are also totipotent. These cells can differentiate into embryonic and extraembryo nic cell types. Pluripotent stem cells are the descendants of totipotent cells and can differentiate into cells derived from any of the three germ layers. Multipotent stem cells can produce only cells of a closely related family of cells (e.g., hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.). Unipotent cells can produce only one cell type, but have the property of self-renewal, which distinguishes them from non-stem cells.
  • Induced pluripotent stem cells are a type of pluripotent stem cell derived from adult cells that have been reprogrammed into an embryonic-like pluripotent state.
  • Induced pluripotent stem cells can be derived, for example, from adult somatic cells such as peripheral blood mononuclear cells, fibroblasts, keratinocytes, epithelial cells, endothelial progenitor cells, mesenchymal stem cells, adipose cells, leukocytes, hematopoietic stem cells, bone marrow cells, or hepatocytes.
  • reprogramming factors refers to one or more, i.e., a cocktail, of biologically active factors that act on a cell to alter transcription, thereby reprogramming a cell to multipotency or to pluripotency.
  • Reprogramming factors may be provided individually or as a single composition, that is, as a premixed composition, of reprogramming factors to the cells, e.g., somatic cells from an individual with a family history or genetic make-up of interest, such as a patient who has a neurological disorder or a neurodegenerative disease.
  • the factors may be provided at the same molar ratio or at different molar ratios.
  • the reprogramming factor is a transcription factor, including without limitation, Oct3/4; Sox2; Klf4; c-Myc; Nanog; and Lin-28.
  • the somatic cells may include, without limitation, peripheral blood mononuclear cells, fibroblasts, keratinocytes, epithelial cells, endothelial progenitor cells, mesenchymal stem cells, adipose cells, leukocytes, hematopoietic stem cells, bone marrow cells, or hepatocytes, etc., which are contacted with reprogramming factors, as defined above, in a combination and quantity sufficient to reprogram the cell to pluripotency.
  • Reprogramming factors may be provided to the somatic cells individually or as a single composition, that is, as a premixed composition, of reprogramming factors. In some embodiments the reprogramming factors are provided as a plurality of coding sequences on a vector.
  • container is meant a glass, plastic, or metal vessel that can provide an aseptic environment for culturing cells.
  • animal is used herein to include all vertebrate animals, except humans.
  • the term also includes animals at all stages of development, including embryonic, fetal, neonate, and adult stages.
  • Animals may include any member of the subphylum Chordata, including, without limitation, non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; fish, including zebrafish and medaka; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.
  • transgenic animal is meant a non-human animal, usually a mammal, having a non- endogenous (i.e., heterologous or foreign) nucleic acid sequence present as an extrachromosomal element in a portion of its cells or stably integrated into its germ line DNA (i.e., in the genomic sequence of most or all of its cells).
  • a heterologous nucleic acid is introduced into the germ line of such transgenic animals by genetic manipulation of, for example, embryos or embryonic stem cells of the host animal according to methods well known in the art.
  • a “transgene” is meant to refer to such a heterologous nucleic acid, e.g., heterologous nucleic acid in the form of an expression construct (e.g., for the production of a "knock-in” transgenic animal) or a heterologous nucleic acid that upon insertion within or adjacent a target gene results in a decrease in target gene expression (e.g., for production of a "knock-out” transgenic animal).
  • a DNA molecule is artificially introduced into the cells of an animal, a “transgenic animal” is produced.
  • the DNA molecule is called a "transgene” and may contain one or many genes.
  • subject any member of the subphylum Chordata, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; fish, including zebrafish and medaka; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.
  • chimeric refers to cells (e.g., microglia) from a different species than a host animal embryo or animal.
  • transfection is used to refer to the uptake of foreign DNA or RNA by a cell.
  • a cell has been "transfected” when exogenous DNA or RNA has been introduced inside the cell membrane.
  • transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001 ) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981 ) Gene 13:197.
  • a “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas") genes.
  • one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system.
  • one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.
  • a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence.
  • Cas9 encompasses type II clustered regularly interspaced short palindromic repeats (CRISPR) system Cas9 endonucleases from any species, and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks).
  • CRISPR type II clustered regularly interspaced short palindromic repeats
  • a Cas9 endonuclease binds to and cleaves DNA at a site comprising a sequence complementary to its bound guide RNA (gRNA).
  • a gRNA may comprise a sequence "complementary" to a target sequence (e.g., major or minor allele), capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence).
  • the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.
  • a gRNA will bind to a substantially complementary sequence and not to unrelated sequences.
  • a gRNA that "selectively binds" to a particular allele, such as a particular mutant allele e.g., allele comprising a substitution, insertion, or deletion) denotes a gRNA that binds preferentially to the particular target allele, but to a lesser extent to a wild-type allele or other sequences.
  • a gRNA that selectively binds to a particular target DNA sequence will selectively direct binding of Cas9 to a substantially complementary sequence at the target site and not to unrelated sequences.
  • donor polynucleotide refers to a polynucleotide that provides a sequence of an intended edit to be integrated into the genome at a target locus by homology directed repair (HDR).
  • a "target site” or “target sequence” is the nucleic acid sequence recognized (i.e., sufficiently complementary for hybridization) by a guide RNA (gRNA) or a homology arm of a donor polynucleotide.
  • gRNA guide RNA
  • the target site may be allele-specific (e.g., a major or minor allele).
  • homology arm is meant a portion of a donor polynucleotide that is responsible for targeting the donor polynucleotide to the genomic sequence to be edited in a cell.
  • the donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence comprising the intended edit to the genomic DNA.
  • the homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide.
  • the 5' and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence” and "3' target sequence,” respectively.
  • the nucleotide sequence comprising the intended edit is integrated into the genomic DNA by HDR or recombineering at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3' homology arms.
  • administering comprises transducing, transfecting, electroporating, translocating, fusing, phagocytosing, shooting or ballistic methods, etc., i.e., any means by which a nucleic acid can be transported across a cell membrane.
  • microglia disorder is used herein to refer to any central nervous system (CNS) disorder, peripheral nervous system disorder, or other disorder associated with dysregulation of microglia (e.g., hyperactivation or hypoactivation) or impaired function of microglia.
  • CNS central nervous system
  • peripheral nervous system disorder or other disorder associated with dysregulation of microglia (e.g., hyperactivation or hypoactivation) or impaired function of microglia.
  • Microglia disorders include, but are not limited to, chronic neuroinflammation, neuropathic pain, CNS- related injuries such as stroke, epilepsy, traumatic brain injury, and spinal cord injury, neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington disease, and Nasu- Hakola disease, glioma, meningitis, psychiatric diseases such as schizophrenia, autism spectrum disorder, and affective disorders, autonomic nerve dysfunction, cardiovascular disorders, such as hypertension, myocardial infarction, heart failure, cardiac ischemia/reperfusion injury, and ventricular arrhythmias, glaucoma, and infections, including infections of retroviruses such as human immunodeficiency virus (HIV) and human T lymphotropic virus type 1 , herpesviruses such as herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus, human herpesvirus 6,
  • pathological pain any pain resulting from a pathology, such as from functional disturbances and/or pathological changes, lesions, burns and the like.
  • pathological pain is “neuropathic pain” which is pain thought to initially result from nerve damage but extended or exacerbated by other mechanisms including glial cell activation.
  • pathological pain examples include, but are not limited to, thermal or mechanical hyperalgesia, thermal or mechanical allodynia, diabetic pain, pain arising from irritable bowel or other internal organ disorders, endometriosis pain, phantom limb pain, complex regional pain syndromes, fibromyalgia, low back pain, fibrodysplasia ossificans progressiva (FOP) pain, cancer pain, pain arising from infection, inflammation or trauma to peripheral nerves or the central nervous system, multiple sclerosis pain, entrapment pain, and the like.
  • thermal or mechanical hyperalgesia thermal or mechanical allodynia
  • diabetic pain pain arising from irritable bowel or other internal organ disorders
  • endometriosis pain arising from irritable bowel or other internal organ disorders
  • endometriosis pain arising from irritable bowel or other internal organ disorders
  • endometriosis pain arising from irritable bowel or other internal organ disorders
  • endometriosis pain
  • Hyperalgesia refers to an abnormally increased sensitivity to pain, including pain that results from excessive sensitivity to stimuli. Hyperalgesia can result from damage to nociceptors or nerves. Primary hyperalgesia refers to pain sensitivity that occurs in damaged tissues. Secondary hyperalgesia refers to pain sensitivity that occurs in undamaged tissue surrounding damaged tissue. Examples of hyperalgesia include, without limitation, thermal hyperalgesia (i.e., hypersensitivity to cold or heat) and opioid-induced hyperalgesia (e.g., hypersensitivity to pain as a result of long-term opioid use such as caused by treatment of chronic pain).
  • thermal hyperalgesia i.e., hypersensitivity to cold or heat
  • opioid-induced hyperalgesia e.g., hypersensitivity to pain as a result of long-term opioid use such as caused by treatment of chronic pain.
  • “Hypalgesia” or “hypoalgesia” refers to decreased sensitivity to pain.
  • Allodynia means pain that results from a normally non-painful, non-noxious stimulus to the skin or body surface.
  • examples of allodynia include, but are not limited to, thermal (hot or cold) allodynia (e.g., pain from normally mild temperatures), tactile or mechanical allodynia (e.g., static mechanical allodynia (pain triggered by pressure), punctate mechanical allodynia (pain when touched), or dynamic mechanical allodynia (pain in response to stroking or brushing)), movement allodynia (pain triggered by normal movement of joints or muscles), and the like.
  • thermal (hot or cold) allodynia e.g., pain from normally mild temperatures
  • tactile or mechanical allodynia e.g., static mechanical allodynia (pain triggered by pressure), punctate mechanical allodynia (pain when touched), or dynamic mechanical allodynia (pain in response to stroking or brushing)
  • Nociception is defined herein as pain sense.
  • Nociceptor herein refers to a structure that mediates nociception. The nociception may be the result of a physical stimulus, such as, a mechanical, electrical, thermal, or a chemical stimulus. Nociceptors are present in virtually all tissues of the body.
  • Analgesia is defined herein as the relief of pain without the loss of consciousness.
  • An “analgesic” is an agent or drug useful for relieving pain, again, without the loss of consciousness.
  • modulating pain refers to the modulation (e.g., inhibition or diminishment) of pain or the perception of pain in a given subject and includes absence from pain sensations as well as states of reduced or absent sensitivity to pain stimuli.
  • altering the activity of a target neuron refers to changing the activity level of a cell function.
  • altering the activity of a target neuron may include changing the membrane potential of a neuron, wherein the membrane potential of a neuron is important for its function (e.g., action potential firing).
  • the activity of the neuron is altered such that the membrane potential is increased (e.g., hyperpolarized).
  • the activity of the neuron is altered such that the membrane potential is decreased below a threshold potential, resulting in an action potential (e.g., depolarized).
  • the firing rate of the neuron is altered.
  • treatment used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease.
  • treatment encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and/or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease and/or symptom(s), i.e., arresting their development; or (c) relieving the disease symptom(s), i.e., causing regression of the disease and/or symptom(s).
  • Those in need of treatment include those already inflicted (e.g., those with a microglia disorder) as well as those in which prevention is desired (e.g., those with increased susceptibility or a genetic predisposition to developing a microglia disorder).
  • a therapeutic treatment is one in which the subject is inflicted prior to administration and a prophylactic treatment is one in which the subject is not inflicted prior to administration.
  • the subject has an increased likelihood of becoming inflicted or is suspected of being inflicted prior to treatment.
  • the subject is suspected of having an increased likelihood of becoming inflicted.
  • a chimeric animal is created by genetically modifying a non-human animal host embryo by deleting or inactivating a CSF1 R gene to create a microglia-deficient host.
  • Stem cells having a functional CSF1 R gene are transplanted into the non-human animal host embryo and differentiated into microglia, which populate the central nervous system as the non-human animal host embryo grows.
  • An adult chimeric animal having high microglia chimerism in the spinal cord is produced from the host embryo.
  • the non-human animal can be any non-human animal known in the art that can be used in the methods as described herein.
  • Such animals include, without limitation, non-human primates such as chimpanzees, gorillas, orangutans, and other apes and monkey species, cattle, sheep, pigs, goats, horses, deer, dogs, cats, ferrets, and rodents such as mice, rats, guinea pigs, hamsters, and rabbits.
  • the genome of the non-human animal host embryo is genetically modified to delete or inactivate a CSF1 R gene (i.e. , gene knockout) using standard methods in the art.
  • the non-human animal host embryo is genetically modified at the zygote stage.
  • a site-specific nuclease is used to create a DNA break that can be repaired by homology directed repair (HDR) or non-homologous end joining (NHEJ) to produce a knockout of a CSF1 R gene.
  • HDR homology directed repair
  • NHEJ non-homologous end joining
  • a donor polynucleotide is used comprising an intended edit sequence to be integrated into the genomic target locus.
  • the donor polynucleotide is used, for example, to delete all or a portion of the CSF1 R gene or introduce a frameshift mutation.
  • NHEJ the two DNA ends at the DNA break, produced by a site-specific nuclease, are ligated together imperfectly, resulting in incorporation of insertions or deletions of base pairs to create a frameshift mutation.
  • a DNA break may be created by a site-specific nuclease, such as, but not limited to, a Cas nuclease (e.g., Cas9, Cpf1 , or C2c1 ), an engineered RNA-guided Fokl nuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), a restriction endonuclease, a meganuclease, a homing endonuclease, and the like.
  • a site-specific nuclease such as, but not limited to, a Cas nuclease (e.g., Cas9, Cpf1 , or C2c1 ), an engineered RNA-guided Fokl nuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), a restriction endonu
  • Genome Editing Using Site-Specific Nucleases: ZFNs, TALENs, and the CRISPR/Cas9 System (T. Yamamoto ed., Springer, 2015); Genome Editing: The Next Step in Gene Therapy (Advances in Experimental Medicine and Biology, T. Cathomen, M. Hirsch, and M. Porteus eds., Springer, 2016); Aachen Press Genome Editing (CreateSpace Independent Publishing Platform, 2015); herein incorporated by reference.
  • genome modification is performed using HDR with a donor polynucleotide comprising a sequence comprising an intended genome edit flanked by a pair of homology arms responsible for targeting the donor polynucleotide to the target locus to be edited in a cell.
  • the donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence.
  • the homology arms are referred to herein as 5' and 3' (i.e.
  • upstream and downstream homology arms which relates to the relative position of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide.
  • the 5' and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence” and "3' target sequence,” respectively.
  • the homology arm must be sufficiently complementary for hybridization to the target sequence to mediate homologous recombination between the donor polynucleotide and genomic DNA at the target locus.
  • a homology arm may comprise a nucleotide sequence having at least about 80-100% sequence identity to the corresponding genomic target sequence, including any percent identity within this range, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the nucleotide sequence comprising the intended edit is integrated into the genomic DNA by HDR at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3' homology arms.
  • the corresponding homologous nucleotide sequences in the genomic target sequence flank a specific site for cleavage and/or a specific site for introducing the intended edit.
  • the distance between the specific cleavage site and the homologous nucleotide sequences can be several hundred nucleotides. In some embodiments, the distance between a homology arm and the cleavage site is 200 nucleotides or less (e.g., 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides).
  • the donor polynucleotide is substantially identical to the target genomic sequence, across its entire length except for the sequence changes to be introduced to a portion of the genome that encompasses both the specific cleavage site and the portions of the genomic target sequence to be altered.
  • a homology arm can be of any length, e.g., 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 300 nucleotides or more, 350 nucleotides or more, 400 nucleotides or more, 450 nucleotides or more, 500 nucleotides or more, 1000 nucleotides (1 kb) or more, 5000 nucleotides (5 kb) or more, 10000 nucleotides (10 kb) or more, etc.
  • the 5' and 3' homology arms are substantially equal in length to one another, e.g., one may be 30% shorter or less than the other homology arm, 20% shorter or less than the other homology arm, 10% shorter or less than the other homology arm, 5% shorter or less than the other homology arm, 2% shorter or less than the other homology arm, or only a few nucleotides less than the other homology arm.
  • the 5' and 3' homology arms are substantially different in length from one another, e.g., one may be 40% shorter or more, 50% shorter or more, sometimes 60% shorter or more, 70% shorter or more, 80% shorter or more, 90% shorter or more, or 95% shorter or more than the other homology arm.
  • the donor polynucleotide is used in combination with a site-specific nuclease.
  • the site-specific nuclease is an RNA-guided nuclease, which is targeted to a particular genomic sequence (i.e., genomic target sequence to be modified) by a guide RNA (gRNA).
  • gRNA guide RNA
  • a target-specific guide RNA comprises a nucleotide sequence that is complementary to a genomic target sequence, and thereby mediates binding of the nuclease-gRNA complex by hybridization at the target site.
  • the gRNA can be designed with a sequence complementary to a target sequence in a gene of interest.
  • a CRISPR system is used to knockdown or knockout a CSF1 R gene in the non-human animal host embryo.
  • the RNA-guided nuclease used for genome modification is a CRISPR system Cas nuclease. Any RNA-guided Cas nuclease capable of catalyzing site-directed cleavage of DNA to allow integration of donor polynucleotides by the HDR mechanism can be used in genome editing, including CRISPR system type I, type II, or type III Cas nucleases.
  • Cas proteins include Cas1 , Cas1 B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1 , Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Casl Od, Cas12a (Cpf1 ), Cas12b (C2c1 ), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), CasF, CasG, CasH, Csy1 , Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1 , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm
  • a type II CRISPR system Cas9 endonuclease is used.
  • Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks
  • the Cas9 need not be physically derived from an organism, but may be synthetically or recombinantly produced.
  • Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database.
  • NCBI National Center for Biotechnology Information
  • sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein. See also Fonfara et al. (2014) Nucleic Acids Res. 42(4):2577-90; Kapitonov et al. (2015) J. Bacteriol.
  • the CRISPR-Cas system naturally occurs in bacteria and archaea where it plays a role in RNA-mediated adaptive immunity against foreign DNA.
  • the bacterial type II CRISPR system uses the endonuclease, Cas9, which forms a complex with a guide RNA (gRNA) that specifically hybridizes to a complementary genomic target sequence, where the Cas9 endonuclease catalyzes cleavage to produce a double-stranded break.
  • gRNA guide RNA
  • Targeting of Cas9 typically further relies on the presence of a 5' protospacer-adjacent motif (PAM) in the DNA at or near the gRNA-binding site.
  • PAM 5' protospacer-adjacent motif
  • the genomic target site will typically comprise a nucleotide sequence that is complementary to the gRNA, and may further comprise a protospacer adjacent motif (PAM).
  • the target site comprises 20-30 base pairs in addition to a 3 base pair PAM.
  • the first nucleotide of a PAM can be any nucleotide, while the two other nucleotides will depend on the specific Cas9 protein that is chosen.
  • Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide.
  • the allele targeted by a gRNA comprises a mutation that creates a PAM within the allele, wherein the PAM promotes binding of the Cas9- gRNA complex to the allele.
  • the gRNA is 5-50 nucleotides, 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length, or any length between the stated ranges, including, for example, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35 nucleotides in length.
  • the guide RNA may be a single guide RNA comprising crRNA and tracrRNA sequences in a single RNA molecule, or the guide RNA may comprise two RNA molecules with crRNA and tracrRNA sequences residing in separate RNA molecules.
  • Cpf1 the CRISPR nuclease from Prevotella and Francisella 1
  • Cas12a the CRISPR nuclease from Prevotella and Francisella 1
  • Cpf1 is another class II CRISPR/Cas system RNA-guided nuclease with similarities to Cas9 and may be used analogously. Unlike Cas9, Cpf1 does not require a tracrRNA and only depends on a crRNA in its guide RNA, which provides the advantage that shorter guide RNAs can be used with Cpf1 for targeting than Cas9.
  • Cpf 1 is capable of cleaving either DNA or RNA.
  • the PAM sites recognized by Cpf 1 have the sequences 5'-YTN-3' (where "Y” is a pyrimidine and “N” is any nucleobase) or 5'-TTN-3', in contrast to the G-rich PAM site recognized by Cas9.
  • Cpf1 cleavage of DNA produces double-stranded breaks with a sticky- ends having a 4 or 5 nucleotide overhang.
  • Cpf 1 see, e.g., Ledford et al. (2015) Nature. 526 (7571 ) :17-17, Zetsche et al. (2015) Cell. 163 (3):759-771 , Murovec et al. (2017) Plant BiotechnoL J. 15(8):917-926, Zhang et al. (2017) Front. Plant Sci. 8:177, Fernandes et al. (2016) Postepy Biochem. 62(3):315-326; herein incorporated by reference.
  • Cas12b (C2c1) is another class II CRISPR/Cas system RNA-guided nuclease that may be used.
  • C2c1 similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites.
  • For a description of Cas12b see, e.g., Shmakov et al. (2015) Mol Cell. 60(3):385-397, Zhang et al. (2017) Front Plant Sci. 8:177; herein incorporated by reference.
  • RNA-guided Fokl nucleases comprise fusions of inactive Cas9 (dCas9) and the Fokl endonuclease (Fokl-dCas9), wherein the dCas9 portion confers guide RNA-dependent targeting on Fokl.
  • dCas9 inactive Cas9
  • Fokl-dCas9 Fokl endonuclease
  • engineered RNA-guided Fokl nucleases see, e.g., Havlicek et al. (2017) Mol. Ther. 25(2):342-355, Pan et al. (2016) Sci Rep. 6:35794, Tsai et al. (2014) Nat BiotechnoL 32(6):569-576; herein incorporated by reference.
  • RNA-guided nuclease can be provided in the form of a protein, such as the nuclease complexed with a gRNA, or provided by a nucleic acid encoding the RNA-guided nuclease, such as an RNA (e.g., messenger RNA) or DNA (expression vector).
  • RNA e.g., messenger RNA
  • DNA expression vector
  • the RNA- guided nuclease and the gRNA are both provided by vectors. Both can be expressed by a single vector or separately on different vectors.
  • the vector(s) encoding the RNA-guided nuclease an gRNA may be included in a CRISPR expression system to target a CSF1 R gene in the nonhuman animal host embryo.
  • Codon usage may be optimized to improve production of an RNA-guided nuclease in a particular cell or organism.
  • a nucleic acid encoding an RNA-guided nuclease or reverse transcriptase can be modified to substitute codons having a higher frequency of usage in a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence.
  • CRISPR interference is used to repress gene expression of a CSF1 R gene in the non-human animal host embryo.
  • CRISPRi is performed with a complex of a catalytically inactive Cas9 (dCas9) with a guide RNA that targets the gene of interest.
  • An engineered nuclease-deactivated Cas9 is used to allow sequence-specific targeting without cleavage.
  • Nuclease-deactivated forms of Cas9 may be engineered by mutating catalytic residues at the active site of Cas9 to destroy nuclease activity. Any such nuclease deficient Cas9 protein from any species may be used as long as the engineered dCas9 retains gRNA-mediated sequence-specific targeting.
  • the nuclease activity of Cas9 from Streptococcus pyogenes can be deactivated by introducing two mutations (D10A and H841 A) in the RuvC1 and HNH nuclease domains.
  • Other engineered dCas9 proteins may be produced by similarly mutating the corresponding residues in other bacterial Cas9 isoforms.
  • the dCas9 protein can be designed to target a gene of interest by altering its guide RNA sequence.
  • a target-specific single guide RNA comprises a nucleotide sequence that is complementary to a target site, and thereby mediates binding of the dCas9-sgRNA complex by hybridization at the target site.
  • CRISPRi can be used to sterically repress transcription by blocking either transcriptional initiation or elongation by designing a sgRNA with a sequence complementary to a promoter or exonic sequence.
  • the sgRNA may be complementary to the non-template strand or the template strand, but preferably is complementary to the non-template strand to more strongly repress transcription.
  • the target site will typically comprise a nucleotide sequence that is complementary to the sgRNA, and may further comprise a protospacer adjacent motif (PAM).
  • the target site comprises 20-30 base pairs in addition to a 3 base pair PAM.
  • the first nucleotide of a PAM can be any nucleotide, while the two other nucleotides will depend on the specific Cas9 protein that is chosen.
  • Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide.
  • the sgRNA comprises 5-50 nucleotides, 10-30 nucleotides, 15- 25 nucleotides, 18-22 nucleotides, 19-21 nucleotides, and any length between the stated ranges, including, for example, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.
  • the sgRNAs are readily synthesized by standard techniques, e.g., solid phase synthesis via phosphoramidite chemistry, as disclosed in U.S. Patent Nos. 4,458,066 and 4,415,732, incorporated herein by reference; Beaucage et aL, Tetrahedron (1992) 48:2223-2311 ; and Applied Biosystems User Bulletin No. 13 (1 April 1987).
  • Other chemical synthesis methods include, for example, the phosphotriester method described by Narang et aL, Meth. Enzymol. (1979) 68:90 and the phosphodiester method disclosed by Brown et aL, Meth. Enzymol. (1979) 68:109.
  • the dCas9 is fused to a transcriptional repressor domain capable of further repressing transcription of the gene of interest, e.g., by inducing heterochromatinization.
  • a Kruppel associated box KRAB
  • KRAB Kruppel associated box
  • dCas9 can be used to introduce epigenetic changes that reduce expression of a CSF1 R gene by fusion of dCas9 to an epigenetic modifier such as a chromatin-modifying epigenetic enzyme.
  • the promoter for the gene of interest can be silenced, for example, by methylation or acetylation (e.g., histone H3 lysine 9 [H3K9] methylation, histone H3 lysine 27 [H3K27] methylation, and/or DNA methylation).
  • fusion of dCas9 to a DNA methyltransferase such as DNA methyltransferase 3 alpha (DNMT3A) or a chimeric Dnmt3a/Dnmt3L methyltransferase (DNMT3A3L) allows targeted DNA methylation.
  • Fusion of dCas9 to histone demethylase LSD1 allows targeted histone demethylation (see, e.g., Liu et aL (2016) Cell 167(1 ):233-247, Lo et al. (2017) FWOORes. 6. pii: F1000 Faculty Rev-747, and Stepper et al. (2017) Nucleic Acids Res. 45(4):1703-1713; herein incorporated by reference).
  • an RNA-targeting CRISPR-Cas13 system is used to perform RNA interference to reduce expression of a CSF1 R gene.
  • Members of the Cas13 family are RNA- guided RNases containing two HEPN domains having RNase activity.
  • Cas13a (C2c2), Cas13b (C2c6), and Cas13d can be used for RNA knockdown.
  • Cas13 proteins can be made to target and cleave transcribed RNA using a gRNA with complementarity to the target transcript sequence.
  • the gRNA is typically about 64 nucleotides in length with a short hairpin crRNA and a 28-30 nucleotide spacer that is complementary to the target site on the RNA transcript.
  • Cas13 recognition and cleavage of a target transcript results in degradation of the transcript as well as nonspecific degradation of any nearby transcripts. See, e.g., Abudayyeh et al. (2017) Nature 550:280-284, Hameed et al. (2019) Microb. Pathog. 133:103551 , Wang et al. (2019) Biotechnol Adv. 37(5):708-729, Aman et al. (2016) Viruses 10(12). pii: E732, and Zhang et al. (2016) Cell 175(1 ):212-223; herein incorporated by reference.
  • the stem cells can be introduced into the animal host embryo at the blastocyst or morula stage.
  • transplantation of the stem cells is performed in utero to a conceptus or to an embryo in in vitro culture.
  • stem cells can be injected into a blastocyst cavity near the inner cell mass or aggregated with morula-stage embryo cells.
  • at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 stem cells or more are introduced into the animal host embryo.
  • 5-10 stem cells are introduced into the animal host embryo, including any number of stem cells within this range such as 5, 6, 7, 8, 9, or 10 stem cells.
  • the stem cells transplanted into the animal host embryo may be any type of stem cell, including, without limitation, embryonic stem cells, adult stem cells, or induced pluripotent stem cells (IPSCs).
  • the stem cells are mammalian stem cells.
  • the mammalian stem cells are human stem cells.
  • IPSCs can be generated by reprogramming somatic cells into pluripotent stem cells. Somatic cells can be induced into forming pluripotent stem cells, for example, by treating them with reprograming factors such as Yamanaka factors, including but not limited to, OCT3, OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN28 (see, e.g., Takahashi et al. (2007) Cell. 131 (5):861 - 872; herein incorporated by reference in its entirety).
  • reprograming factors such as Yamanaka factors, including but not limited to, OCT3, OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN28 (see, e.g., Takahashi et al. (2007) Cell. 131 (5):861 - 872; herein incorporated by reference in its entirety).
  • somatic cells that may be converted into IPSCs include, without limitation, peripheral blood mononuclear cells, fibroblasts, keratin ocytes, epithelial cells, endothelial progenitor cells, mesenchymal stem cells, adipose derived stem cells, leukocytes, hematopoietic stem cells, bone marrow cells, and hepatocytes.
  • Somatic cells are contacted with reprogramming factors in a combination and quantity sufficient to reprogram the cells to pluripotency.
  • Reprogramming factors may be provided to the somatic cells individually or as a single composition, that is, as a premixed composition, of reprogramming factors. In some embodiments the reprogramming factors are provided as a plurality of coding sequences on a vector.
  • Methods for "introducing a cell reprogramming factor into somatic cells are not limited in particular, and known procedures can be selected and used as appropriate.
  • methods include ones using protein introducing reagents, fusion proteins with protein transfer domains (PTDs), electroporation, and microinjection.
  • a nucleic acid(s), such as cDNA(s), encoding the cell reprogramming factor can be inserted in an appropriate expression vector comprising a promoter that functions in somatic cells, which then can be introduced into somatic cells by procedures such as infection, lipofection, liposomes, electroporation, calcium phosphate coprecipitation, DEAE-dextran, microinjection, and electroporation.
  • an "expression vector” examples include viral vectors, such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, and herpes viruses; and expression plasmids for animal cells.
  • viral vectors such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, and herpes viruses
  • expression plasmids for animal cells For example, retroviral or Sendai virus (SeV) vectors are commonly used to introduce a nucleic acid(s) encoding a cell reprogramming factor as described above into somatic cells.
  • SeV Sendai virus
  • a sample comprising somatic cells is obtained from the subject.
  • the somatic cells may include, without limitation, peripheral blood mononuclear cells, fibroblasts, keratinocytes, epithelial cells, endothelial progenitor cells, mesenchymal stem cells, adipose derived stem cells, leukocytes, hematopoietic stem cells, bone marrow cells, and hepatocytes, and other cell types capable of generating patient-derived IPSCs,
  • the biological sample comprising somatic cells is typically whole blood, buffy coat, peripheral blood mononucleated cells (PBMCS), skin, fat, or a biopsy, but can be any sample from bodily fluids, tissue or cells that contain suitable somatic cells.
  • a biological sample can be obtained from a subject by conventional techniques. For example, blood can be obtained by venipuncture, and solid tissue samples can be obtained by surgical techniques according to methods well known in the art.
  • the stem cells that are transplanted into the non-human animal host embryo are adult stem cells.
  • exemplary adult stem cells include, without limitation, mesenchymal stem cells (e.g., from placenta, adipose tissue, lung, bone marrow, or blood), hematopoietic stem cells, mammary stem cells, intestinal stem cells, endothelial stem cells, and neural stem cells.
  • the stem cells or somatic cells from which IPSCs are generated are preferably obtained from the mammalian subject that will be receiving the chimeric microglia transplant.
  • the cells can be obtained directly from a donor, a culture of cells from a donor, or from established cell culture lines. Cells are preferably of the same immunological profile as the subject receiving the transplant.
  • Adult stem cells and somatic cells can be obtained, for example, by biopsy from a close relative or matched donor.
  • the stem cells express a functional or wild-type CSF1 R gene (i.e., the CSF1 R gene that is deficient in the non-human animal host embryo where the CSF1 R gene is deleted or inactivated).
  • the stem cells are genetically modified to overexpress the CSF1 R gene.
  • Overexpression of CSF1 R can be accomplished, for example, by cloning a nucleic acid encoding the colony-stimulating factor 1 receptor into an expression vector to create an expression cassette and transfecting the stem cells with the expression vector.
  • NCBI National Center for Biotechnology Information
  • Expression cassettes typically include control elements operably linked to the coding sequence, which allow for the expression of the gene in mammalian cells.
  • typical promoters for mammalian cell expression include the SV40 early promoter, a CMV promoter such as the CMV immediate early promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), and the herpes simplex virus promoter, among others.
  • Other nonviral promoters such as a promoter derived from the murine metallothionein gene, will also find use for mammalian expression.
  • a promoter can be selected that overexpresses the CSF1 R gene.
  • transcription termination and polyadenylation sequences will also be present, located 3' to the translation stop codon.
  • a sequence for optimization of initiation of translation located 5' to the coding sequence, is also present.
  • transcription terminator/polyadenylation signals include those derived from SV40, as described in Sambrook et al., supra, as well as a bovine growth hormone terminator sequence.
  • Enhancer elements may also be used herein to increase expression levels of the mammalian constructs. Examples include the SV40 early gene enhancer, as described in Dijkema et al., EMPO J. (1985) 4:761 , the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et aL, Proc. Natl. Acad. Sci. USA (1982b) 79:6777 and elements derived from human CMV, as described in Boshart et aL, Cell (1985) 41 :521 , such as elements included in the CMV intron A sequence.
  • LTR long terminal repeat
  • a number of viral based systems have been developed for gene transfer into mammalian cells. These include adenoviruses, retroviruses (y-retroviruses and lentiviruses), poxviruses, adeno-associated viruses, baculoviruses, and herpes simplex viruses (see e.g., Warnock et al. (201 1 ) Methods Mol. Biol. 737:1-25; Walther et al. (2000) Drugs 60(2):249-271 ; and Lundstrom (2003) Trends BiotechnoL 21 (3) :117-122; herein incorporated by reference).
  • retroviruses provide a convenient platform for gene delivery systems. Selected sequences can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems have been described (U.S. Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1 :5-14; Scarpa et al. (1991 ) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci.
  • Lentiviruses are a class of retroviruses that are particularly useful for delivering polynucleotides to mammalian cells because they are able to infect both dividing and nondividing cells (see e.g., Lois et al (2002) Science 295:868-872; Durand et al. (201 1 ) Viruses 3(2):132-159; herein incorporated by reference).
  • adenovirus vectors have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267- 274; Bett et aL, J. Virol. (1993) 67:5911 -5921 ; Mittereder et aL, Human Gene Therapy (1994) 5:717-729; Seth et aL, J. Virol. (1994) 68:933-940; Barr et aL, Gene Therapy (1994) 1 :51-58; Berkner, K. L.
  • AAV vector systems have been developed for gene delivery.
  • AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941 ; International Publication Nos. WO 92/01070 (published 23 January 1992) and WO 93/03769 (published 4 March 1993); Lebkowski et aL, Molec. Cell. Biol.
  • Another vector system useful for delivering the polynucleotides encoding the colonystimulating factor 1 receptor is the enterically administered recombinant poxvirus vaccines described by Small, Jr., P. A., et al. (U.S. Pat. No. 5,676,950, issued Oct. 14, 1997, herein incorporated by reference).
  • Additional viral vectors which will find use for delivering the nucleic acid molecules encoding the colony-stimulating factor 1 receptor include those derived from the pox family of viruses, including vaccinia virus and avian poxvirus.
  • vaccinia virus recombinants expressing the colony-stimulating factor 1 receptor can be constructed as follows. The DNA encoding the colony-stimulating factor 1 receptor coding sequence is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia.
  • TK thymidine kinase
  • Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the coding sequences of interest into the viral genome.
  • the resulting TK-recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto.
  • avipoxviruses such as the fowlpox and canarypox viruses
  • Recombinant avipox viruses expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species.
  • the use of an avipox vector is particularly desirable in human and other mammalian species since members of the avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells.
  • Methods for producing recombinant avipoxviruses are known in the art and employ genetic recombination, as described above with, respect to the production of vaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.
  • Molecular conjugate vectors such as the adenovirus chimeric vectors described in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery.
  • Sindbis virus SIN
  • Semliki Forest virus SSV
  • Venezuelan Equine Encephalitis virus VEE
  • Sindbis-virus derived vectors useful for the practice of the instant methods, see, Dubensky et al. (1996) J. Virol. 70:508-519; and International Publication Nos. WO 95/07995, WO 96/17072; as well as, Dubensky, Jr., T. W., et al., U.S. Pat. No.
  • chimeric alphavirus vectors comprised of sequences derived from Sindbis virus and Venezuelan equine encephalitis virus. See, e.g., Perri et al. (2003) J. Virol. 77: 10394-10403 and International Publication Nos. WO 02/099035, WO 02/080982, WO 01/81609, and WO 00/61772; herein incorporated by reference in their entireties.
  • a vaccinia-based infection/transfection system can be conveniently used to provide for inducible, transient expression of the coding sequences of interest (for example, a colonystimulating factor 1 receptor expression cassette) in a host cell.
  • coding sequences of interest for example, a colonystimulating factor 1 receptor expression cassette
  • cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays extraordinar specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the polynucleotide of interest, driven by a T7 promoter.
  • the polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA which is then translated into protein by the host translational machinery.
  • the method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.
  • an amplification system can be used that will lead to high level expression following introduction into host cells.
  • a T7 RNA polymerase promoter preceding the coding region for T7 RNA polymerase can be engineered. Translation of RNA derived from this template will generate T7 RNA polymerase which in turn will transcribe more template. Concomitantly, there will be a cDNA whose expression is under the control of the T7 promoter. Thus, some of the T7 RNA polymerase generated from translation of the amplification template RNA will lead to transcription of the desired gene.
  • T7 RNA polymerase can be introduced into cells along with the template(s) to prime the transcription reaction.
  • the polymerase can be introduced as a protein or on a plasmid encoding the RNA polymerase.
  • the synthetic expression cassette of interest can also be delivered without a viral vector.
  • the synthetic expression cassette can be packaged as DNA or RNA in liposomes prior to delivery to the subject or to cells derived therefrom.
  • Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid.
  • the ratio of condensed DNA to lipid preparation can vary but will generally be around 1 :1 (mg DNA:micromoles lipid), or more of lipid.
  • Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations, with cationic liposomes particularly preferred.
  • Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner et aL, Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081 ); and purified transcription factors (Debs et al., J. Biol. Chem. (1990) 265:10189-10192), in functional form.
  • Cationic liposomes are readily available.
  • N[1 -2,3-dioleyloxy)propyl]-N,N,N- triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Feigner et aL, Proc. NatL Acad. Sci. USA (1987) 84:7413- 7416).
  • Other commercially available lipids include (DDAB/DOPE) and DOTAP/DOPE (Boerhinger).
  • Other cationic liposomes can be prepared from readily available materials using techniques well known in the art.
  • anionic and neutral liposomes are readily available, such as, from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials.
  • Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others.
  • DOPC dioleoylphosphatidyl choline
  • DOPG dioleoylphosphatidyl glycerol
  • DOPE dioleoylphoshatidyl ethanolamine
  • the liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs).
  • MLVs multilammelar vesicles
  • SUVs small unilamellar vesicles
  • LUVs large unilamellar vesicles
  • the various liposome-nucleic acid complexes are prepared using methods known in the art. See, e.g., Straubinger et aL, in METHODS OF IMMUNOLOGY (1983), Vol. 101 , pp. 512-527; Szoka et aL, Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198; Papahadjopoulos et aL, Biochim. Biophys.
  • DNA and/or peptide(s) can also be delivered in cochleate lipid compositions similar to those described by Papahadjopoulos et aL, Biochem. Biophys. Acta (1975) 394:483-491. See, also, U.S. Pat. Nos. 4,663,161 and 4,871 ,488.
  • the expression cassette of interest may also be encapsulated, adsorbed to, or associated with, particulate carriers.
  • particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co- glycolides), known as PLG. See, e.g., Jeffery et aL, Pharm. Res. (1993) 10:362-368; McGee J. P., et aL, J MicroencapsuL 14(2):197-210, 1997; O'Hagan D. T., et aL, Vaccine 11 (2):149-54, 1993.
  • particulate systems and polymers can be used for delivery of the nucleic acid of interest.
  • polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules, are useful for transferring a nucleic acid of interest.
  • DEAE dextran-mediated transfection, calcium phosphate precipitation or precipitation using other insoluble inorganic salts, such as strontium phosphate, aluminum silicates including bentonite and kaolin, chromic oxide, magnesium silicate, talc, and the like will find use with the present methods. See, e.g., Feigner, P.
  • Peptoids Zuckerman, R. N., et aL, U.S. Pat. No. 5,831 ,005, issued Nov. 3, 1998, herein incorporated by reference
  • biolistic delivery systems employing particulate carriers such as gold and tungsten, are especially useful for delivering synthetic expression cassettes of the present invention.
  • the particles are coated with the synthetic expression cassette(s) to be delivered and accelerated to high velocity, generally under a reduced atmosphere, using a gun powder discharge from a "gene gun.”
  • a gun powder discharge from a "gene gun” For a description of such techniques, and apparatuses useful therefore, see, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006; 5,100,792; 5,179,022; 5,371 ,015; and 5,478,744.
  • needle-less injection systems can be used (Davis, H. L., et al, Vaccine 12:1503- 1509, 1994; Bioject, Inc., Portland, Oreg.).
  • Disease-relevant mutations can be introduced into the genome of the non-human animal or chimeric microglia using any method known in the art to produce a disease model.
  • one or more disease-relevant mutations are introduced into the non-human animal embryo, the non-human animal produced from the embryo, the chimeric microglia, or a stem cell from which the chimeric microglia are derived.
  • somatic cells, from which IPSCs are derived are obtained from normal individuals and subsequently genetically modified to introduce a disease-relevant mutation such as a mutation linked to a microglia disorder, then transplanted into a CSF1 R-deficient non-human animal host embryo to produce chimeric microglia comprising the disease-relevant mutation.
  • the stem cells or somatic cells, from which IPSCs are derived are obtained from an individual comprising at least one allele encoding a mutation associated with a disease such as a mutation linked to a microglia disorder, wherein the IPSCs comprising the disease-relevant mutation are transplanted into a CSF1 R-deficient non-human animal host embryo to produce chimeric microglia comprising the disease-relevant mutation.
  • the disease-relevant mutation is associated with a microglia disorder such as, but not limited to, chronic neuroinflammation, neuropathic pain, CNS-related injuries such as stroke, epilepsy, traumatic brain injury, and spinal cord injury, neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington disease, and Nasu-Hakola disease, glioma, meningitis, psychiatric diseases such as schizophrenia, autism spectrum disorder, and affective disorders, autonomic nerve dysfunction, cardiovascular disorders, such as hypertension, myocardial infarction, heart failure, cardiac ischemia/reperfusion injury, and ventricular arrhythmias, glaucoma, and infections, including infections of retroviruses such as human immunodeficiency virus (HIV) and human T lymphotropic virus type 1 , herpesviruses such as herpes simplex virus (HSV), cytomegal
  • retroviruses such as
  • the stem cells are genetically modified to introduce two or more disease-relevant mutations.
  • disease-relevant mutations see, e.g., The Human Gene Mutation Database (HGMD®, hgmd.cf.ac.uk/ac/), MalaCards: The human disease database (malacards.org/), Gene4PD database (genemed.tech/gene4pd/home), Alz forum database (alzforum.org/mutations), MSgene database (msgene.org/), ALSOD: the Amyotrophic Lateral Sclerosis Online Database (alsod.org/), the AD/FTD Mutation database
  • the disease-relevant mutation is in a gene selected from the group consisting of TREM2, TYROBP, CR3, CD33, CR1, SPI1, MS4A4A, MS4A4E, MS4A6A, MS4A6E, ABCA7, INPP5D, CD2AP, SOD1, GRN, PAX2, LRRK2, RIPK1, FMR1, DNMT3A, BIN1, TET2, AXL, APOE, CLEC7A, ITGAX, LGALS3, and CST7.
  • TREM2 encodes the triggering receptor expressed on myeloid cells 2, a transmembrane receptor that modulates microglial activity and survival.
  • TREM2 Nasu-Hakola disease
  • AD Alzheimer’s disease
  • FDD frontotemporal dementia
  • PD Parkinson’s disease
  • ALS amyotrophic lateral sclerosis
  • TYROBP encodes a transmembrane adaptor protein.
  • TYROBP may affect the function of various receptors expressed on the plasma membrane including TREM2, CD33, and CR3 and downstream signaling of SIRPi p.
  • Mutations in TYROBP, CD33, and CR3 are linked to AD.
  • the MS4A gene family encodes a class of tetraspanin proteins.
  • MS4A gene family including MS4A4A, MS4A4E, MS4A6A, MS4A6E, ABCA7 have been implicated in AD.
  • INPP5D encodes inositol polyphosphate-5-phosphatase D. Mutations in INPP5D have been linked to AD and cancer.
  • a CRISPR/Cas system is used to make genetic changes to a gene of interest to produce a non-human animal useful for disease modeling and drug screening.
  • a CRISPR/Cas system can be used to delete, inactivate, or mutate a gene, or eliminate or reduce gene expression or protein activity.
  • Genome modification can be performed, for example, using homology directed repair (HDR) with a donor polynucleotide comprising a sequence comprising an intended genome edit flanked by a pair of homology arms responsible for targeting the donor polynucleotide to the target locus to be edited in a cell.
  • HDR homology directed repair
  • the donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence.
  • the homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide.
  • the 5' and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence" and "3' target sequence,” respectively.
  • the homology arm must be sufficiently complementary for hybridization to the target sequence to mediate homologous recombination between the donor polynucleotide and genomic DNA at the target locus.
  • a homology arm may comprise a nucleotide sequence having at least about 80-100% sequence identity to the corresponding genomic target sequence, including any percent identity within this range, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the nucleotide sequence comprising the intended edit is integrated into the genomic DNA by HDR at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3' homology arms.
  • the corresponding homologous nucleotide sequences in the genomic target sequence flank a specific site for cleavage and/or a specific site for introducing the intended edit.
  • the distance between the specific cleavage site and the homologous nucleotide sequences can be several hundred nucleotides. In some embodiments, the distance between a homology arm and the cleavage site is 200 nucleotides or less (e.g., 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides).
  • the donor polynucleotide is substantially identical to the target genomic sequence, across its entire length except for the sequence changes to be introduced to a portion of the genome that encompasses both the specific cleavage site and the portions of the genomic target sequence to be altered.
  • the donor polynucleotide is used in combination with an RNA-guided nuclease, which is targeted to a particular genomic sequence (i.e., genomic target sequence to be modified) by a guide RNA (gRNA).
  • gRNA guide RNA
  • a target-specific guide RNA comprises a nucleotide sequence that is complementary to a genomic target sequence, and thereby mediates binding of the nuclease- gRNA complex by hybridization at the target site.
  • the gRNA can be designed with a sequence complementary to a target sequence in a gene of interest where a disease-relevant mutation will be introduced.
  • Chimeric microglia may be isolated from a non-human animal, produced as described herein, using methods known in the art.
  • methods of isolating microglia typically involve dissociation of brain tissue by enzymatic treatment (e.g., treatment of brain tissue with dispase II, papain, and DNase I) followed by mechanical dissociation and cell separation by centrifugation with percoll gradients of various densities, fluorescence-activated cell sorting using microglia-specific surface markers, magnetic sorting, and/or immunopanning.
  • enzymatic treatment e.g., treatment of brain tissue with dispase II, papain, and DNase I
  • mechanical dissociation and cell separation by centrifugation with percoll gradients of various densities fluorescence-activated cell sorting using microglia-specific surface markers, magnetic sorting, and/or immunopanning.
  • Microglia cell lines may be derived from such isolated cells, and immortalized using standard techniques, e.g., through use of viruses.
  • the isolated microglia are useful for testing candidate agents to determine their effects on, e.g., morphology, gene expression, and/or activity of the microglia.
  • isolated microglia comprising one or more disease-relevant mutations, which allow screening of candidate agents for their ability to treat a microglia disorder. Screening Assays
  • a non-human animal comprising chimeric microglia, produced by the methods described herein, can be subjected to a plurality of candidate agents or other therapeutic intervention.
  • candidate agents encompass numerous chemical classes, e.g., small organic compounds having a molecular weight of more than 50 daltons and less than about 10,000 daltons, less than about 5,000 daltons, or less than about 2,500 daltons.
  • Test agents can comprise functional groups necessary for structural interaction with proteins, e.g., hydrogen bonding, and can include at least an amine, carbonyl, hydroxyl or carboxyl group, or at least two of the functional chemical groups.
  • test agents can comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Test agents are also found among biomolecules including peptides, peptide fragments, receptor fragments, co-receptor fragments, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • Test agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. Moreover, screening may be directed to known pharmacologically active compounds and chemical analogs thereof, or to new agents with unknown properties such as those created through rational drug design.
  • test agents are synthetic compounds.
  • a number of techniques are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. See for example WO 94/24314, hereby expressly incorporated by reference, which discusses methods for generating new compounds, including random chemistry methods as well as enzymatic methods.
  • test agents are provided as libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts that are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, including enzymatic modifications, to produce structural analogs.
  • test agents are organic moieties.
  • test agents are synthesized from a series of substrates that can be chemically modified. “Chemically modified” herein includes traditional chemical reactions as well as enzymatic reactions. These substrates generally include, but are not limited to, alkyl groups (including alkanes, alkenes, alkynes and heteroalkyl), aryl groups (including arenes and heteroaryl), alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds (including purines, pyrimidines, benzodiazepins, beta-lactams, tetracylines, cephalosporins, and carbohydrates), steroids (including estrogens, androgens, cortisone, ecodysone, etc.), alkaloids (including ergots, vinca, curare, pyrollizdine, and mitomycines), organometallic compounds, hetero
  • a test agent is assessed for any cytotoxic activity it may exhibit toward a living eukaryotic cell, using well-known assays, such as trypan blue dye exclusion, an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide) assay, and the like. Agents that do not exhibit significant cytotoxic activity are considered candidate agents.
  • the screening methods are used for identifying agents that promote the neuroprotective effects of microglia.
  • candidate agents may be screened for their effects on phagocytosis and clearance of foreign material, toxins, pathogens, damaged cells, apoptotic cells, synaptic remnants, myelin debris, DNA fragments, neurofibrillary tangles, and/or plaques in the central nervous system.
  • the screening methods are used for identifying inhibitors of microglial activation and/or the production of cytotoxic mediators by microglia which damage cells and cause neuronal cell death.
  • a variety of assays may be used, and in many embodiments, a candidate agent will be tested in different assays to confirm the effects on microglia activity as well as efficacy in treating a microglia disorder.
  • Activated microglia can be detected based on morphology, proliferation, expression of activation markers (e.g., IBA-1 , TLR4, CD14, CD16 CD32, CD86, macrophage receptor with collagenous structure (MARCO), or major histocompatibility complex II), secretion of effector molecules such as cytokines (e.g., interleukin (IL)-4, IL-5, IL-10, IL-13, IL-1 b, IL-6, IL-12, IL-17, IL-18, IL-23, transforming growth factor (TGF)-
  • microglia Different morphological subtypes of microglia have been correlated with functional activity and extent of activation.
  • the resting phenotype of microglia is characterized by a ramified morphology.
  • the “activated” state is characterized by a larger cell body and shorter, thick processes.
  • a “reactive state” is characterized by a small spherical morphology, rod-like morphology, or amoeboid-like morphology.
  • Microglia may also exhibit a phagocytic “reactive phenotype” with processes containing pyknotic fragments (see, e.g., Fernandez-Arjona et al. (2017) Front. Cell Neurosci.
  • Microglial activation may be evaluated, for example, using a Shell analysis (see, e.g., Catalin et al. (2013). Curr. Health Sci. J. 39 (Suppl 4):1 -5) or skeleton analysis (see, e.g., Morrison et al. (2013) J. Neuroinflammation 10(1 ):1 -20; herein incorporated by reference).
  • Shell analysis see, e.g., Catalin et al. (2013). Curr. Health Sci. J. 39 (Suppl 4):1 -5)
  • skeleton analysis see, e.g., Morrison et al. (2013) J. Neuroinflammation 10(1 ):1 -20; herein incorporated by reference.
  • Morphology, phagocytosis, synaptic pruning, and scavenging activity of microglia can be monitored using microscopy techniques. Any suitable method known in the art may be used for imaging microglia including, without limitation, fluorescence microscopy, confocal microscopy, two-photon microscopy, multi-photon microscopy, light-field microscopy, expansion microscopy, light sheet microscopy, and electron microscopy.
  • Activation of microglia leads to proliferation of microglia. Therefore, activation of a population of microglia can be detected by the increase in numbers of microglia.
  • Cell proliferation can be detected and quantified, for example, by flow cytometry, using a cell counter, or staining of microglia with a fluorescent tracking dye, such as carboxyfluorescein succinimidyl ester (CFSE).
  • CFSE carboxyfluorescein succinimidyl ester
  • activation of microglia can also be detected by expression of activation markers.
  • Transmembrane and surface proteins associated with activation of microglia include, but are not limited to, CD11 b, CD18, CR3, CD68, CD16, CD14, CD45, CA115, CX3CR1 , F4/80, and FCER1 G.
  • Intracellular markers associated with activation include, but are not limited to, iNOS, I BA-1 , and ferritin.
  • iNOS is an enzyme that produces NO, which promotes synthesis of IL- 6 and expression of the transcription factors IRF-1 and NF-KB that are involved in the microglia inflammatory response.
  • IBA-1 is an intracellular actin-binding protein involved in reorganization of the microglial cytoskeleton and functions in promoting phagocytosis.
  • Ferritin is involved in iron storage and is upregulated in activated microglia.
  • Activation markers can be detected by standard methods, including, without limitation, immunohistochemistry, immunofluorescence, Western blot, or flow cytometry.
  • changes in gene expression in response to a candidate agent can be detected, for example, by performing microarray analysis, RNA sequencing, or quantitative polymerase chain reaction.
  • Microglia can also be characterized as having an anti-inflammatory phenotype, pro- inflammatory phenotype, or resting/surveilling phenotype which can be distinguished by detection of biomarkers (see, e.g., Xu et al. (2021) Neural Regen Res. 16(2): 270-280).
  • Biomarkers of the anti-inflammatory phenotype include IL-13, TGF-
  • Biomarkers of the pro-inflammatory phenotype include IL-6, IL-1 p, iNOS, TNF-a, and major histocompatibility complex II (MHCII).
  • Biomarkers of the resting/surveilling phenotype include CD47, CXCR2, CX3CR1 , and CD200R1.
  • Cytotoxicity of activated microglia involves secretion of hydrogen peroxide, nitric oxide, proteases, including matrix metallopeptidases, cathepsins, and tissue-type plasminogen activator, and cytokines and apoptotic factors such as TNF-a that induce apoptosis of target cells.
  • Secretion of cytokines, chemokines, apoptotic factors, and proteases can be measured, for example, using a multiplexed enzyme-linked immunosorbent assay (ELISA). Surface markers can be detected, for example using standard immunohistochemistry and fluorescence microscopy techniques.
  • Cytolysis can be assayed in vitro based on the release of compounds containing radioactive isotopes such as 51 Cr from radiolabeled target cells.
  • a membrane- permeable live-cell labeling dye such as calcein acetoxymethyl ester of calcein (Calcein/AM) can be used to distinguish live cells from dead cells.
  • Calcein/AM intracellular esterases cleave the acetoxymethyl (AM) ester group to produce a membrane-impermeable calcein fluorescent dye that is retained in live cells. Apoptotic and dead cells without intact cell membranes do not retain the calcein fluorescent dye.
  • a lactate dehydrogenase (LDH) assay can also be used to evaluate cytotoxicity.
  • LDH is a cytoplasmic enzyme, which is released into the extracellular space when the plasma membrane is damaged. Cytotoxicity is monitored by detecting LDH release from cells. See, e.g., Lieberman (2003) Nat Rev Immunol 3(5):361 -370, Neri et al. (2001 ) Clin Diagn Lab Immunol 8(6):1131 -1135, Smith et al. (201 1) PLoS One 6(11 ):e26908, Chan et al. (2013) Methods Mol Biol 979:65-70; herein incorporated by reference in their entireties. [00146] Flow cytometry can also be used to assess cell proliferation, activation, and cytotoxicity.
  • the percentage of target cells that are live, apoptotic, or dead can be determined by staining target cells with viability dyes such that the live and dead cell populations can be distinguished based on differences in fluorescence.
  • viability dyes such that the live and dead cell populations can be distinguished based on differences in fluorescence.
  • Annexin V-FITC can be used to label target cells that are at an early stage of apoptosis.
  • Propidium iodide can be used to label target cells that are at a late stage of apoptosis or dead.
  • Lipophilic dyes, such as PKH67 and PKH26 can be used to label the cell membranes of target cells for measuring proliferation of microglia by flow cytometry. See, e.g., Zaritskaya et al.
  • Microglia have been implicated in neuroinflammation and the pathogenesis of pain.
  • Pro- inflammatory and anti-inflammatory cytokines secreted by microglia, regulate synaptic transmission and pain via neuron-glial interactions. Therefore, in certain embodiments, candidate agents are tested in a non-human animal to determine if an agent reduces neuroinflammation or relieves microglia-mediated pain.
  • a candidate agent can be administered to a non- human animal experiencing neuroinflammation and/or pain to determine if the candidate agent is anti-inflammatory or analgesic.
  • a variety of screening methods may be used for assessing whether an agent relieves pain and/or reduces pain affective-motivational behavior including sensory perception of pain, pain avoidance behavior, hyperalgesia, and allodynia.
  • Exemplary screening methods include, without limitation, stimulus-evoked behavioral tests such as a mechanical withdrawal test, an electronic Von Frey test, a manual Von Frey test, a Randall-Selitto test, a Hargreaves test, a hot plate test, a cold plate test, a thermal probe test, an acetone evaporation test, cold plantar test, and a temperature preference test; and non-stimulus-evoked behavioral tests such as a grimace scale test, weight bearing and gait analysis, locomotive activity test (e.g., still, walking, trotting, running, distance traveled, velocity, eating/drinking and foraging behavior frequencies), and burrowing behavior test.
  • locomotive activity test e.g., still, walking, trotting, running, distance traveled, velocity, eating/drinking and foraging behavior frequencies
  • Pain induced by mechanical stimuli may include mechanical hyperalgesia or allodynia, which can be subdivided into dynamic (triggered by brushing), punctate (triggered by touch) and static (triggered by pressure) subtypes of hyperalgesia or allodynia.
  • Testing for dynamic mechanical allodynia and hyperalgesia may include, for example, brushing the skin of a subject with a cotton ball or paintbrush. Punctate mechanical allodynia and hyperalgesia can be tested, for example, with a pinprick or von Frey filaments of varying forces (0.08-2940 mN).
  • Static hyperalgesia can be tested, for example, by applying pressure to the skin or underlying tissue by pressing a finger or using a pressure algometer.
  • Pain induced by heat or cold stimuli may include thermal hyperalgesia or allodynia.
  • Thermal hyperalgesia or allodynia may be tested, for example, by applying a metal probe to the skin that increases or decreases in temperature to determine a threshold temperature at which pain is experienced. Pain induced by heat is typically experienced at temperatures of 42-48°C, and pain induced by cold is typically experienced at temperatures of 23.7-1 ,5°C.
  • pain is inferred from “pain-like” behaviors, such as withdrawal from a nociceptive stimulus.
  • An animal is considered to have allodynia if the animal withdraws from an innocuous stimulus that does not normally evoke a withdrawal response.
  • An animal is considered to have hyperalgesia if an animal withdraws with an exaggerated response to a stimulus that does normally evoke a withdrawal response.
  • Responses of animals to mechanical stimuli can be tested using a manual or electronic Von Frey test or the Randall Selitto test.
  • Responses of animals to heat stimuli can be tested, for example, using the tail flick test, the Hargreaves test, a hot plate test, or a thermal probe test.
  • Thermal hyperalgesia or allodynia can be tested in animals for example by using a temperature preference test. For example, an animal is allowed to choose between two adjacent areas maintained at different temperatures or a preferred position along a continuous temperature gradient (either in linear or circular form).
  • a grimace scales test can be used to score the subjective intensity of pain based on facial expressions of a subject.
  • facial features can be scored, including orbital tightening, nose/cheek bulge or flattening, ear position, and whisker position.
  • Burrowing which is a self-motivated behavior, can also be used as a measure of spontaneous or nonstimulus evoked nociception in mice and rats. Gait and weight bearing of rodents also can be analyzed as an indicator of nociception.
  • Other behavior that can be analyzed in test subjects include locomotive activity (still, walking, trotting, running), distance traveled, velocity, grooming, posture, eating/drinking and foraging. The frequencies of these behaviors in animal models of pain are compared to control states to determine if an agent alleviates pain or pain-motivated behavior.
  • Methods of analysis at the single cell level are also of interest, such as live imaging (including confocal or light-sheet microscopy), single cell gene expression or single cell RNA sequencing, calcium imaging, immunocytochemistry, patch-clamping, flow cytometry and the like.
  • live imaging including confocal or light-sheet microscopy
  • single cell gene expression or single cell RNA sequencing single cell gene expression or single cell RNA sequencing
  • calcium imaging including confocal or light-sheet microscopy
  • immunocytochemistry including confocal or light-sheet microscopy
  • patch-clamping single cell RNA sequencing
  • flow cytometry flow cytometry
  • chimeric microglia can be isolated from the non-human animal and used in screening assays.
  • microglia in culture may be tested with one or a panel of cellular environments, where the cellular environment includes one or more of: exposure to a candidate agent of interest, contact with other cells such as neurons, electrical stimulation including alterations in ionicity, contact with pro-inflammatory or anti-inflammatory agents, contact with infectious agents, e.g. bacterial, viral, fungal, or parasitic infectious agents, and the like, and where cells may vary in genotype, in prior exposure to an environment of interest, in the dose of agent that is provided, etc.
  • infectious agents e.g. bacterial, viral, fungal, or parasitic infectious agents, and the like
  • infectious agents e.g. bacterial, viral, fungal, or parasitic infectious agents, and the like
  • infectious agents e.g. bacterial, viral, fungal, or parasitic infectious agents, and the like
  • infectious agents e.g. bacterial, viral, fungal, or parasitic infectious agents, and the
  • chimeric microglia is typically performed in a sterile environment, for example, at 37°C. in an incubator containing a humidified 92-95% air/5-8% CO 2 atmosphere.
  • Cell culture may be carried out in nutrient mixtures containing undefined biological fluids such as fetal calf serum, or media which is fully defined and serum free. The effect of the altering of the environment is assessed by monitoring multiple output parameters, including morphological, functional and genetic changes.
  • the agents are conveniently added in solution, or readily soluble form, to the medium used in culturing the chimeric microglia.
  • the agents may be added in a flow-through system, as a stream, intermittent or continuous, or alternatively, adding a bolus of the compound, singly or incrementally, to an otherwise static solution.
  • a flow-through system two fluids are used, where one is a physiologically neutral solution, and the other is the same solution with the test compound added. The first fluid is passed over the multi-spheroid tissue, followed by the second.
  • a bolus of the test compound is added to the volume of medium surrounding the multi-spheroid tissue.
  • preferred agent formulations do not include additional components, such as preservatives, that may have a significant effect on the overall formulation.
  • preferred formulations consist essentially of a biologically active compound and a physiologically acceptable carrier, e.g., water, ethanol, DMSO, etc.
  • a physiologically acceptable carrier e.g., water, ethanol, DMSO, etc.
  • the formulation may consist essentially of the compound itself.
  • a plurality of assays may be run in parallel with different agent concentrations to obtain a differential response to the various concentrations.
  • determining the effective concentration of an agent typically uses a range of concentrations resulting from 1 :10, or other log scale, dilutions.
  • the concentrations may be further refined with a second series of dilutions, if necessary.
  • one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection of the agent or at or below the concentration of agent that does not give a detectable change in the phenotype.
  • Various methods can be utilized for quantifying the presence of selected parameters, in addition to the functional parameters described above.
  • a convenient method is to label a molecule with a detectable moiety, which may be fluorescent, luminescent, radioactive, enzymatically active, etc., particularly a molecule specific for binding to the target analyte with high affinity.
  • Fluorescent moieties are readily available for labeling virtually any biomolecule, structure, or cell type. Immunofluorescent moieties can be directed to bind not only to specific proteins but also specific conformations, cleavage products, or site modifications like phosphorylation.
  • Individual peptides and proteins can be engineered to fluoresce, e.g., by expressing them as green fluorescent protein chimeras inside cells (for a review see Jones et al. (1999) Trends BiotechnoL 17(12):477-81 ).
  • Cells can be genetically modified to provide fusions of an antibody to a fluorescent or bioluminescent protein.
  • parameters may be measured using immunoassay techniques such as a radioimmunoassay (RIA) or enzyme linked immunosorbance assay (ELISA), homogeneous enzyme immunoassays, and related non-enzymatic techniques.
  • RIA radioimmunoassay
  • ELISA enzyme linked immunosorbance assay
  • these techniques utilize specific antibodies as reporter molecules, which are particularly useful due to their high degree of specificity for attaching to a single molecular target.
  • U.S. Pat. No. 4,568,649 describes ligand detection systems, which employ scintillation counting. These techniques are particularly useful for protein or modified protein parameters or epitopes, or carbohydrate determinants. Cell readouts for proteins and other cell determinants can be obtained using fluorescent or otherwise tagged reporter molecules.
  • Cell based ELISA or related non-enzymatic or fluorescence-based methods enable measurement of cell surface parameters and secreted parameters.
  • Capture ELISA and related non-enzymatic methods usually employ two specific antibodies or reporter molecules and are useful for measuring parameters in solution.
  • Flow cytometry methods are useful for measuring cell surface and intracellular parameters, as well as shape change and granularity and for analyses of beads used as antibody- or probe-linked reagents. Readouts from such assays may be the mean fluorescence associated with individual fluorescent antibody-detected cell surface molecules or cytokines, or the average fluorescence intensity, the median fluorescence intensity, the variance in fluorescence intensity, or some relationship among these.
  • Quantitative readouts of parameters may include baseline measurements in the absence of agents or a pre-defined genetic control condition and test measurements in the presence of a single or multiple agents or a genetic test condition. Furthermore, quantitative readouts of parameters may include long-term recordings and may therefore be used as a function of time (change of parameter value). Readouts may be acquired either spontaneously or in response to stimulation or perturbation of the microglia. The quantitative readouts of parameters may further include a single determined value, the mean or median values of parallel, subsequent or replicate measurements, the variance of the measurements, various normalizations, the cross-correlation between parallel measurements, etc. and every statistic used to a calculate a meaningful and informative factor.
  • Co-cultures of microglia and neurons can be treated with pro-inflammatory agents to study neuroinflammation and microglia-mediated neurotoxicity in vitro, and to test candidate agents for neuroprotective effects and anti-inflammatory activity.
  • the assays described herein are used to evaluate changes in function in response to optogenetic perturbation.
  • optogenetics is used to induce cell-specific perturbations in the non-human animal test subject in vivo.
  • optogenetics can be used to excite or inhibit one or more selected neurons of interest using light to test the effects on microglia activation and microglia-mediated neuroinflammation and neurotoxicity.
  • the screening methods described above are applied to a nonhuman animal comprising one or more disease-relevant mutations such as mutations associated with a microglia disorder. Methods are also provided for determining the activity of a candidate agent on chimeric microglia, obtained from such a non-human animal. In some embodiments, the method comprises contacting microglia comprising at least one allele encoding a mutation associated with a microglia disorder with the candidate agent; and determining the effect of the agent on morphologic, genetic, or functional parameters.
  • the disease-relevant mutation is in a gene selected from the group consisting of TREM2, TYROBP, CR3, CD33, CR1, SPI1, MS4A4A, MS4A4E, MS4A6A, MS4A6E, ABCA7, INPP5D, CD2AP, SOD1, GRN, PAX2, LRRK2, RIPK1, FMR1, DNMT3A, BIN1, TET2, AXL, APOE, CLEC7A, ITGAX, LGALS3, and CST7.
  • TREM2 encodes the triggering receptor expressed on myeloid cells 2, a transmembrane receptor that modulates microglial activity and survival.
  • TREM2 Nasu-Hakola disease
  • AD Alzheimer’s disease
  • FDD frontotemporal dementia
  • PD Parkinson’s disease
  • ALS amyotrophic lateral sclerosis
  • TYROBP encodes a transmembrane adaptor protein.
  • TYROBP may affect the function of various receptors expressed on the plasma membrane including TREM2, CD33, and CR3 and downstream signaling of SIRPi p.
  • Mutations in TYROBP, CD33, and CR3 are linked to AD.
  • the MS4A gene family encodes a class of tetraspanin proteins.
  • MS4A gene family including MS4A4A, MS4A4E, MS4A6A, MS4A6E, ABCA7 have been implicated in AD.
  • INPP5D encodes inositol polyphosphate-5-phosphatase D. Mutations in INPP5D have been linked to AD and cancer.
  • Agents, identified by the screening methods described herein, as useful for treating a microglia disorder can be formulated into pharmaceutical compositions optionally comprising one or more pharmaceutically acceptable excipients.
  • excipients include, without limitation, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.
  • Excipients suitable for injectable compositions include water, alcohols, polyols, glycerine, vegetable oils, phospholipids, and surfactants.
  • a carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may be present as an excipient.
  • carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.
  • the excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphat
  • a composition of the invention can also include an antimicrobial agent for preventing or deterring microbial growth.
  • antimicrobial agents suitable for the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.
  • An antioxidant can be present in the composition as well. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of the agent, or other components of the preparation. Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.
  • a surfactant can be present as an excipient.
  • exemplary surfactants include: polysorbates, such as “Tween 20” and “Tween 80,” and pluronics such as F68 and F88 (BASF, Mount Olive, New Jersey); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; steroids, such as cholesterol; chelating agents, such as EDTA; and zinc and other such suitable cations.
  • Acids or bases can be present as an excipient in the composition.
  • acids that can be used include those acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof.
  • Suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and combinations thereof.
  • the amount of the agent (e.g., when contained in a drug delivery system) in the composition will vary depending on a number of factors but will optimally be a therapeutically effective dose when the composition is in a unit dosage form or container (e.g., a vial).
  • a therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the composition in order to determine which amount produces a clinically desired endpoint.
  • the amount of any individual excipient in the composition will vary depending on the nature and function of the excipient and particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects. Generally, however, the excipient(s) will be present in the composition in an amount of about 1% to about 99% by weight, preferably from about 5% to about 98% by weight, more preferably from about 15 to about 95% by weight of the excipient, with concentrations less than 30% by weight most preferred.
  • compositions encompass all types of formulations and in particular those that are suited for injection, e.g., powders or lyophilates that can be reconstituted with a solvent prior to use, as well as ready for injection solutions or suspensions, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration.
  • suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof.
  • solutions and suspensions are envisioned.
  • compositions include those for intraneural, intracerebral, intrathecal, intraspinal, or localized delivery such as by stereotactic injection into the dorsal root ganglion (DRG).
  • DRG dorsal root ganglion
  • the pharmaceutical preparations herein can also be housed in a syringe, an implantation device, or the like, depending upon the intended mode of delivery and use.
  • the compositions comprising the agent are in unit dosage form, meaning an amount of a conjugate or composition of the invention appropriate for a single dose, in a premeasured or pre-packaged form.
  • Chimeric microglia produced by a non-human animal donor according to the methods described herein can be used for transplantation.
  • Healthy chimeric microglia may be used, for example, to replace or supplement dysfunctional microglia or a lack of microglia in a subject to restore neurological function.
  • chimeric microglia may be tested to determine that the microglia are suitable for transplant. For example, chimeric microglia may be screened for normal morphology, phagocytic ability, expression of microglia-derived cytokines and chemokines, ability to prune synapses and neuronal connections, and ability to promote neurogenesis and angiogenesis.
  • chimeric microglia are transplanted into a subject for treatment of a microglia disorder.
  • Microglia disorders include, but are not limited to, chronic neuroinflammation, neuropathic pain, CNS-related injuries such as stroke, traumatic brain injury, and spinal cord injury, neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, frontotemporal dementia, amyotrophic lateral sclerosis, and Huntington disease, glioma, meningitis, psychiatric diseases such as schizophrenia, autism spectrum disorder, autonomic nerve dysfunction, cardiovascular disorders, such as hypertension, myocardial infarction, heart failure, cardiac ischemia/reperfusion injury, and ventricular arrhythmias, glaucoma, and infections, including infections of retroviruses such as human immunodeficiency virus (HIV) and human T lymphotropic virus type 1 , herpesviruses such as herpes simplex virus (HSV), cytomegalovirus (CM
  • At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more of the cells in the chimeric microglia are derived from mammalian stem cells transplanted into the non-human animal host embryo.
  • 70-100% of the cells in the chimeric organ or tissue are derived from mammalian stem cells transplanted into the non-human animal host embryo, including any percent within this range, such as 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100%.
  • Chimeric microglia can be harvested from the chimeric donor and transplanted to a mammalian recipient.
  • Microglia may be transplanted from the chimeric donor to a recipient such that the microglia are placed into the appropriate position in the central nervous system of the recipient’s body.
  • the microglia are preferably from a living chimeric animal donor but, in some cases, may be from a deceased chimeric animal donor as long as the microglia remain viable.
  • the mammalian recipient of the transplant will typically be human. However, the methods described herein may also find use in veterinarian applications such as for treatment of farm animals such as cattle, sheep, pigs, goats and horses and domestic mammals (e.g., pets) such as dogs and cats.
  • an immune response may be mounted against the microglia after transplantation. During such episodes, the transplanted microglia may suffer diminished function or damage. The function and survival of the transplanted microglia may be improved by administration of an immunosuppressive agent.
  • immunosuppressive agents include, without limitation, glucocorticoids, such as prednisone, dexamethasone, and hydrocortisone; calcineurin inhibitors such as tacrolimus and ciclosporin; mTOR inhibitors such as sirolimus, everolimus, and zotarolimus; cytostatics such as methotrexate, dactinomycin, anthracyclines, mitomycin C, bleomycin, and mithramycin; and antibodies such as anti-CD20, anti-CD25, and anti-CD3 monoclonal antibodies.
  • glucocorticoids such as prednisone, dexamethasone, and hydrocortisone
  • calcineurin inhibitors such as tacrolimus and ciclosporin
  • mTOR inhibitors such as sirolimus, everolimus, and zotarolimus
  • cytostatics such as methotrexate, dactinomycin, anthracyclines, mito
  • Diagnosis of a rejection episode may utilize clinical data, markers for activation of immune function, markers for tissue damage, and the like. Histological signs include infiltrating T cells, perhaps accompanied by infiltrating eosinophils, plasma cells, and neutrophils, particularly in telltale ratios, structural compromise of tissue anatomy, varying by tissue type transplanted, and injury to blood vessels. Tissue biopsy is restricted, however, by sampling limitations and risks/complications of the invasive procedure. Cellular magnetic resonance imaging (MRI) of immune cells radiolabeled in vivo may provide noninvasive testing.
  • MRI Cellular magnetic resonance imaging
  • a method of producing a non-human animal comprising chimeric microglia comprising: a) genetically modifying a non-human animal host embryo by deleting or inactivating a CSF1 R gene; b) transplanting a stem cell comprising a functional CSF1 R gene into the non-human animal host embryo to produce a chimeric non-human animal host embryo; and c) producing a non-human animal from the chimeric non-human animal host embryo, wherein differentiation of the stem cell generates the chimeric microglia in the nervous system of the non-human animal.
  • stem cell is an embryonic stem cell, an adult stem cell, or an induced pluripotent stem cell.
  • stem cell is a mammalian stem cell.
  • any one of aspects 1 -10 wherein said genetically modifying the non-human animal host embryo comprises using a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector nuclease (TALEN), or a zinc-finger nuclease to delete or inactivate the CSF1 R gene.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • TALEN transcription activator-like effector nuclease
  • zinc-finger nuclease to delete or inactivate the CSF1 R gene.
  • the CRISPR system comprises a single guide RNA (sgRNA) targeting the CSF1 R gene.
  • sgRNA single guide RNA
  • stem cell comprises a mutation linked to a microglia disorder.
  • a non-human animal comprising chimeric microglia produced by the method of any one of aspects 1 -19.
  • a method of screening a candidate agent comprising: administering the candidate agent to the non-human animal of aspect 20; and detecting an effect of the candidate agent on morphology, gene expression, or activity of the chimeric microglia, or any combination thereof.
  • detecting the morphology of the chimeric microglia comprises detecting ramified, reactive, activated, amoeboid, or rod-like morphology.
  • measuring the activity of the chimeric microglia comprises measuring secretion of cytokines, chemokines, NO, glucocorticoids, proteases, or glutamate, phagocytosis, synaptic pruning, or production of reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • measuring the secretion by the chimeric microglia comprises detecting interleukin (IL)-4, IL-5, IL-10, IL-13, IL-10, IL-6, IL-12, IL-17, IL-18, IL-23, transforming growth factor (TGF)-0, tumor necrosis factor (TNF)-a, interferon (IFN)-y, CCL2, CX3CL1 , or CXCL10, or any combination thereof.
  • TGF transforming growth factor
  • TGF tumor necrosis factor
  • IFN interferon
  • measuring gene expression comprises performing microarray analysis, RNA sequencing, or quantitative polymerase chain reaction.
  • measuring gene expression comprises detecting expression of a microglia activation marker.
  • the genome of the non-human animal or the chimeric microglia comprises a mutation linked to a microglia disorder.
  • the mutation is in a gene selected from the group consisting of TREM2, TYROBP, CR1, SPI1, MS4A4A, MS4A4E, MS4A6A, MS4A6E, ABCA7, CD33, INPP5D, CD2AP, SOD1, GRN, PAX2, LRRK2, RIPK1, FMR1, DNMT3A, BIN1, and TET2.
  • a method of transplanting microglia into a mammalian recipient subject comprising transplanting chimeric microglia from the non-human animal of aspect 21 to the mammalian recipient subject.
  • stem cell is an adult stem cell from the mammalian recipient subject.
  • stem cell is an induced pluripotent stem cell derived from a somatic cell from the mammalian recipient subject.
  • a non-human animal host embryo comprising: a) a genetically modified genome comprising a knockout of a CSF1 R gene; and b) transplanted stem cells having a wild-type CSF1 R gene, wherein a non-human animal can be produced from the chimeric non-human animal host embryo, wherein differentiation of the transplanted stem cells generates chimeric microglia in the nervous system of the non- human animal during development.
  • 41 The non-human animal host embryo of aspect 40, wherein the non-human animal host embryo is a vertebrate.
  • a method of screening a candidate agent comprising: contacting the isolated microglia of aspect 54 with the candidate; and detecting an effect of the candidate agent on morphology, gene expression, or activity of the chimeric microglia, or any combination thereof.
  • detecting the morphology of the chimeric microglia comprises detecting ramified, reactive, activated, amoeboid, or rod-like morphology.
  • measuring the activity of the chimeric microglia comprises measuring secretion of cytokines, chemokines, NO, glucocorticoids, proteases, or glutamate, phagocytosis, synaptic pruning, or production of reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • measuring the secretion by the chimeric microglia comprises detecting interleukin (IL)-4, IL-5, IL-10, IL-13, IL-1 (3, IL-6, IL-12, IL-17, IL-18, IL-23, transforming growth factor (TGF)-p, tumor necrosis factor (TNF)-a, interferon (IFN)-y, CCL2, CX3CL1 , or CXCL10, or any combination thereof.
  • IL interleukin
  • TGF tumor necrosis factor
  • IFN interferon
  • measuring gene expression comprises performing microarray analysis, RNA sequencing, or quantitative polymerase chain reaction.
  • measuring gene expression comprises detecting expression of a microglia activation marker.
  • the activation marker is IBA-1 , TLR4, CD14, CD16 CD32, CD86, macrophage receptor with collagenous structure (MARCO), or major histocompatibility complex II.
  • FIG. 4 A schematic of the protocol for blastocyst complementation is shown in FIG. 4.
  • CRISPR was used to knockout the CSF1 R gene in mouse embryos at the 1 cell stage.
  • donor mouse GFP-ESCs were injected into the embryos, which differentiated into microglia.
  • Flow cytometry analysis showed that adult chimera had high microglia chimerism in the spinal cord (FIG. 5).
  • the donor ESCs may be genetically modified to carry any desired mutation of interest, including mutations associated with microglia disorders. These results indicate that blastocyst microglia complementation may be used effectively to produce a nonhuman animal model carrying microglia mutations of interest for gene validation and therapeutic screening.

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Abstract

Un animal non humain comprenant une microglie chimérique et des procédés de réalisation d'une complémentation de microglie de blastocyste pour produire un tel animal chimérique non humain sont divulgués. En particulier, les procédés divulgués peuvent être utilisés afin de produire un modèle animal non humain comportant des mutations de microglie d'intérêt pour la validation de gènes et le criblage thérapeutique.
PCT/US2023/071778 2022-08-08 2023-08-07 Complémentation de microglie embryonnaire pour manipulation in vivo de microglie et production d'un modèle animal non humain pour la validation de la fonction génique et le criblage thérapeutique WO2024036106A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140351965A1 (en) * 2011-02-25 2014-11-27 Recombinetics Swine genetically modified with specificity for ldl-r knockout
US20200315148A1 (en) * 2013-01-29 2020-10-08 The University Of Tokyo Method for producing chimeric animal
WO2021221879A1 (fr) * 2020-05-01 2021-11-04 Albert Einstein College Of Medicine Compositions et procédés d'utilisation de microglie transplantée comme véhicule permettant l'administration étendue de cellules et d'autres agents biologiques au cerveau
WO2021239308A1 (fr) * 2020-05-27 2021-12-02 Universität Zürich Vecteurs viraux exprimant des protéines thérapeutiques spécifiquement dans les cellules myéloïdes et la microglie
WO2022067105A1 (fr) * 2020-09-25 2022-03-31 The Board Of Trustees Of The Leland Stanford Junior University Procédé de remplacement efficace de microglies

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140351965A1 (en) * 2011-02-25 2014-11-27 Recombinetics Swine genetically modified with specificity for ldl-r knockout
US20200315148A1 (en) * 2013-01-29 2020-10-08 The University Of Tokyo Method for producing chimeric animal
WO2021221879A1 (fr) * 2020-05-01 2021-11-04 Albert Einstein College Of Medicine Compositions et procédés d'utilisation de microglie transplantée comme véhicule permettant l'administration étendue de cellules et d'autres agents biologiques au cerveau
WO2021239308A1 (fr) * 2020-05-27 2021-12-02 Universität Zürich Vecteurs viraux exprimant des protéines thérapeutiques spécifiquement dans les cellules myéloïdes et la microglie
WO2022067105A1 (fr) * 2020-09-25 2022-03-31 The Board Of Trustees Of The Leland Stanford Junior University Procédé de remplacement efficace de microglies

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