WO2023220639A2 - Methods and compositions for improved memory in the aging - Google Patents

Methods and compositions for improved memory in the aging Download PDF

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WO2023220639A2
WO2023220639A2 PCT/US2023/066832 US2023066832W WO2023220639A2 WO 2023220639 A2 WO2023220639 A2 WO 2023220639A2 US 2023066832 W US2023066832 W US 2023066832W WO 2023220639 A2 WO2023220639 A2 WO 2023220639A2
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csf
fgfl7
antibody
doi
cells
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PCT/US2023/066832
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French (fr)
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WO2023220639A3 (en
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Anton Wyss-Coray
Tal IRAM
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The Board Of Trustees Of The Leland Stanford Junior University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]

Definitions

  • compositions and method for treating cells, tissues, and subjects to improve memory in the aging brain, and to study or to treat age-related diseases and conditions associated with memory loss are provided herein.
  • Fgf 17 Fibroblast growth factor 17
  • oligodendrocyte progenitor cells OPCs
  • Rejuvenation of oligodendrocytes in aging individuals is a desired therapeutic strategy to treat or prevent cognitive aging, neurodegenerative disease such as Alzheimer’s disease, well as demyelinating diseases such as multiple sclerosis.
  • Cerebrospinal fluid nourishes the brain and provides it with growth factors that sustain progenitor function. Changes in the brain microenvironment that occur with age result in lower progenitor cell support thereby leading to reduced myelin turnover and axonal damage.
  • oligodendrocytes were identified as the cells most prominently responding to the young CSF environment. Infusion of CSF from young brains directly into aging brains induced OPC proliferation and maturation to myelin-forming cells in the aging hippocampus and in primary OPC cultures.
  • SRF serum response factor
  • FGFs Fibroblast growth factors
  • the present invention provides compositions, methods, systems, kits and uses to restore OPC function in the aging and diseased brain to a more youthful state.
  • compositions and method for treating cells, tissues, and subjects to improve memory in the aging brain, and to study or to treat age-related diseases and conditions associated with memory loss are provided herein.
  • Fgf 17 Fibroblast growth factor 17
  • an age-related disease or condition comprising exposing one or more of a subject’s central nervous system cells to Fgfl7 and/or to a Fgfl7 agonist wherein the exposing prevents and or treats the age-related disease or condition.
  • the age-related disease or condition is selected from the group of cognitive aging, neurodegeneration, or demyelination.
  • the subject is a human subject.
  • the Fgf 17 agonist is a Fgfl7 peptide or fragment thereof.
  • the Fgfl7 agonist is an agonist antibody.
  • the Fgfl7 agonist is a nucleic acid.
  • the nucleic acid is delivered to the choroid plexus.
  • the nucleic acid is an aptamer.
  • the Fgfl7 agonist increases Fgfl7 expression.
  • the exposing is in vivo exposing, ex vivo exposing or in vitro exposing.
  • the exposing is selected from the group consisting of local administration, topical administration, intrathecal administration, intraparenchymal administration, intracerebroventrical administration, intravenous administration, intraarterial administration, intrapulmonary administration, and oral administration.
  • exposing comprises combination therapy with an agent increases Fgfl7 function.
  • compositions comprising a Fgfl7 peptide and/or a Fgfl7 agonist, and a pharmaceutically acceptable carrier.
  • FIG. 1A-1O show that young CSF improves memory consolidation and promotes OPC proliferation and differentiation.
  • FIG. 1A is an overview of the experimental paradigm.
  • FIG. 1C shows that GSEA of hippocampal bulk RNA-seq data identifies oligodendrocyte genes as highly upregulated following 6 d of infusion with YM-CSF.
  • FIG. IF shows the representative images of the experiment in FIG. IE .
  • FIG. 1H is representative images of the experiment in FIG. 1G. Scale bars, 50 pm.
  • FIG. 1 J is representative images of the experiment in FIG. II.
  • FIG. IM is representative images of the experiment in FIG. IL. Scale bars, 50 pm.
  • FIG. 10 is representative images of the experiment in FIG. IN. Scale bars, 20 pm.
  • FIGS. 2A-2I show that serum response factor (Srf) is induced by young CSF and mediates CSF- induced OPC proliferation.
  • FIG. 2D is representative images of the experiment in FIG. 2E. Scale bars, 20 pm.
  • FIG. 2F is representative images of the experiment in FIG 2G. Scale bars, 10 pm.
  • FIG. 2H is a schematic of mouse OPC primary cultures from Srf pups infected with AAVs encoding Cre-GFP to induce recombination or ACre-GFP as a control.
  • FIGS. 3A-3G show that Srf signaling is downregulated in hippocampal OPCs with aging and induced following acute young CSF injection.
  • FIG. 3B is representative images of the experiment in a. Scale bars, 10 pm (5 pm in insets).
  • FIG. 3D shows pathways enriched (red) or depleted (blue) in hippocampal OPCs with age.
  • FIGS. 4A-4S show that Fgfl7 induces OPC proliferation and improves memory.
  • FIG. 4A is a diagram of the SRE-GFP reporter in HEK293 cells.
  • FIG. 4A is a diagram of the SRE-GFP reporter in HEK293 cells.
  • FIG. 4D is representative images of the experiment in FIG. 4C. Scale bars, 400 pm.
  • RFU relative fluorescence units.
  • FIG. 4G is representative images of the experiment in FIG. 4F. Scale bars, 5 pm.
  • FIG. 41 is representative images of the experiment in FIG. 4H. Scale bars, 20 pm.
  • FIG. 4K is representative images of the experiment in FIG. 4H. Scale bars, 20 pm.
  • FIG. 4M is representative images of the experiment in 1. Arrowheads point to proliferating OPCs. Scale bars, 50 pm.
  • FIG. 4M is representative images of the experiment in 1. Arrowheads point to proliferating OPCs.
  • FIG. 4R is representative images of the experiment in FIG. 4Q. Scale bars, 100 pm.
  • FIGS. 5A-5C show that Fgfl7 is predominantly expressed in the brain by a subset of neurons and choroid plexus epithelial cells.
  • FIG. 5 A shows that Fgfl7 is predominantly expressed in the brain based on the human protein atlas.
  • FIG. 5B shows that Fgfl7 is lowly expressed by neurons but not glial cells in the adult human cortex (Allen Brain Atlas).
  • FIG. 5C shows that Fgfl7 is lowly expressed by neurons and choroid plexus epithelial cells in a human COVID19 brain and choroid plexus dataset.
  • FIGS. 6A-6I show the bulk RNAseq, infusion site details and overall overview of proliferating cells.
  • FIG. 6C shows the effect size of the subset of oligodendrocyte genes in FIG.
  • FIG. 6D shows the location of the infusion site.
  • Image source Allen Institute, Mouse brain atlas (coronal).
  • FIG. 6E shows the location of the analysis site.
  • Image source Allen Institute, Mouse brain atlas (coronal).
  • FIG. 6G is representative images of EdU (red) and BrdU (green) cells in mice with no surgery or infused with aCSF or YM-CSF. Scale bar, 500 pm.
  • FIG. 61 is representative images of analysis in panel FIG. 6H. Arrows pointing to Pdgfra+EdU+ cells. Scale bar, 100 pm.
  • FIGS. 7A-7I show cortical Pdgfra+EdU+ cells and identity of Pdgfra- EDU+ cells.
  • FIG. 7C shows location of region of interest in the cortex. Scale bar, 100 pm.
  • FIG. 7G shows the percentage of Pdgfra+ EdU+ / EdU+ in the hippocampus of aged mice infused with YM-CSF
  • FIGS. 8A-8E show young CSF increases number of myelinated axons in the molecular layer.
  • FIG. 8A is a representative overview of 1mm diameter biopsy punch in the hippocampus.
  • FIG. 8A is a representative overview of 1mm diameter biopsy punch in the hippocampus.
  • FIG. 8B is a representative overview of molecular layer (MoL, between dashed lines) before and after TEM imaging of three 10x10 montage
  • FIGS. 9A-9D show young CSF boosts OPC differentiation in vitro and validation of OPC culture purity.
  • FIGS. 10A-10G show SLAMseq QC and principal component analysis.
  • FIG. 10A shows the overall conversion rates in all SLAMseq samples, showing an enrichment for T>C mutation rate (orange bar) which increases with longer incubation time (6 h).
  • FIGS. 10B and 10C is the distribution of T>C mutations across read position (FIG. 10B) and 3’UTR position indicating an equal distribution of s4U incorporation along the positive strand (FIG. 10C).
  • FIGS. 10D and 10E are UMAP of aCSF and YH-CSF samples in both time points by all genes detected in the total (FIG. 10D) and nascent (FIG.
  • FIG. 10E is gene set enrichment analysis (GSEA) of 6hr genes sorted by log2-transformed fold change (log2FC) showing an enrichment for SRF target genes by TRANSFAC.
  • GSEA gene set enrichment analysis
  • FIG. 10G shows the overall log2FC enrichment indicating upregulation of SRF target genes (TRANSFAC and curated list) and actin cytoskeleton genes in YH-CSF treated OPCs over aCSF.
  • SRF TRANSFAC - 423 genes validated SRF targets from literature (74 genes) and actin genes (212 genes); Wilcoxon rank sum test; box show the median and the 25— 75th percentiles, and the whiskers indicate values up to 1.5-times the interquartile range).
  • FIGS. 11 A-l II show YH-CSF induces actin cytoskeleton alterations in vitro.
  • FIG. 11C is representative images of experiment quantified in FIGS. 11 A and 1 IB. Scale bar 200 pm.
  • FIG. 1 ID shows OPC coverslips treated with YH-CSF for 6 h and stained for phalloidin.
  • FIG. 1 IE is mouse OPC primary cultures from SRF-fl/fl pups infected with CRE-GFP and ACRE-GFP AAVs to induce recombination. Representative images of infected cells (green) 48 h after infection. Scale bar, 100 pm.
  • FIG. 11G is representative images of data presented in FIG. 2H. Scale bar, 20 pm.
  • FIGS. 12A-12D show bulk RNAseq of hippocampal OPC and OL nuclei from young and aged mice.
  • FIG. 12A shows the gating strategy for sorting of hippocampal OPC and OL nuclei.
  • FIG. 12D shows pathways enriched (red) or depleted (blue) in hippocampal OLs with age (unweighted Kolmogorov- Smimow test).
  • FIGS. 13A-13H show bulk RNAseq of hippocampal OPC and OL nuclei from aged mice following acute injection and Srf levels in neurons.
  • FIG. 13F is representative images of FIG. 13E. Scale bar, 70 pm.
  • FIG. 13H is representative images of FIG. 13G. Scale bar, 70 pm.
  • FIGS. 14A-14F show that Fgf8 induces OPC proliferation and Fgfl7 induces SRF reporter activation mediated by actin dynamics and Fgfr3.
  • FIG. 14B shows the percentage of BRDU+/DAPI primary rat OPCs treated with 10, 20, 40 ng/ml Fgf8.
  • FIGS. 15A-15H show Fgfl7 is predominantly expressed in the brain by a subset of neurons and is downregulated with age.
  • FIG. 15A shows Fgfl7 is expressed by cortical glutamatergic neurons in the young adult mouse (Allen brain atlas).
  • FIG. 15B shows sub-clustering of mouse cortical layer 4/5 neurons indicates expression by a subset of cortical neurons (Allen brain atlas).
  • FIG. 15C is gene set enrichment analysis of genes mostly correlated with Fgfl7 in layer 4/5 neurons (Allen brain atlas).
  • FIG. 15D shows Fgfl7 is expressed by cortical glutamatergic and GABAergic neurons in the human cortex (Allen brain atlas).
  • FIG. 15E is a representative image of analysis in FIG.
  • FIG. 15F Scale bar, 100 pm.
  • FIG. 15H is representative images of analysis in FIGS. 15G and 4F. Scale bar, 20 pm.
  • FIGS. 16A-16G show perfusion of labeled YH-CSF and mouse Fgfl7 to the brain parenchyma and working model.
  • FIG. 16G is a schematic of a working model. OPC proliferation and differentiation (termed oligodendrogenesis) slow down with age. Re-exposure of the aged brain to young CSF or the brainspecific growth factor Fgfl 7, boost hippocampal oligodendorgenesis, concomitant with improvement in long term memory recall.
  • FIGS. 17A and 17B show a subset of Srf targets present in CSF proteomic datasets.
  • FIG. 17A provides exemplary proteins tested in the SRE reported assay (related to FIGS. 4A and 4B).
  • FIG. 17B provides exemplary Srf targets in CSF datasets that were not tested in the SRE reporter assay. DEFINITIONS
  • the term “subject” refers to any animal (e.g, a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment.
  • the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
  • non-human animals refers to all non-human animals including, but not limited to, vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.
  • cell culture refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro.
  • zzz vitro refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell culture.
  • the term “/// vivo” refers to the natural environment (e.g, an animal or a cell) and to processes or reaction that occur within a natural environment.
  • test compound and “candidate compound” refer to any chemical entity, pharmaceutical, drug, and the like that is a candidate for use to treat or prevent a disease, illness, sickness, or disorder of bodily function (e.g., Alzheimer’s disease, Parkinson’s disease, atherosclerosis, cancer).
  • Test compounds comprise both known and potential therapeutic compounds.
  • a test compound can be determined to be therapeutic by screening using the screening methods of the present disclosure.
  • sample is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum, and the like. Environmental samples include environmental material such as surface matter, soil, water, and industrial samples. Such examples are not however to be construed as limiting the sample types applicable to the present disclosure.
  • an effective amount refers to the amount of a compound (e.g., a compound described herein) sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages and is not limited to or intended to be limited to a particular formulation or administration route.
  • co-administration refers to the administration of at least two agent(s) or therapies to a subject. In some embodiments, the co-administration of two or more agents/therapies is concurrent. In other embodiments, a first agent/therapy is administered prior to a second agent/therapy.
  • the appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents/therapies are co-administered, the respective agents/therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents/therapies lowers the requisite dosage of a known potentially harmful (e.g, toxic) agent(s).
  • the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo, or ex vivo.
  • the term “antigen binding agent e.g., “antigen-binding protein” or protein mimetic such as an aptamer
  • Antigen-binding proteins include, but are not limited to, immunoglobulins, including polyclonal, monoclonal, chimeric, single chain, single domain, scFv, minibody, nanobody, and humanized antibodies, Fab fragments, F(ab’)2 fragments, and Fab expression libraries.
  • single-chain variable fragment refers to an antibody fragment that comprises a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin.
  • VH and VL are connected with a short linker peptide.
  • minobodies refers to an antibody fragment that retains antigen binding activity.
  • minobodies comprise an scFv fused to an Fc region e.g., an IgG Fc region).
  • polyclonal antibodies various procedures known in the art are used for the production of polyclonal antibodies.
  • various host animals can be immunized by injection with the peptide or protein containing the desired epitope including but not limited to rabbits, mice, rats, sheep, goats, llamas, alpacas, etc.
  • the peptide is conjugated to an immunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyhole limpet hemocyanin (KLH)).
  • an immunogenic carrier e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyhole limpet hemocyanin (KLH).
  • adjuvants are used to increase the immunological response, depending on the host species, including but not limited to Freund’s (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, Gerbu adjuvant and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.
  • Freund complete and incomplete
  • mineral gels such as aluminum hydroxide
  • surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol
  • Gerbu adjuvant and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.
  • any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used (See e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). These include, but are not limited to, the hybridoma technique originally developed by Kohler and Milstein (Kohler and Milstein, Nature, 256:495-497 [1975]), as well as the trioma technique, the human B-cell hybridoma technique (See e.g., Kozbor et al., Immunol.
  • An additional embodiment of the invention utilizes the techniques known in the art for the construction of Fab expression libraries (e.g., Huse et al., Science, 246:1275-1281 [1989]) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • monoclonal antibodies are generated using the ABL-MYC method (See e.g., U.S. Patent 5,705,150 and 5,244,656, each of which is herein incorporated by reference) (Neoclone, Madison, WI).
  • ABL-MYC is a recombinant retrovirus that constitutively expresses v-abl and c-myc oncogenes. When used to infect antigen-activated splenocytes, this retroviral system rapidly induces antigen-specific plasmacytomas.
  • ABL-MYC targets antigen-stimulated (Ag-stimulated) B-cells for transformation.
  • biopanning as described in Pardon etal., Nat Protoc. 2014 Mar;9(3):674- 93 is used to generate single domain antibodies.
  • phage-based biopanning strategies of which there are several published protocols available, are used.
  • Antibody fragments that contain the idiotype (antigen binding region) of the antibody molecule can be generated by known techniques.
  • fragments include but are not limited to: the F(ab’)2 fragment that can be produced by pepsin digestion of an antibody molecule; the Fab’ fragments that can be generated by reducing the disulfide bridges of an F(ab’)2 fragment, and the Fab fragments that can be generated by treating an antibody molecule with papain and a reducing agent.
  • Genes encoding antigen-binding proteins can be isolated by methods known in the art. In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art (e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), Western Blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein A assays, phage display biopanning, and immunoelectrophoresis assays, etc.).
  • radioimmunoassay e.g., ELISA (enzyme-linked immunosorbent assay),
  • the term “toxic” refers to any detrimental or harmful effects on a cell or tissue as compared to the same cell or tissue prior to the administration of the toxicant.
  • compositions and method for treating cells, tissues, and subjects to improve memory in the aging brain, and to study or to treat age-related diseases and conditions associated with memory loss are provided herein.
  • Fgf 17 Fibroblast growth factor 17
  • CSF Cerebrospinal fluid
  • mice were then randomly split into 2 groups and infused with either artificial CSF (aCSF) or young mouse CSF (YM-CSF) for 1 week and remote memory recall was tested 3 weeks after memory acquisition.
  • aCSF artificial CSF
  • YM-CSF young mouse CSF
  • remote memory recall was tested 3 weeks after memory acquisition.
  • YM-CSF infusion resulted in higher average freezing rates following exposure to the tone and light, indicating improved preservation of remote fear memory (FIG. IB).
  • This paradigm allowed testing of interventions that affect remote memory formation and consolidation, a process implicated in ageing-related cognitive decline.
  • young CSF promoted upregulation of transcription factors driving oligodendrocyte differentiation and maj or myelin protein components (for example, Oligl, Myrf, Mag, Mbp and Mobp) (FIG. ID).
  • transcription factors driving oligodendrocyte differentiation and maj or myelin protein components for example, Oligl, Myrf, Mag, Mbp and Mobp
  • OPC proliferation induced by optogenetic tools or by learning tasks, promotes OPC proliferation and differentiation, and regulates myelin plasticity in mature oligodendrocytes (termed activitydependent myelination.
  • OPC proliferation underlies the transcriptomic signature observed following young CSF infusion, dividing cells were labeled in the last 2 days of young CSF infusion with the thymidine analogue EdU. While overall cell proliferation was very low in the aged brains, a surge in overall cell proliferation specifically in the hippocampus relatively distant from the infusion site was discovered (FIGS. 6D-6I).
  • Young CSF induced a 2.35-fold increase in the percentage of proliferating OPCs (EdU + PdgfraV Pdgfra + ) in the CAI region of the hippocampus, but not in the cortex (FIGS. 1E-1F and 6D-6I).
  • aged mice were infused with human CSF pooled from healthy young donors (YH-CSF, mean age of 24.6 years) or aged donors (AH-CSF, mean age of 69 years).
  • YH-CSF induced OPC proliferation at comparable levels to YM-CSF, whereas AH-CSF induced proliferation for only half as many cells (FIGS. 1G and 1H).
  • CSF infusions also triggered EdU incorporation in astrocytes and microglia (FIGS. 7G-7I).
  • the proliferating cells were allowed to mature for 3 weeks to assess effects of young CSF on hippocampal myelination by MBP staining and transmission electron microscopy. Increases in MBP intensity in the molecular layer of the hippocampus (FIGS. II and 1 J) and in the number of myelinated axons (FIGS. IK and 8) in the molecular layer of the hippocampus were observed.
  • nascent mRNA was metabolically labeled with 4-thiouridine (s 4 U) using thiol(SH)-linked alkylation and sequenced RNA (SLAMseq) from cultured OPCs 1- or 6 hr after exposure to YH-CSF.
  • the top gene induced after 1 hr was Serum Response Factor (Srf) (FIG. 2A), a transcription factor in skeletal muscle, heart and in neurons in the brain.
  • Srf binds to serum response element (SRE) promoter sequences to induce cell motility, proliferation, and differentiation through modulation of immediate early genes (such as Egrf) and the actin cytoskeleton.
  • Srf Serum Response Factor
  • SRE serum response element
  • SiR-actin intensity increased within hours of OPC stimulation with YH-CSF, without a change in total area, indicating an increase in cellular actin filament levels (FIGS. 11 A-l 1C).
  • OPCs exposed to YH-CSF for 6 h expressed twice as much phalloidin per cell as controls (FIGS. 2D and 2E).
  • the increase in phalloidin intensity was confirmed in hippocampal OPCs in aged mice infused with YM-CSF for 6 d (FIGS. 2F and 2G).
  • SRF is necessary for the formation of actin filaments in axonal growth cones in neurons it was tested whether SRF has a similar role in OPCs and quantified the number of growth cones per OPC.
  • YH-CSF induced significantly more growth cones per cell compared with aCSF (FIG. 1 ID).
  • AAVs adeno-associated viruses
  • Cre-GFP to create SRF-knockout (SRF -KO) OPCs
  • SRF-WT truncated Cre-GFP as a control
  • oligodendrocyte transcription factor Olig2 (Olig2 high for OPCs and Olig2 low for OLs) from young (3-month-old) and aged (25-month old) mice and performed bulk RNAseq (FIGS. 12A and 12B).
  • the top pathways downregulated in OPCs with aging were related to oligodendrocyte cell markers, regulation of glial cell differentiation, cellular respiration and metabolism and protein folding.
  • immune related pathways and microglial specific genes were upregulated as previously reported for OPCs in aging and multiple sclerosis (FIGS. 3C, 3D, 12C, and 12D).
  • a focused analysis of SRF TRANSFAC target genes in aging OPCs indicated overall downregulation with age (FIG. 3E, left box plot).
  • genes upregulated in OPCs were linked to SRF-related pathways such as “regulation of GTPase activity,” “chromatin organization,” “transcription factor binding,” “cell cycle,” and “regulation of cytoskeleton organization,” respectively (FIGS. 3F and 13B- 13D).
  • SRF target genes were also downregulated in other published human and murine datasets of OPCs in aging and Alzheimer’s disease (AD) (FIG. 3G).
  • Srf mRNA levels in neurons decreased with ageing and were not changed following acute injection with CSF (FIGS. 13E-13H).
  • Fgfl7 When added to primary rat OPCs, Fgfl7 (40 ng/ml) induced OPC proliferation (FIGS. 4H-4I) and differentiation (FIGS. 4J and 4K). Earlier work with cultured OPCs suggested that Fgfl7 slightly promoted proliferation, although it may inhibit OPC differentiation in some contexts. To determine the in vivo activity of Fgf8 and Fgfl7 recombinant proteins were infused over 7 days similar to administration of CSF (FIG. 1). Fgfl7 induced OPC proliferation in the aged hippocampus, but Fgf8 did not (FIGS. 4L, 4M and 14C).
  • mice were infused with an anti-Fgfl7 blocking antibody ICV to test whether Fgfl7 is necessary for normal memory function.
  • Mice infused with anti-Fgfl7, but not with control antibody showed impaired performance in 2 hippocampal-dependent cognitive tests (Y maze and contextual fear conditioning; FIGS. 40 and 4P), and impaired neuronal plasticity measured by lower c-Fos levels in dentate gyrus granule cells following behavioral tests (FIGS. 4Q and 4R).
  • the same concentration of anti-Fgfl7 antibody inhibited OPC proliferation induced by young CSF or Fgfl7.
  • Fgfl7 whose levels decrease with age in mouse neurons and in human CSF, is sufficient and necessary to improve cognition in aged mice and promotes OPC proliferation in vivo and in vitro, suggesting that it constitutes a major component of the rejuvenating effects of young CSF (FIG. 16G).
  • the CSF proteome comprises proteins secreted by the choroid plexus or transferred through it from the blood plasma, as well as proteins secreted from parenchymal and immune cells.
  • improper signaling cues derived from the aging choroid plexus led to neuronal stem cell quiescence with aging.
  • OPCs which account for the largest population of stem cells in the aged brain have been less extensively investigated.
  • Studies in young rodents reveal that oligodendrogenesis, the formation of myelinating oligodendrocytes from OPCs, facilitates consolidation of newly formed memories, implicating their active role in cognitive function.
  • Hippocampal oligodendrogenesis is inhibited with age and boosting it improves performance in learning and memory tasks in aged mice and in AD mouse models in keeping with data showing that aged OPCs in white matter regions, are slow to proliferate and to differentiate following demyelination in diseases such as multiple sclerosis, and that local or systemic environmental manipulations restored their myelination capacity.
  • Fgfl7 was infused into the CSF of aged mice and it recapitulates the effects of young CSF on OPC proliferation and long-term memory recall.
  • blocking Fgfl7 by infusing mice with an inhibitory antibody resulted in impaired function in hippocampal-dependent memory tests.
  • Fgfr signaling is critical for oligodendrocyte development, with complex and diverse functions in disease processes such as demyelination and remyelination in multiple sclerosis.
  • Fg/3r-null mice studies using Fg/3r-null mice have shown a delay in the terminal differentiation of pro-oligodendrocytes and transient expression of Fgfr3 in subventricular zone progenitors drives oligodendrogenesis and promotes remyelination following a demyelinating injury.
  • Young mice lacking Fgfl7 have a diversity of social behavior abnormalities coinciding with lower c-fos expressing cells in the prefrontal cortex following a novel social interaction test indicating that Srf, which regulates c-fos expression, may participate in these circuits and in neuropsychiatric disorders.
  • Fgfl7 is critical for normal embryonic brain development but little is certain about its function in the adult nervous system.
  • Fgfl7 is a brain-derived protein, that in the adult mouse and human brain is expressed by a small subset of cortical neurons and by the choroid plexus epithelial cells immediately accessible to the CSF (FIG. 5).
  • Fgfl7 is a brain-derived protein, that in the adult mouse and human brain is expressed by a small subset of cortical neurons and by the choroid plexus epithelial cells immediately accessible to the CSF (FIG. 5).
  • methods and compositions of the present invention comprise de novo peptide targeted therapeutics as described, for example, by Chevalier A. el al. Nature Publishing Group 2017:550;74-79 incorporated by reference herein in its entirety.
  • the present disclosure provides peptides that directly or indirectly enhance Fgfl7 function.
  • the peptide is an agonist to, for example, the receptor for Fgfl7.
  • the peptide may comprise a fragment or portion of Fgfl7 which binds to the receptor for Fgfl7.
  • compositions comprise oligomeric antisense compounds, particularly oligonucleotides used to modulate the function of nucleic acid molecules encoding Fgfl7, ultimately modulating the amount of Fgfl7 expressed. This is accomplished by providing antisense compounds that specifically hybridize with one or more nucleic acids encoding Fgfl7 or that hybridize to a nucleic acid that encodes a specific direct or indirect inhibitor of Fgfl7. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid.
  • This modulation of function of a target nucleic acid by compounds that specifically hybridize to it is generally referred to as “antisense.”
  • the functions of DNA to be interfered with include replication and transcription.
  • the functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity that may be engaged in or facilitated by the RNA.
  • the overall effect of such interference with a target nucleic acid function is modulation of Fgfl7.
  • modulation means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene.
  • Fgfl7 expression may be stimulated to treat or prevent dementia, cognitive impairment, cognitive aging, or a white matter disorder, particularly in an aged subject.
  • nucleic acids are small RNAs, for example, siRNAs.
  • RNA interference is the process of sequence-specific, post-transcriptional gene silencing initiated by a small interfering RNA (siRNA). During RNAi, siRNA induces degradation of target mRNA with consequent sequence-specific inhibition of gene expression.
  • siRNA small interfering RNA
  • An “RNA interference,” “RNAi,” “small interfering RNA” or “short interfering RNA” or “siRNA” or “short hairpin RNA” or “shRNA” molecule, or “miRNA” is an RNA duplex of nucleotides that is targeted to a nucleic acid sequence of interest, for example, Fgfl7.
  • RNA duplex refers to the structure formed by the complementary pairing between two regions of an RNA molecule.
  • siRNA is “targeted” to a gene in that the nucleotide sequence of the duplex portion of the siRNA is complementary to a nucleotide sequence of the targeted gene.
  • the siRNAs are targeted to the sequence encoding Fgfl7.
  • the length of the duplex of siRNAs is less than 30 base pairs.
  • the duplex can be 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 base pairs in length.
  • the length of the duplex is 19 to 32 base pairs in length. In certain embodiment, the length of the duplex is 19 or 21 base pairs in length.
  • the RNA duplex portion of the siRNA can be part of a hairpin structure. In addition to the duplex portion, the hairpin structure may contain a loop portion positioned between the two sequences that form the duplex. The loop can vary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides in length. In certain embodiments, the loop is 18 nucleotides in length.
  • the hairpin structure can also contain 3' and/or 5' overhang portions. In some embodiments, the overhang is a 3' and/or a 5' overhang 0, 1, 2, 3, 4 or 5 nucleotides in length.
  • Dicer- substrate RNAs are chemically synthesized asymmetric 25- mer/27-mer duplex RNAs that have increased potency in RNA interference compared to traditional siRNAs.
  • Traditional 21-mer siRNAs are designed to mimic Dicer products and therefore bypass interaction with the enzyme Dicer.
  • Dicer has been recently shown to be a component of RISC and involved with entry of the siRNA duplex into RISC.
  • Dicer- substrate siRNAs are designed to be optimally processed by Dicer and show increased potency by engaging this natural processing pathway. Using this approach, sustained knockdown has been regularly achieved using sub-nanomolar concentrations. (U.S. Pat. No. 8,084,599; Kim et al., Nature Biotechnology 23:222 2005; Rose et al., Nucleic Acids Res., 33:4140 2005).
  • the transcriptional unit of a “shRNA” is comprised of sense and antisense sequences connected by a loop of unpaired nucleotides.
  • shRNAs are exported from the nucleus by Exportin-5, and once in the cytoplasm, are processed by Dicer to generate functional siRNAs.
  • miRNAs stem-loops are comprised of sense and antisense sequences connected by a loop of unpaired nucleotides typically expressed as part of larger primary transcripts (pri -miRNAs), which are excised by the Drosha-DGCR8 complex generating intermediates known as pre-miRNAs, which are subsequently exported from the nucleus by Exportin-5, and once in the cytoplasm, are processed by Dicer to generate functional miRNAs or siRNAs.
  • the term “artificial” arises from the fact the flanking sequences ( ⁇ 35 nucleotides upstream and ⁇ 40 nucleotides downstream) arise from restriction enzyme sites within the multiple cloning site of the siRNA.
  • miRNA encompasses both the naturally occurring miRNA sequences as well as artificially generated miRNA shuttle vectors.
  • the siRNA can be encoded by a nucleic acid sequence, and the nucleic acid sequence can also include a promoter.
  • the nucleic acid sequence can also include a polyadenylation signal.
  • the polyadenylation signal is a synthetic minimal polyadenylation signal or a sequence of six Ts.
  • the present disclosure contemplates the use of any genetic manipulation for use in modulating the expression of Fgfl7.
  • genetic manipulation include, but are not limited to, gene knockout or knock-in (e.g, removing or adding the Fgfl7 gene from the chromosome using, for example, recombination), expression of antisense constructs with or without inducible promoters, and the like.
  • Delivery of nucleic acid construct to cells in vitro or in vivo may be conducted using any suitable method.
  • a suitable method is one that introduces the nucleic acid construct into the cell such that the desired event occurs (e.g, expression of an antisense construct or stimulation of Fgfl7 expression).
  • exemplary methods use gene delivery vehicles derived from viruses, including, but not limited to, adenoviruses, retroviruses, vaccinia viruses, and adeno-associated viruses. Because of the higher efficiency as compared to retroviruses, vectors derived from adenoviruses are of use as gene delivery vehicles for transferring nucleic acid molecules into host cells in vivo.
  • Adenoviral vectors have been shown to provide very efficient in vivo gene transfer into a variety of solid tumors in animal models and into human solid tumor xenografts in immune-deficient mice. Examples of adenoviral vectors and methods for gene transfer are described in PCT publications WO 00/12738 and WO 00/09675 and U.S. Pat. Appl. Nos. 6,033,908, 6,019,978, 6,001,557, 5,994,132, 5,994,128, 5,994,106, 5,981,225, 5,885,808, 5,872,154, 5,830,730, and 5,824,544, each of which is herein incorporated by reference in its entirety.
  • Vectors may be administered to the subject in a variety of ways.
  • vectors are administered into nervous system tissue or other tissue associated with aging using direct injection.
  • administration is via the blood or lymphatic circulation (See e.g., PCT publication 1999/02685 herein incorporated by reference in its entirety).
  • Exemplary dose levels of adenoviral vector are preferably 10 8 to 10 11 vector particles added to the perfusate.
  • the present disclosure provides antibodies that directly or indirectly enhance Fgfl7 expression and or function.
  • Any suitable antibody e.g., monoclonal, polyclonal, or synthetic
  • the antibodies are humanized antibodies. Methods for humanizing antibodies are well known in the art (See e.g., U.S. Patents 6,180,370, 5,585,089, 6,054,297, and 5,565,332; each of which is herein incorporated by reference).
  • the antibody is an agonist antibody to, for example, and the receptor for Fgfl7.
  • Receptor agonism may be a critical step in the transmission of a signal from the outside to the inside of a cell.
  • Agonist activity may occur when an antibody binds a receptor, for example the receptor for Fgfl7, such that it mimics binding of the natural ligand resulting in antibody-mediated downstream signaling or agonism.
  • Antibody-mediated agonist activity may occur, for example, when 2 FAb arms of an IgG each bind to a half-receptor of a homo-dimeric receptor pair, causing the receptors to link and mimic the activity of a natural ligand.
  • the targeting unit is an antigen binding protein.
  • Antigen binding proteins include, but are not limited to an immunoglobulins, a Fab, F(ab')2, Fab' single chain antibody, Fv, single chain (scFv), mono-specific antibody, bi-specific antibody, tri-specific antibody, multivalent antibody, chimeric antibody, humanized antibody, human antibody, CDR-grafted antibody, shark antibody, an immunoglobulin single variable domain (e.g., a nanobody or a single variable domain antibody), minibody, camelid antibody (e.g., from the Camelidae family) microbody, intrabody (e.g., intracellular antibody), and /or de-fucosylated antibody and I or derivative thereof. Mimetics of binding agents and/or antibodies are also provided.
  • scFv polypeptides described herein are fused to Fc regions to generate minibodies.
  • fragment crystallizable region refers to the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system.
  • the Fc region is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains; IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.
  • the Fc regions of IgGs bear a highly conserved N-glycosylation site.
  • the Fc region is derived from an IgG.
  • the IgG is human IgGl, although other suitable Fc regions derived from other organisms or antibody frameworks may be utilized.
  • scFv polypeptides described herein are fused to chimeric antigen receptors.
  • Chimeric antigen receptors CARs
  • CARs also known as chimeric immunoreceptors, chimeric T cell receptors, artificial T cell receptors or CAR-T
  • these receptors are used to graft the specificity of an antibody (e.g., an scFv described herein) onto a T cell, with transfer of their coding sequence facilitated by retroviral vectors.
  • the receptors are called chimeric because they are composed of parts from different sources.
  • the present invention also envisages expression vectors comprising nucleic acid sequences encoding any of the above polypeptides or fusion proteins thereof or functional fragments thereof, as well as host cells expressing such expression vectors.
  • Suitable expression systems include constitutive and inducible expression systems in bacteria or yeasts, virus expression systems, such as baculovirus, Semliki forest virus and lentiviruses, or transient transfection in insect or mammalian cells.
  • Suitable host cells include E. co/i. Lactococcus lactis, Saccharomyces cerevisiae, Schizosaccharomyces pomhe. Pichia pastoris, and the like.
  • Suitable animal host cells include HEK 293, COS, S2, CHO, NSO, DT40 and the like. The cloning, expression and/or purification of the antibodies can be done according to techniques known by the skilled person in the art.
  • polypeptides described herein may be identified with reference to the nucleotide and /or amino acid sequence corresponding to the variable and/or complementarity determining regions (“CDRs”) thereof.
  • immunoglobulin single variable domains of the invention are natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (herein collectively referred to as “variants”) of the immunoglobulin single variable domains of the invention as defined herein.
  • variants natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (herein collectively referred to as “variants”) of the immunoglobulin single variable domains of the invention as defined herein.
  • the term “immunoglobulin single variable domain of the invention” or “nanobody” in their broadest sense also covers such variants, in particular variants of the antibodies described herein.
  • one or more amino acid residues may have been replaced, deleted and/or added compared to the antibodies of the invention as defined herein.
  • substitutions, insertions or deletions may be made in one or more of the framework regions and/or in one or more of the CDRs.
  • Variants are sequences wherein each or any framework region and each or any complementarity determining region shows at least 80% identity, preferably at least 85% identity, more preferably 90% identity, even more preferably 95% identity or, still even more preferably 99% identity with the corresponding region in the reference sequence (i.e., FRl variant versus FR1 reference, CDR1 variant versus CDRl reference, FR2_variant versus FR2_reference, CDR2_variant versus CDR2_reference, FR3_variant versus FR3_reference, CDR3_variant versus CDR3_reference, FR4_variant versus FR4_reference), as can be measured electronically by making use of algorithms such as PILEUP and BLAST.
  • a “deletion” is defined here as a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent as compared to an amino acid sequence or nucleotide sequence of a parental polypeptide or nucleic acid.
  • a deletion can involve deletion of about two, about five, about ten, up to about twenty, up to about thirty or up to about fifty or more amino acids.
  • a protein or a fragment thereof may contain more than one deletion.
  • an “insertion” or “addition” is that change in an amino acid or nucleotide sequences which has resulted in the addition of one or more amino acid or nucleotide residues, respectively, as compared to an amino acid sequence or nucleotide sequence of a parental protein.
  • “Insertion” generally refers to addition to one or more amino acid residues within an amino acid sequence of a polypeptide, while “addition” can be an insertion or refer to amino acid residues added at an N- or C-terminus, or both termini.
  • an insertion or addition is usually of about one, about three, about five, about ten, up to about twenty, up to about thirty or up to about fifty or more amino acids.
  • a protein or fragment thereof may contain more than one insertion.
  • substitution results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a parental protein or a fragment thereof. It is understood that a protein or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on the protein's activity. By conservative substitutions is intended combinations such as gly, ala; val, ile, leu, met; asp, glu; asn, gin; ser, thr; lys, arg; cys, met; and phe, tyr, trp.
  • a substitution may, for example, be a conservative substitution (as described herein) and/or an amino acid residue may be replaced by another amino acid residue that naturally occurs at the same position in another variable domain.
  • any one or more substitutions, deletions or insertions, or any combination thereof, that either improve the properties of the antibody of the invention or that at least do not detract too much from the desired properties or from the balance or combination of desired properties of the antibody of the invention (i.e., to the extent that the antibody is no longer suited for its intended use) are included within the scope of the invention.
  • a skilled person will generally be able to determine and select suitable substitutions, deletions or insertions, or suitable combinations of thereof, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may, for example, involve introducing a limited number of possible substitutions and determining their influence on the properties of the antibodies thus obtained.
  • deletions and/or substitutions may be designed in such a way that one or more sites for post-translational modification (such as one or more glycosylation sites) are removed, as will be within the ability of the person skilled in the art.
  • substitutions or insertions may be designed to introduce one or more sites for attachment of functional groups (as described herein), for example, to allow site-specific PEGylation.
  • modifications as well as examples of amino acid residues within the immunoglobulin single variable domain, that can be modified (z.e., either on the protein backbone but preferably on a side chain), methods and techniques that can be used to introduce such modifications and the potential uses and advantages of such modifications will be clear to the skilled person.
  • a modification may involve the introduction (e.g., by covalent linking or in another suitable manner) of one or more functional groups, residues or moieties into or onto the immunoglobulin single variable domain of the invention, and in particular of one or more functional groups, residues or moieties that confer one or more desired properties or functionalities to the immunoglobulin single variable domain of the invention.
  • Such functional groups can generally comprise all functional groups and techniques mentioned in the general background art cited hereinabove as well as the functional groups and techniques known per se for the modification of pharmaceutical proteins, and in particular for the modification of antibodies or antibody fragments (including ScFvs and single domain antibodies), for which reference is, for example, made to Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980).
  • Such functional groups may, for example, be linked directly (for example, covalently) to an immunoglobulin single variable domain of the invention, or optionally via a suitable linker or spacer, as will again be clear to the skilled person.
  • PEG poly(ethyleneglycol)
  • mPEG poly(ethyleneglycol)
  • any suitable form of PEGylation can be used, such as the PEGylation used in the art for antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFvs); reference is made to, for example, Chapman, Nat. Biotechnol., 54, 531- 545 (2002); by Veronese and Harris, Adv. Drug Deliv. Rev.
  • reagents for PEGylation of proteins are also commercially available, for example, from Nektar Therapeutics, USA.
  • site-directed PEGylation is used, in particular via a cysteine-residue (see, for example, Yang et al., Protein Engineering, 16, 10, 761-770 (2003).
  • PEG may be attached to a cysteine residue that naturally occurs in an antibody of the invention
  • an antibody of the invention may be modified so as to suitably introduce one or more cysteine residues for attachment of PEG, or an amino acid sequence comprising one or more cysteine residues for attachment of PEG may be fused to the N- and/or C-terminus of an antibody of the invention, all using techniques of protein engineering known per se to the skilled person.
  • a PEG is used with a molecular weight of more than 5000, such as more than 10,000 and less than 200,000, such as less than 100,000; for example, in the range of 20,000-80,000.
  • Another, usually modification comprises N-linked or O-linked glycosylation, usually as part of co-translational and/or post-translational modification, depending on the host cell used for expressing the immunoglobulin single variable domain or polypeptide of the invention.
  • Another technique for increasing the half-life of an immunoglobulin single variable domain may comprise the engineering into bifunctional constructs or into fusions of immunoglobulin single variable domains with peptides (for example, a peptide against a serum protein such as albumin).
  • Yet another modification may comprise the introduction of one or more detectable labels or other signal-generating groups or moieties, depending on the intended use of the labeled antibody.
  • Suitable labels and techniques for attaching, using and detecting them will be clear to the skilled person and, for example, include, but are not limited to, fluorescent labels (such as fluorescein, isothiocyanate, 1 rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine and fluorescent metals such as Eu or others metals from the lanthanide series), phosphorescent labels, chemiluminescent labels or bioluminescent labels (such as luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogs), radio-isotopes, metals, metals chelates or metallic cations or other
  • labeled antibodies and polypeptides of the invention may, for example, be used for in vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA, RIA, EIA and other “sandwich assays,” etc.), as well as in vivo diagnostic and imaging purposes, depending on the choice of the specific label.
  • another modification may involve the introduction of a chelating group, for example, to chelate one of the metals or metallic cations referred to above.
  • Suitable chelating groups include, without limitation, diethylenetriaminepentaacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA).
  • DTP A diethylenetriaminepentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • Yet another modification may comprise the introduction of a functional group that is one part of a specific binding pair, such as the biotin-(strept)avidin binding pair.
  • a functional group may be used to link the antibody of the invention to another protein, polypeptide or chemical compound that is bound to the other half of the binding pair, i.e., through formation of the binding pair.
  • an agonist antibody of the invention may be conjugated to biotin, and linked to another protein, polypeptide, compound, or carrier conjugated to avidin or streptavidin.
  • such a conjugated antibody may be used as a reporter, for example, in a diagnostic system where a detectable signal -producing agent is conjugated to avidin or streptavidin.
  • binding pairs may, for example, also be used to bind the antibody of the invention to a carrier, including carriers suitable for pharmaceutical purposes.
  • a carrier including carriers suitable for pharmaceutical purposes.
  • One non-limiting example is the liposomal formulations described by Cao and Suresh, lournal of Drug Targeting, 8, 4, 257 (2000).
  • Such binding pairs may also be used to link a therapeutically active agent to the antibody of the invention.
  • the immunoglobulin single variable domain of the present invention is fused to a detectable label, either directly or through a linker.
  • the detectable label is a radioisotope or radioactive tracer, which is suitable for medical applications, such as in in vivo nuclear imaging. Examples include, without the purpose of being limitative, "mTc, 123 1, 125 I, in In, 18 F, 64 Cu, 67 Ga, 68 Ga, and any other radio-isotope which can be used in animals, in particular mouse or human.
  • the immunoglobulin single variable domain of the present invention is fused to a moiety selected from the group consisting of a toxin, or to a cytotoxic drug, or to an enzyme capable of converting a prodrug into a cytotoxic drug, or to a radionuclide, or coupled to a cytotoxic cell, either directly or through a linker.
  • the present invention provides an antibody-drug conjugate and/or an antibody-enzyme conjugate comprising, for example, a Fgfl7 agonist.
  • the antibody drug conjugates are administered to cells expressing Fgfl7.
  • linkers are peptides of 1 to 50 amino acids length and are typically chosen or designed to be unstructured and flexible. These include, but are not limited to, synthetic peptides rich in Gly, Ser, Thr, Gin, Glu or further amino acids that are frequently associated with unstructured regions in natural proteins. (See, e.g., Dosztanyi Z., V. Csizmok, P. Tompa, and I. Simon (2005). lUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics (Oxford, England), 21(16), 3433-4.)
  • the therapeutic polypeptide is an immunoglobulin or fragment thereof.
  • immunoglobulins include, but are not limited to, aptamers and immunoglobulins.
  • Immunoglobulins are proteins generated by the immune system to provide a specific molecule capable of complexing with an invading molecule commonly referred to as an antigen. Natural antibodies have two identical antigenbinding sites, both of which are specific to a particular antigen. The antibody molecule recognizes the antigen by complexing its antigen-binding sites with areas of the antigen termed epitopes. The epitopes fit into the conformational architecture of the antigen-binding sites of the antibody, enabling the antibody to bind to the antigen.
  • the immunoglobulin molecule is composed of two identical heavy and two identical light polypeptide chains, held together by interchain disulfide bonds. Each individual light and heavy chain folds into regions of about 110 amino acids, assuming a conserved three-dimensional conformation.
  • the light chain comprises one variable region (termed VL) and one constant region (CL), while the heavy chain comprises one variable region (VH) and three constant regions (CHI, CH2 and CH3). Pairs of regions associate to form discrete structures.
  • the light and heavy chain variable regions, VL and VH associate to form an “Fv “ area that contains the antigen-binding site.
  • variable regions of both heavy and light chains show variability in structure and amino acid composition from one antibody molecule to another, whereas the constant regions show little variability.
  • Each antibody recognizes and binds an antigen through the binding site defined by the association of the heavy and light chain, variable regions into an Fv area.
  • the light-chain variable region VL and the heavychain variable region VH of a particular antibody molecule have specific amino acid sequences that allow the antigen-binding site to assume a conformation that binds to the antigen epitope recognized by that particular antibody.
  • variable regions are found regions in which the amino acid sequence is extremely variable from one antibody to another.
  • three of these so-called “hypervariable” regions or “complementarity-determining regions” (CDR's) are found in each of the light and heavy chains.
  • the three CDRs from a light chain and the three CDRs from a corresponding heavy chain form the antigenbinding site.
  • Fab's for Fragment, antigen binding site
  • CL, VL, CHI and VH regions of the antibody are composed of the CL, VL, CHI and VH regions of the antibody.
  • Fab fragment, antigen binding site
  • Monoclonal antibodies against target antigens are produced by a variety of techniques including conventional monoclonal antibody methodologies such as the somatic cell hybridization techniques of Kohler and Milstein, Nature, 256:495 (1975). Although in some embodiments, somatic cell hybridization procedures are of use, other techniques for producing monoclonal antibodies are contemplated as well (e.g., viral or oncogenic transformation of B lymphocytes).
  • hybridomas An animal system for preparing hybridomas is the murine system. Hybridoma production in the mouse is a well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.
  • Human monoclonal antibodies (mAbs) directed against human proteins can be generated using transgenic mice carrying the complete human immune system rather than-the mouse system. Splenocytes from the transgenic mice are immunized with the antigen of interest, which are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein.
  • Splenocytes from the transgenic mice are immunized with the antigen of interest, which are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein.
  • Monoclonal antibodies can also be generated by other methods known to those skilled in the art of recombinant DNA technology.
  • An alternative method referred to as the “combinatorial antibody display” method, has been developed to identify and isolate antibody fragments having a particular antigen specificity, and can be utilized to produce monoclonal antibodies.
  • a stry et al. Proc. Nat. Acad. Sci. USA, 86:5728 [1989]
  • Huse et al. Science, 246:1275 [1989]
  • Orlandi et al. Proc. Nat. Acad. Sci. USA, 86:3833 [1989]).
  • the antibody repertoire of the resulting B-cell pool is cloned.
  • Methods are generally known for obtaining the DNA sequence of the variable regions of a diverse population of immunoglobulin molecules by using a mixture of oligomer primers and PCR.
  • mixed oligonucleotide primers corresponding to the 5' leader (signal peptide) sequences and/or framework 1 (FR1) sequences, as well as primer to a conserved 3' constant region primer can be used for PCR amplification of the heavy and light chain variable regions from a number of murine antibodies.
  • a similar strategy can also be used to amplify human heavy and light chain variable regions from human antibodies (See e.g., Larrick et al., Methods: Companion to Methods in Enzymology, 2:106-110 [1991]).
  • modified antibody is also intended to include antibodies, such as monoclonal antibodies, chimeric antibodies, and humanized antibodies which have been modified by, for example, deleting, adding, or substituting portions of the antibody.
  • an antibody can be modified by deleting the hinge region, thus generating a monovalent antibody. Any modification is within the scope of the invention so long as the antibody has at least one antigen binding region specific.
  • Chimeric mouse-human monoclonal antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted.
  • the chimeric antibody can be further humanized by replacing sequences of the Fv variable region that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions.
  • General reviews of humanized chimeric antibodies are provided by S.L. Morrison, Science, 229: 1202-1207 (1985) and by Oi et al., Bio. Techniques, 4:214 (1986). Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain.
  • Suitable humanized antibodies can alternatively be produced by CDR substitution (e.g, US 5,225,539 (incorporated herein by reference in its entirety); Jones et al., Nature, 321:552-525 [1986]; Verhoeyan et al., Science, 239:1534 [1988]; and Beidler et rz/., J. Immunol., 141:4053 [1988]). All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to the Fc receptor.
  • CDR substitution e.g, US 5,225,539 (incorporated herein by reference in its entirety); Jones et al., Nature, 321:552-525 [1986]; Verhoeyan et al., Science, 239:1534 [1988];
  • An antibody can be humanized by any method that is capable of replacing at least a portion of a CDR of a human antibody with a CDR derived from a non-human antibody.
  • the human CDRs may be replaced with non-human CDRs; using oligonucleotide site-directed mutagenesis.
  • humanized antibodies in which specific amino acids have been substituted, deleted, or added.
  • humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen.
  • amino acids located in the human framework region can be replaced with the amino acids located at the corresponding positions in the mouse antibody. Such substitutions are known to improve binding of humanized antibodies to the antigen in some instances.
  • the antibodies can be of various isotypes, including, but not limited to: IgG (e.g., IgGl, IgG2, IgG2a, IgG2b, IgG2c, IgG3, IgG4); IgM; IgAl; IgA2; IgAsec; IgD; and IgE.
  • IgG e.g., IgGl, IgG2, IgG2a, IgG2b, IgG2c, IgG3, IgG4
  • IgM IgAl
  • IgA2 IgAsec
  • IgD and IgE.
  • the antibody is an IgG isotype.
  • the antibody is an IgM isotype.
  • the antibodies can be full-length (e.g., an IgGl, IgG2, IgG3, or IgG4 antibody) or can include only an antigen-binding portion (e.g., a Fab, F(ab')2, Fv or a single chain Fv fragment).
  • an antigen-binding portion e.g., a Fab, F(ab')2, Fv or a single chain Fv fragment.
  • the immunoglobulin is a recombinant antibody (e.g., a chimeric or a humanized antibody), a subunit, or an antigen binding fragment thereof (e.g, has a variable region, or at least a complementarity determining region (CDR)).
  • a recombinant antibody e.g., a chimeric or a humanized antibody
  • a subunit e.g., a subunit
  • an antigen binding fragment thereof e.g, has a variable region, or at least a complementarity determining region (CDR)
  • the immunoglobulin is monovalent (e.g, includes one pair of heavy and light chains, or antigen binding portions thereof). In other embodiments, the immunoglobulin is a divalent (e.g., includes two pairs of heavy and light chains, or antigen binding portions thereof).
  • recombinant Fgfl7 fusion proteins that agonize the Fgfl7 receptor are provided.
  • the fusion proteins are prepared as part of a pharmaceutical composition in a form appropriate for the intended application. Generally, this entails preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals. However, in some embodiments of the present invention, a fusion protein composition formulation may be administered using one or more of the routes described herein.
  • the fusion protein compositions are used in conjunction with appropriate salts and buffers to render delivery of the compositions in a stable manner to allow for uptake by target cells. Buffers also are employed when the compositions are introduced into a patient.
  • Aqueous compositions comprise an effective amount of composition dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula.
  • candidate Fgfl7 agonists are screened for activity (e.g, using the methods described in the experimental methods or another suitable assay).
  • cell permeation is enhanced by administration of a high affinity and selectivity glycan ligand as a prodrug, by replacing, for example, the carboxylate with a bioisostere, or by administering high affinity Fgfl7 receptor ligands on the surface of liposomal nanoparticles.
  • BBB blood brain barrier
  • the methods and compositions comprise one or more bi-specific antibodies comprising, for example, antibodies to highly expressed proteins, including basigin, Glutl, and CD98hc. Antibodies to these targets are significantly enriched in the brain after administration in vivo. In particular, antibodies against CD98hc show robust accumulation in the brain after systemic dosing. Accordingly, in specific embodiments, methods and compositions of the present invention comprise, for example, use of CD98hc as a robust receptor-mediated transcytosis pathway for antibody delivery to the brain. (Zuchero et al.
  • transfer across the BBB is enhanced by transient disruption, for example, osmotic or pharmacologic disruption, and/or by other membrane protein pathways using receptor-mediate transcytosis comprising, for example, antibodies against the transferrin receptor.
  • the present invention provides methods and compositions for increasing Fgfl7 activity comprising, for example, methods and compositions that enhance Fgfl7 transcription, translation, and expression, which inhibit Fgfl7 degradation, and/or that agonize Fgfl7 activity comprising, for example, a nucleic acid, an antibody, a small molecule, or a combination thereof.
  • methods and compositions that increase Fgfl7 provided in combination.
  • the present disclosure further provides pharmaceutical compositions (e.g, comprising the compounds described above).
  • the pharmaceutical compositions of the present disclosure may be administered in a number of ways depending upon whether local or systemic treatment, for example, treatment to the central nervous system (CNS), the autonomic nervous system and/or the peripheral nervous system is desired, and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including oral and nasal delivery), pulmonary (e.g, by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, epidermal and transdermal), or parenteral.
  • Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intracerebroventricular, administration.
  • Fgfl7 agonists and agents that increase Fgfl7 activity are administered by methods that bypass the BBB including, for example, direct application to the surface of the CNS, to the parenchyma of the CNS, to the ventricles of the CNS, and to the cerebrospinal fluid (CSF) of the CNS.
  • intrathecal and epidural administration may be achieved by single shot, a series of single shots, and/or by continuous administration to the CSF.
  • continuous administration to the CSF is provided by a programmable external pump, for example, an osmotic pump.
  • continuous administration is provided by a programmable implantable pump.
  • compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, or binders may be desirable.
  • compositions and formulations for parenteral, intrathecal or intracerebroventricular administration may include sterile aqueous solutions that may also contain buffers, diluents, and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
  • Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids.
  • the pharmaceutical formulations of the present disclosure may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • compositions of the present disclosure may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas.
  • the compositions of the present disclosure may also be formulated as suspensions in aqueous, non-aqueous, or mixed media.
  • Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • cationic lipids such as lipofectin (U.S. Pat. No. 5,705,188), cationic glycerol derivatives, nanoparticles and polycationic molecules, such as polylysine (WO 97/30731), also enhance the cellular uptake of oligonucleotides.
  • compositions of the present disclosure may additionally contain other adjunct components conventionally found in pharmaceutical compositions.
  • the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • such materials when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure.
  • the formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until prevention or a cure is effected or a diminution of the disease state is achieved.
  • Optimal dosing schedules can
  • the administering physician can determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of, for example, individual peptides, antibodies, oligonucleotides, and the like, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models or based on the examples described herein. In general, dosage is from 0.01 pg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly, or yearly. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues.
  • the subject undergo maintenance therapy to prevent the recurrence of the disease state, wherein the Fgfl7 agonist is administered in maintenance doses, ranging from 0.01 pg to 100 g per kg of body weight, once or more daily, to once every 20 years.
  • mice were anesthetized intraperitoneally with Ketamine (120mg/kg) and xylazine (8mg/kg), and then placed in a stereotactic instrument (KOPF) with the head secured at a 45-degree angle facing downwards. An incision was made above the neck and muscles were held separated with microretractors allowing exposure of the cisterna magna by blunt forceps without any bleeding.
  • Ketamine 120mg/kg
  • xylazine 8mg/kg
  • KOPF stereotactic instrument
  • CSF was pulled out of the cisterna magna cavity with a 20pl pipettor connected through an aspirator tube assembly (Sigma, A5177) to a pooled glass capillary (Borosilicate glass tubes, ID 1.30 mm, OD 1.70 mm, Length 4.00 in, type 8250, King precision glass) held secure by Model 1769 90° Electrode Holder (KOPF).
  • CSF was kept in a low-protein bind tube on ice and spun in a cold centrifuge for 10 min at 1500 rpm to exclude CSF immune cells. Supernatant was collected and kept in -80 until use.
  • the pellet was resuspended in 6ul of milli-q ultra-pure water for blood contamination quality control using the Nanodrop UV-vis setting with a 415nm wavelength for detection of oxyhemoglobin. A cutoff of below than 0.02 AU was used for CSF infusions.
  • lOOul osmotic pumps (Alzet, 1007D) with a 7-day infusion at a rate of 0.5 pl/hr.
  • Osmotic pumps were connected to a cannula (Brain infusion kit III, Alzet) and incubated overnight in a 37°C water bath.
  • a cannula was inserted at +1 mm medio-lateral, 0 mm anterior-posterior, and -3mm dorso-ventral relative to bregma in order to target the right lateral ventricle.
  • the pump was placed subcutaneously and mice received post-surgical buprenorphine and Baytril.
  • mice were split into groups to achieve an equal average body weight in all groups.
  • a pool of 3 young or 3 aged human CSF samples, pooled YM-CSF or aCSF was loaded into an osmotic pump (without a coil) and surgery was performed as described above.
  • Recombinant carrier free human /mouse Fgf8b (423-F8/CF, R&D) and mouse Fgfl7 (7400-FG-025/CF, R&D) were resuspended in aCSF (Tocris) to a concentration of 25 pg/ml and loaded to an osmotic pump (Alzet, 1007D) with a 7-day infusion at a rate of 0.5ul I hr.
  • mice Acute intracerebroventricular infusion
  • mice Eighteen-month old mice were anesthetized with 2.5% isoflurane and then placed in a stereotactic instrument (KOPF).
  • 3 pl of a pool of young mouse CSF (YM-CSF) (as described above) or aCSF were injected to the right lateral ventricle using a digital pump (WPI syringe pump with Micro4t controller model UMP3T-1) at a rate of I pl/min.
  • WPI syringe pump with Micro4t controller model UMP3T-1 Micro4t controller model UMP3T-1
  • mice were trained to associate cage context or an audiovisual cue with an aversive stimulus (foot shock).
  • foot shock On day 1, mice were placed in a cage and exposed to 3 periods of 30 s of paired cue light and 1,000-Hz tone followed by a 2s foot shock (0.6 mA), with a 60s interval.
  • mice On day 2 and day 22, mice were subjected to 2 trials.
  • mice In the first trial assessing contextual memory, mice were re-exposed to the same cage context, and freezing behavior was measured during minute 390s using a FreezeScan tracking system (Cleversys).
  • mice were placed in a novel context and exposed to the same cue light and tone from day 1 on min 2, 3 and 4 of the trial. Freezing behavior was averaged across min 3-5. No significant differences in contextual fear conditioning were observed between groups at day 22. All experiments were performed by a blinded researcher
  • the forced-alternation Y-maze and contextual fear conditioning tests were performed as previously described (Pluvinage JV et al. CD22 blockade restores homeostatic microglial phagocytosis in ageing brains. Nature 568, 187-192, doi: 10.1038/s41586-019- 1088-4 (2019)).
  • the forced-alteration Y-maze test consisted of a 5-min training trial followed by a 5-min retrieval trail, with a 1-h intertrial interval. For the training trial, one arm of the Y maze was blocked off and mice were allowed to explore the 2 open arms.
  • mice were again placed in the Y maze with all 3 arms open and a black-and-white pattern placed at the end of the novel arm. Between mice and trials, the maze was wiped with ethanol to remove odor cues. For analysis, video was analyzed by a blinded observer and both the number of arm entries and the time spent in each arm were quantified.
  • mice were trained to associate cage context or an audiovisual cue with an aversive stimulus (foot shock). The test was administered over 2 d. On day 1, mice were placed in a cage and exposed to two periods of 30 s of paired cue light and 1,000-Hz tone followed by a 2-s foot shock (0.6 mA), with a 180-s interval.
  • mice were subjected to 2 trials.
  • mice were re-exposed to the same cage context and freezing behavior was measured during minutes 1-3 using a FreezeScan tracking system (Cleversys).
  • mice were placed in a novel context and exposed to the same cue light and tone from day 1 after 2 min of exploration. Freezing behavior was measured for 1-3 min following the cue. No significant differences in cued fear conditioning were observed between groups.
  • mice were anesthetized with Avertin (2,2,2-tribromoethanol: T48402, Sigma-Aldrich; 2-methyl-2- butanol: 240486, Sigma-Aldrich) (0.018 ml (2.5%) per gram of body weight) and perfused with 20 ml cold PBS. Brains were collected and divided sagittally. One hemisphere was used to dissect the hippocampus for RNA sequencing, which was snap frozen and stored at -80 °C. The second hemisphere was fixed in phosphate-buffered 4% paraformaldehyde overnight at 4 °C before transfer to 30% sucrose in PBS at 4 °C until sectioning.
  • Avertin 2,2,2-tribromoethanol: T48402, Sigma-Aldrich; 2-methyl-2- butanol: 240486, Sigma-Aldrich
  • Brains were frozen at -30 °C and cryosectioned coronally at 40 pm with a microtome (Leica, SM2010R). Brain sections were stored in cryoprotectant (40% PBS, 30% glycerol, 30% ethylene glycol) and kept at -20 °C until staining.
  • cryoprotectant 50% PBS, 30% glycerol, 30% ethylene glycol
  • PDGF receptor a D1E1E
  • XP rabbit mAb (1 :500; Cell Signaling, 3174
  • rabbit anti-MBP 1: 100; Millipore, MAB386
  • rabbit anti-Fgfl7 1 :500; Thermo, PAS- 109722
  • rabbit anti-c-Fos (1 :500; Cell Signaling, 9F6)
  • rabbit anti-GFAP (1 :500; Dako, Z0334
  • goat anti-IBAl (1 :500; Abeam, ab5076
  • rabbit anti-NG2-Alexa488 (1 :200; Millipore, AB5230A4
  • rabbit anti-Olig2 (1:500; Millipore, AB9610)
  • anti-Acta2-Cy3 1 :500; Sigma, C6198
  • mouse anti-NeuN 1:500; Sigma, MAB377).
  • brain sections were washed 3 times for 10 min in TBST, followed by incubation for 1.5 hrs in Alexa Fluor-conjugated secondary antibodies (1 :500).
  • Alexa Fluor-conjugated secondary antibodies (1 :500).
  • phallidin-Alexa488 (Thermo, A12379) was added at a 1 :50 dilution to the secondary antibody mix.
  • Brain sections were washed and mounted on Superfrost microscope slides (12- 550-15, Fisher Scientific) with Vectashield Hardset Antifade Mounting Medium with DAPI (Vector labs, H-1500/ NC9029229).
  • tissue de-lipidation For MBP stains an additional step of tissue de-lipidation was added before blocking: tissues were incubated in 100% EtOH for lOmin in room temperature and then washed twice with PBS.
  • tissue delipidation an additional step of tissue delipidation was performed before blocking: tissues were incubated in 100% ethanol for 10 min at room temperature and then washed twice with PBS. EdU staining
  • BrdU staining- brain slices and 384-well plates Following staining with other primary and secondary antibodies, sections were incubated in 2N HC1 for 30 min at 37 °C and then washed 3times for 10 min in TBST. Sections were blocked for 1.5 hr in TBS++ and then transferred to primary antibody with Rat anti-BRDU antibody (1 :500, ab6326, Abeam) overnight at 4 °C. Secondary staining started with 3 washes for 10 min in TBST, followed by incubation with secondary antibody mix for 1.5 h. After 3 10-min washes in TBST, sections were mounted as described above.
  • nuclei were stained and with Hoechst 33342 (1 :2000, H3570, Thermo) and immediately imaged on a Keyence microscope (BZ-X800).
  • BZ-X800 Keyence microscope
  • the sequence was; permeabilization, HCL antigen retrieval, EDU Click-it reaction, blocking, primary and secondary antibody stain as described above in detail.
  • RNAScope was performed on fresh frozen coronal brain sections (10pm thick) using the Multiplex Fluorescence v.2 kit (Advanced Cell Diagnostics) according to the manufacturer’s protocol with minor modifications. Tissue fixation with 4% PFA was extended to 60 min at RT, and Protease IV treatment was shortened to 20 min to better preserve the hippocampal tissue. Probes for mouse Pdgfira and SRF were commercially available from the manufacturer and secondary Opal 690 and 520 reagents (FP1497001KT and FP1487001KT, Akoya Biosciences) were diluted at 1 :1500 in TSA buffer.
  • RNAscope analysis similar hippocampal tiled z-stacks were acquired (at least 4 hippocampal coronal slices per mouse, 100pm apart). Percentage of SRF ⁇ OPCs was calculated by dividing the number of SRF* PDGFRoC nuclei by PDGFRcC nuclei in the CAI region of the hippocampus.
  • MBP analysis confocal images of the molecular layer were acquired using the 20x magnification (Keyence microscope model BZ-X800),MBP intensity was measured using batch analysis in ImageJ.
  • MBP analysis z stacks at x63 magnification of individual OPCs were obtained by staining for Pdgfra. Using Imaris, three-dimensional surface rendering was reconstructed by Pdgfra signal and phalloidin intensity was measured only inside the surface. All analyses were performed by a blinded observer.
  • mice were perfused with 20 ml cold EM fixation buffer consisting of EM-grade 2% glutaraldehyde (EMS/Fisher, 50-262-08) and 4% PFA (EMS/Fisher, 50-980-486) in 0.2 M sodium cacodylate (EMS/Fisher, 50-980-279) and kept in fixation buffer until sectioning.
  • Brains were sectioned coronally to 100-pm sections using a Leica VT1200S vibratome and kept in EM fixative until TEM processing.
  • Vibratome sections of 100 pm were stained using an osmium-thiocarbohydrazide-osmium (OTO) method in combination with microwave-assisted processing, followed by high-pressure freezing and freeze substitution (HPF-FS), as previously described (Ewald AJ et al. JCellSci 125, 2638-2654, doi: 10.1242/jcs.096875 (2012)).
  • Samples were stained with OTO, incubated with 2% aqueous uranyl acetate overnight and then subjected to HPF, followed by super-rapid FS with 4% osmium tetroxide, 0.1% uranyl acetate and 5% ddH2O in acetone.
  • Sections were imaged using a Tecnai 12 120-kV transmission electron microscope (FEI), data were recorded using an UltraScan 1000 with Digital Micrograph 3 software (Gatan) and SerialEM was used to collect montages covering an area of 143 x 143 pm.
  • FEI transmission electron microscope
  • HEK293 cells were plated at 50K cells/ well in a 96-well plate in full media (DMEM, 10% FCS and 1% P/S) and transfected on day 2 with Cignal SRE Reporter Assay Kit (GFP) (CCS-010G, Qiagen) using lipofectamine P3000 in experimental media (DMEM, 0.5% FCS, 1% non-essential amino acids (M7145, Sigma)), following the manufacturer’s instructions.
  • Tissue was lysed in a RIPA lysis buffer (ThermoFisher, 89901) cocktail solution containing protease inhibitor (Roche, 11836153001) and Halt phosphatase inhibitor (ThermoFisher, 78420).
  • the protein concentration of each sample was then measured using the Pierce BCA Protein Assay kit (ThermoFisher, 23225). 30ug of protein per sample were then heated at 95°C for lOmin before loading on a 4-12% 10-well gel (ThermoFisher, NP0321BOX). The gel was run at 80V for lOmin and then 150V until samples ran through gel. Transfer was completed at 100V for 1.5hrs.
  • Membranes were blocked in 5% BSA for Jackpot and then stained overnight at 4°C with 1 : 1000 dilutions of mouse a-GAPDH (Origene, TA802519) and rabbit a-FGF17 (ThermoFisher, PA5-109722). Then, membranes were washed and stained with 1 :20,000 Li-Cor a-Mouse (Li-Cor, 926-68072) and a-Rabbit (Li-Cor, 926-32213) secondary antibodies for Cup covered with aluminum foil. Finally, they were washed 3 times with TBST for 5 mins and 2 times with TBS for 5 mins before being imaged using a Li-Cor Odyssey CLx imager.
  • OPCs were isolated from P7-P8 brains by immunopanning and grown in serum-free defined medium, as previously described (Emery B. & Dugas JC Cold Spring Harb Protoc 2013, 854-868, doi: 10.1101/pdb.prot073973 (2013)).
  • Cell culture for proliferation and differentiation experiments was done following the protocol with several modifications. To use the least possible CSF of young healthy human subjects, the culture conditions were minimized to 384 well plates. In addition, to account for inter-subject variability, CSF of 3 subjects with similar ages were pooled, and 3 such pools were used in each experiment in triplicates.
  • 2,500 cells were plated in a total volume of 50pl of full proliferation medium supplemented with 10 ng/ml PDGF (Peprotech 100- 13 A), 10 ng/ml CNTF Peprotech 450-02, 4.2 pg/ml forskolin (Sigma-Aldrich F6886) and Ing/ml NT3 (Peprotech 450-03) with 10% of YH-CSF or aCSF.
  • Actin filaments were visualized by live imaging by addition of 500nM of SiR-Actin (Cytoskeleton, CY-SC002) added with 10% YH-CSF. Wells were imaged every hour for the remaining 6 hours of the experiment.
  • BRDU experiments 18hrs after plating in 384-well plates (with 10% CSF), 5ul of 200pM BRDU (20pM final concentration) was added for a pulse of 6 hrs followed by fixation with 4% PFA for 20 min.
  • BrdU experiments were performed with the indicated concentrations of YH-CSF or Fgfl 7 and in FIG. 4S in combination with rabbit anti-Fgf!7 (Thermo, PA5- 109722) or IgG isotype control (Thermo, 31235) to achieve a final concentration of 5 pg ml -1 antibody, 40 ng ml -1 Fgfl7 and 10% YH-CSF in full proliferation medium, as indicated above.
  • Primary antibodies were as follows; rabbit-anti-MBP (1 :100, abeam, ab7349, knock-out validated 19 ) and mouse anti-GFAP (1:500, Chemicon, MAB360). Primary antibodies were incubated overnight at 4 °C. Coverslips were washed, stained with Alexa Fluor-conjugated secondary antibodies (1:500) followed by a 15 min stain with Cell mask (1 :1000, Invitrogen, Cl 0046) mounted and set on a coverslip before imaging on a Keyence microscope (BZ-X800) or confocal laser-scanning microscope (Zeiss LSM880).
  • Mouse OPCs were purified from brains of mice with loxP -flanked SRF (generated by David Ginty and kindly provided by Eric Small) by immunopanning as described above for rat OPCs (Emery B. & Dugas JC Cold Spring Harb Protoc 2013, 854-868, doi: 10.1101/pdb.prot073973 (2013)).
  • the optimal s 4 U concentration was assessed using the SLAMseq Explorer Kit - Cell Viability Titration Module (061, Lexogen) following manufacturer’s recommendations. OPCs were incubated with increasing doubling concentrations of s 4 U (1.95-2000pM), and viability was assessed with live-dead ratio as described above and with ATP incorporation following manufacturer’s recommendations (Promega,G7571). A final concentration of 62.5uM was found to be the highest concentration that did not compromise cell viability within 12 h (twice the duration of the intended experiment). The SLAMseq experiment was conducted following SLAMseq Kinetics Kit - Anabolic Kinetics Module protocol (061, Lexogen).
  • RNA extraction was performed following the protocol for the anabolic kit. All cell culture and RNA extraction steps were done in the dark under red-light, following manufacturer’s recommendations.
  • FASTQ files were then analyzed using the SLAM-DUNK pipeline vO.3.4 and the related alleyoop toolchain.
  • the pipeline comprises 1) read mapping, 2) alignment filtering, 3) SNP calling and correction, and 4) 3'-UTR sequence counting.
  • NextGenMap reads were aligned against the genome of Rattus norvegicus (release 6.0), which was downloaded in FASTA format from Ensembl release v97. For mapping the following parameters were set; '-5 12, -a 4, -n 1, -ss' leaving the rest at default.
  • Resulting Binary sequence Alignment/Map (BAM) format files were filtered to remove low- quality alignments using the parameters '-mq 2, -mi 0.95, -nm -1’ with the rest at default.
  • SNPs in alignments in particular the T to C (T>C) conversions, were called with parameters '-c 10, -f 0.8' and other parameters remaining at default.
  • T>C T to C
  • the statistical independence of distributions of true SNP-callings identified by VarScan2 in relation to the number of T>C reads was assessed with a Mann-Whitney-U test for each sample as initially described in the alleyoop snpeval toolchain module.
  • a genome feature file for the Rattus norvegicus genome (release 6.0) was downloaded from Ensembl release v97, filtered to retain only three_prime_utr features and converted to Browser Extensible Data (BED) format using bedops v2.4.36.
  • the BED file was used in combination with parameters '-c 1, -q 27' for the SLAMDUNK count command, leaving other parameters at default. Resulting 3'-UTR T>C and total read counts were added up on gene-level along all transcripts and alternative 3'-UTRs available per transcript.
  • nuclei were pelleted and resuspended in 100 pl of antibody mix (1:100, Anti-NeuN antibody -Alexa Fluor® 647, EPR12763, and 1 : 100, Anti-Olig2 antibody-Alexa Fluor® 488) with 0.2U/pl RNAse Inhibitor (Takara, 23138) in FACS buffer (0.5% BSA in PBS) and were incubated on ice with intermittent shaking for 30 min. Nuclei were washed with 1ml FACS buffer and pelleted by centrifugation at 500 RCF.
  • RNAse inhibitor for 5 min, resuspended in FACS buffer with 0.2U/pl RNAse inhibitor and Hoechst 33342 (1 :2000, H3570, Thermo), sorted on a Sony Sorter (SH800) based on Houechst + NeuN" Olig2 +high (OPC) and Houechst + NeuN'Olig2 +low (OL) gating to 350pl of RTL buffer and stored in -80°C until RNA extraction. Data were analyzed using FlowJo software (TreeStar).
  • Oligodendrocyte nuclei were isolated by FACS and RNA was extracted with the RNeasy Plus Micro kit (Qiagen, 74034). cDNA was generated with qScriptTM cDNA SuperMix (QuantaBio, 95048). Samples were diluted and mixed with SYBR green master mix before loading as technical triplicates for qPCR on a LightCycler 480 (Roche). AAC T values normalized to Gapdh were used to assess relative gene expression between samples.
  • RNA quantity and quality were assessed by Agilent 2100 Bioanalyzer (Agilent Technologies). All samples passed a quality-control threshold (RIN > 9) to proceed to library preparation and RNA-seq on HiSeq 4000 (Illumina) using paired-end 100-bp reads. Libraries were sequenced to a depth of >20 million reads per sample. Raw sequencing files were demultiplexed with bcl2fastq, reads were aligned using STAR, and counts of technical replicates were summed up using DESeq2 before performing normalization and differential expression analysis with standard settings.
  • RNA-seq data For deconvolution analysis of bulk RNA-seq data, the CIBERSORTx algorithm (Newman AM et a . Nat Biotechnol Nl , 773-782, doi: 10.1038/s41587-019-0114-2 (2019)) was used to deconvolve the bulk RNA-seq data.
  • single-nucleus RNA-seq data describing the brain of 3-month-old young mice Hahn 0. et al. Nucleic Acids Res 49, el l, doi: 10.1093/nar/gkaal 127 (2021) was used to construct a cell-type-specific signature matrix with CIBERSORTx.
  • astrocytes astrocytes, choroid plexus, endothelial cells (BEC), interneurons, microglia, neurons of the trisynaptic loop (neuron CA), neurons of the dentate gyrus (neuron DG), oligodendrocytes, oligodendrocyte precursors and pericytes.
  • BEC endothelial cells
  • microglia neurons of the trisynaptic loop
  • neurode DG neurons of the dentate gyrus
  • oligodendrocytes oligodendrocyte precursors and pericytes.
  • Sampling was done over the annotated, quality-controlled data to ensure efficiency for CIBERSORTx.
  • CIBERSORTx was ran on the sampled and CPM- normalized dataset with default parameters and inferred a ‘signature matrix’ that provided gene signatures for each noted cell type.
  • the cDNA concentration was measured using the Qubit lx dsDNA HS kit (Q33231) and normalized to 0.4 ng/pl as input for library prep.
  • 0.4 pl of each normalized sample was mixed with 1.2 pl of Tn5 Tagmentation mix (0.64 pl TAPS -PEG buffer (PEG 8000) ( Promega V3011), and TAPS-NaOH pH8.5)(Boston Bioproducts BB-2375), 0.46 pl H2O and 0.1 pl Tn5 enzyme (Illumina 20034198,), followed by incubation at 55 °C for 10 min.
  • the reaction was stopped by adding 0.4 pl 0.1% sodium dodecyle sulfate (Fisher Scientific, BP166-500). Indexing PCR reactions were performed by adding 0.4 pl of 5 pM i5 indexing primer (IDT), 0.4 pl of 5 pM i7 indexing primer (IDT), and 1.2 pl of KAPA HiFi Non-Hot Start Master Mix (Kapa Biosystems) using 12 amplification cycles. Libraries were purified using 2 purification rounds with a ratio of 0.8x and 0.7x AMPure beads. Library quantity and quality was assessed using a Bioanalyzer (Agilent) and Qubit.
  • raw gene expression counts were downloaded for 2 datasets from the Allen Brain Atlas data portal (portal. brainmap. org/).
  • the first comprised human Ml cortex samples profiled with lOx 3' gene expression yielding -77,000 single-nucleus transcriptomes, and the second comprised mouse hippocampus and cortex samples profiled with Smart-seq2 yielding -77,000 single-cell transcriptomes.
  • step 1 normalization
  • step 2 selection of variable features using VST normalization and setting of the number of features to 2,000
  • step 3 scaling and centering of informative features
  • step 4 principal-component analysis with 50 principal components
  • step 5 computation of the nearest-neighbor graph using 20 dimensions
  • step 6 identification of clusters in the graph when setting the resolution to 0.8 and the number of starts and iterations to 10 and 15, respectively
  • step 7 UMAP-based dimension reduction with 20 input dimensions, the number of neighbors set to 30, the minimum distance set to 0.3 and the spread set to 1 (step 7).
  • Fgfl 7-positive cells were defined by a normalized expression count larger than zero.
  • the respective cells were extracted and analysis steps 2-7 from above were repeated.
  • the FindAllMarkers function from Seurat was used together with the MAST package, requiring an absolute log-transformed fold change of at least 0.25 and a minimum percentage expressed of 10%.
  • the expression of Fgfl 7 was correlated to that of all other expressed genes in selected clusters using the cosine similarity. Lists of genes ranked by decreasing correlation were then used to perform GSEA using GeneTrail 3.0.
  • FIGS. 1L-1O, 2E, 21, 4B, 4C, 4F, 4H, 41, 4S, and 7A, 12A, 12B, 12D-12F, 12H, 121, 14A, 14B, 14D, and 14E were successfully replicated in 2 independent experiments.
  • CD22 blockade restores homeostatic microglial phagocytosis in ageing brains. Nature 568, 187-192, doi: 10.1038/s41586-019-1088-4 (2019). Emery, B. & Dugas, J. C. Purification of oligodendrocyte lineage cells from mouse cortices by immunopanning. Cold Spring Harb Protoc 2013, 854-868, doi: 10.1101/pdb.prot073973 (2013). Stockel, D. et al. Multi-omics enrichment analysis using the GeneTrail2 web service. Bioinformatics 32, 1502-1508, doi: 10.1093/bioinformatics/btv770 (2016). Hahn, 0. et al.

Abstract

Provided herein are compositions and method for treating cells, tissues, and subjects to improve memory in the aging brain, and to study or to treat age-related diseases and conditions associated with memory loss. In particular, provided herein are compositions, methods, systems, kits and uses for delivery of Fibroblast growth factor 17 (Fgf17) to restore memory and to rejuvenate oligodendrocyte progenitor cell.

Description

METHODS AND COMPOSITIONS FOR IMPROVED MEMORY IN THE AGING
CROSS-REFERENCE TO RELATED APPLICATIONS
The present Application claims priority to U.S. Provisional Application Serial Number 63/340,023 filed May 10, 2022, the disclosure of which is herein incorporated by reference in its entirety.
SEQUENCE LISTING STATEMENT
The contents of the electronic sequence listing titled STDU2-39617-601_SQL.xml (Size: 2,867 bytes; and Date of Creation: May 9, 2023) is herein incorporated by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with Government support under contract AG064897 awarded by the National Institutes of Health. The Government has certain rights in the invention.
FIELD
Provided herein are compositions and method for treating cells, tissues, and subjects to improve memory in the aging brain, and to study or to treat age-related diseases and conditions associated with memory loss. In particular, provided herein are compositions, methods, systems, kits and uses for delivery of Fibroblast growth factor 17 (Fgf 17) to restore memory and to rejuvenate oligodendrocyte progenitor cells.
BACKGROUND
The systemic environment influences brain structure and function throughout the lifespan. Numerous intervention strategies have been introduced to slow brain aging without consistent success. Myelin degeneration is a feature in brain aging and neurodegenerative diseases including Alzheimer’s Disease. The process by which oligodendrocyte progenitor cells (OPCs) mature to myelinforming oligodendrocytes decreases progressively with age thereby limiting regenerative capacity of the aging brain. Rejuvenation of oligodendrocytes in aging individuals is a desired therapeutic strategy to treat or prevent cognitive aging, neurodegenerative disease such as Alzheimer’s disease, well as demyelinating diseases such as multiple sclerosis.
Cerebrospinal fluid (CSF) nourishes the brain and provides it with growth factors that sustain progenitor function. Changes in the brain microenvironment that occur with age result in lower progenitor cell support thereby leading to reduced myelin turnover and axonal damage. In experiments conducted in the course of development of the present invention using unbiased RNAseq of the hippocampus, oligodendrocytes were identified as the cells most prominently responding to the young CSF environment. Infusion of CSF from young brains directly into aging brains induced OPC proliferation and maturation to myelin-forming cells in the aging hippocampus and in primary OPC cultures. Using SLAMseq to metabolically label nascent mRNA, serum response factor (SRF), a transcription factor that drives actin cytoskeleton rearrangement, mediated OPC proliferation following exposure to young CSF. SRF expression decreased in hippocampal OPCs with aging, and the pathway was induced by young CSF infusion. Following screening for CSF proteins that induce SRF signaling, the strongest induction was achieved by the Fgf8 sub-family of Fibroblast growth factors (FGFs) comprising Fgf8/17/18. The Fgfs play roles in the formation of the brain during development, but their function in the adult brain has been unknown. Infusion of Fgfl7 to the aging brain results in parallel effects seen with young CSF, including OPC proliferation, and improved long-term memory consolidation and recall. Accordingly, the present invention provides compositions, methods, systems, kits and uses to restore OPC function in the aging and diseased brain to a more youthful state.
SUMMARY
Provided herein are compositions and method for treating cells, tissues, and subjects to improve memory in the aging brain, and to study or to treat age-related diseases and conditions associated with memory loss. In particular, provided herein are compositions, methods, systems, kits and uses for delivery of Fibroblast growth factor 17 (Fgf 17) to restore memory and to rejuvenate oligodendrocyte progenitor cells.
For example, provided herein are methods of treating an age-related disease or condition comprising exposing one or more of a subject’s central nervous system cells to Fgfl7 and/or to a Fgfl7 agonist wherein the exposing prevents and or treats the age-related disease or condition. In some embodiments, the age-related disease or condition is selected from the group of cognitive aging, neurodegeneration, or demyelination. In certain embodiments, the subject is a human subject. In other embodiments, the Fgf 17 agonist is a Fgfl7 peptide or fragment thereof. In further embodiments, the Fgfl7 agonist is an agonist antibody. In still further embodiments, the Fgfl7 agonist is a nucleic acid. In given embodiments, the nucleic acid is delivered to the choroid plexus. In certain embodiments, the nucleic acid is an aptamer. In additional embodiments, the Fgfl7 agonist increases Fgfl7 expression. In specific embodiments, the exposing is in vivo exposing, ex vivo exposing or in vitro exposing. In particular embodiments, the exposing is selected from the group consisting of local administration, topical administration, intrathecal administration, intraparenchymal administration, intracerebroventrical administration, intravenous administration, intraarterial administration, intrapulmonary administration, and oral administration. In some embodiments, exposing comprises combination therapy with an agent increases Fgfl7 function.
In some embodiments, provided herein are compositions comprising a Fgfl7 peptide and/or a Fgfl7 agonist, and a pharmaceutically acceptable carrier.
DESCRIPTION OF THE FIGURES
FIG. 1A-1O show that young CSF improves memory consolidation and promotes OPC proliferation and differentiation. FIG. 1A is an overview of the experimental paradigm. FIG. IB shows the percentage of freezing for 20-month-old mice in the remote recall contextual fear conditioning test (aCSF, n = 10; YM-CSF, n = 8; two-sided t test; mean ± s.e.m.). FIG. 1C shows that GSEA of hippocampal bulk RNA-seq data identifies oligodendrocyte genes as highly upregulated following 6 d of infusion with YM-CSF. FIG. ID shows the effect size of oligodendrocyte genes in the hippocampus of mice infused with YM-CSF versus aCSF compared with all genes in the dataset (an asterisk indicates FDR < 0.1) (aCSF, n = 8; YM-CSF, n = 7). FIG. IE shows the quantification of proliferating OPCs in the hippocampus following 6 d of aCSF or YM-CSF infusion into 20-month-old mice (aCSF, n = T, YM- CSF, n = 8; two-sided t test; mean ± s.e.m.). FIG. IF shows the representative images of the experiment in FIG. IE . Arrowheads point to proliferating OPCs. Scale bars, 20 pm (insets, 5 pm). FIG. 1G shows the quantification of proliferating OPCs in the hippocampus as in FIG. IE but for YH-CSF and AH-CSF (n = 5; two-sided / test; mean ± s.e.m.). FIG. 1H is representative images of the experiment in FIG. 1G. Scale bars, 50 pm. FIG. II shows hippocampal MBP staining following the long-term paradigm of infusion with aCSF or YM-CSF (n = 8; two-sided t test; mean ± s.e.m.). FIG. 1 J is representative images of the experiment in FIG. II. Scale bars, 50 pm. FIG. IK shows the quantification of the number of myelinated axons per pm2 in the hippocampus of aged mice following the long-term paradigm of infusion with aCSF or YM-CSF (w = 7; one-sided / test; mean ± s.e.m.). FIG. IL shows the ratio of the percentage of BrdU+/DAPI+ primary rat OPCs treated with the indicated dose of YH-CSF over matching aCSF as control (n = 3; one-way ANOVA followed by Tukey’s post hoc test; mean ± s.e.m ). FIG. IM is representative images of the experiment in FIG. IL. Scale bars, 50 pm. FIG. IN is stacked bar plots of the average number of cells in each differentiation state at day 4 of differentiation with 10% aCSF or 10% YH-CSF (aCSF, // = 28 l cells analyzed on 3 coverslips; YH-CSF, n = 454 cells analyzed on 2 coverslips). FIG. 10 is representative images of the experiment in FIG. IN. Scale bars, 20 pm.
FIGS. 2A-2I show that serum response factor (Srf) is induced by young CSF and mediates CSF- induced OPC proliferation. FIG. 2A is a volcano plot of DEGs at 1 h following addition of YH-CSF (SRF targets in red) (YH-CSF 1 h, n = 4; rest, // = 5; adjusted P value obtained by Wald test in DESeq2; dashed line represents dj = 0.05). FIG. 2B is normalized expression levels of .57/ in nascent mRNA counts and total counts (YH-CSF 1 h, n = 4; rest, n = 5; two-way ANOVA with Sidak’s post hoc test (nascent and total reads separately)). FIG. 2C is a volcano plot of DEGs at 6 h following addition of YH-CSF (SRF targets in red) (n = 5; adjusted P value obtained by Wald test in DESeq2; dashed line represents Adj = 0.05). FIG. 2D is representative images of the experiment in FIG. 2E. Scale bars, 20 pm. FIG. 2E is mean phalloidin intensity in OPCs 6 h following exposure to YH-CSF n = 3 coverslips per condition; two- sided t test; mean ± s.e.m.). FIG. 2F is representative images of the experiment in FIG 2G. Scale bars, 10 pm. FIG. 2G is mean phalloidin intensity in hippocampal OPCs (PdgfroC) following infusion with aCSF or YM-CSF for 6 d n = 3 mice per group, total of 33 42 single cells measured per condition; two- sided t test; mean ± s.e.m.). FIG. 2H is a schematic of mouse OPC primary cultures from Srf pups infected with AAVs encoding Cre-GFP to induce recombination or ACre-GFP as a control. FIG. 21 shows the percentage of proliferating cells (BrdU+GFP+/GFP+) among SRF-WT and SRF -KO cells treated with 10% aCSF or YH-CSF (// = 3; two-way ANOVA followed by Sidak’s post hoc test; mean ± s.e.m.).
FIGS. 3A-3G show that Srf signaling is downregulated in hippocampal OPCs with aging and induced following acute young CSF injection. FIG. 3A shows N/'mRNA quantified in OPCs (Pdgfrof nuclei) in the CAI region of the hippocampus of young (3 months) and aged (22 months) mice (young, n = 6; aged, n = 7; two-sided t test; mean ± s.e.m.). FIG. 3B is representative images of the experiment in a. Scale bars, 10 pm (5 pm in insets). FIG. 3C is a volcano plot of DEGs of aged versus young hippocampal OPC nuclei. The dashed line represents Adj = 0.05 n = 4). FC, fold change. FIG. 3D shows pathways enriched (red) or depleted (blue) in hippocampal OPCs with age. Resource categories: #, CellMarker; ##, WikiPathways; ###, Gene Ontology Biological Processes (n = 4; numbers to the right of the bar represent number of genes identified in the category; unweighted Kolmogorov-Smimow test). FIG. 3E shows box plots of the effect size of SRF targets (TRANSFAC database) in hippocampal OPCs from aged versus young mice and mice treated with YM-CSF versus aCSF at 1- and 6-h time points n = 4; genes prefiltered by a cut-off of P < 0.05, Wilcoxon rank-sum test; boxes show the median and the 25th— 75th percentile range, and whiskers indicate values up to 1.5 times the interquartile range; dashed line indicates effect size = 0). FIG. 3F shows pathways enriched (red) or depleted (blue) in hippocampal OPCs 6 h after injection with aCSF or YM-CSF (n = 4). Resource categories: #, KEGG; ##, Gene Ontology Molecular Function (numbers to the right of the bar represent number of genes identified in the category; unweighted Kolmogorov-Smimow test). FIG. 3G shows meta-analysis of the log2-transformed fold change (log2FC) in expression of SRF target genes (TRANSFAC) in human AD versus control and mouse aged versus young ageing datasets (genes prefiltered by a cut-off of P < 0.05; boxes show the median and 25th— 75th percentile range, and whiskers indicate values up to 1.5 times the interquartile range; dashed line indicates normalized log2FC = 0).
FIGS. 4A-4S show that Fgfl7 induces OPC proliferation and improves memory. FIG. 4A is a diagram of the SRE-GFP reporter in HEK293 cells. FIG. 4B shows SRE-GFP activation by CSF ligands (500 ng ml-1; n = 3; one-way ANOVA with Dunnett’s multiple-comparisons test; mean ± s.e.m.). Conf., confluence; NS, not significant. FIG. 4C shows dose-dependent activation of the SRE-GFP reporter by Fgfl7 (n = 3; one-way ANOVA with Tukey’s post hoc test; mean ± s.e.m.). FIG. 4D is representative images of the experiment in FIG. 4C. Scale bars, 400 pm. FIG. 4E is meta-analysis of FGF17 levels in healthy human CSF (ages 20-40 years, n = 30; ages 40-60 years, n = 23; ages 60-85 years, n = 36; oneway ANOVA with Dunnett’s post hoc test; mean ± s.e.m.). RFU, relative fluorescence units. FIG. 4F shows the number of Fgfl7+ puncta in the cortex and hippocampus of young (3 months) and aged (25 months) mice (young, n = 3; aged, n = 2; mean ± s.e.m.). Ctx, cortex; DG, dentate gyrus; Hipp, hippocampus. FIG. 4G is representative images of the experiment in FIG. 4F. Scale bars, 5 pm. FIG. 4H shows the percentage of proliferating OPCs treated with Fgfl7 under proliferation conditions (n = 3; oneway ANOVA with Tukey’s post hoc test; mean ± s.e.m.). FIG. 41 is representative images of the experiment in FIG. 4H. Scale bars, 20 pm. FIG. 4J shows MBP intensity per area in OPCs treated with Fgfl7 under differentiation conditions (day 3) n = 4; one-way ANOVA with Tukey’s post hoc test; mean ± s.e.m.). FIG. 4K is representative images of the experiment in FIG. 4H. Scale bars, 20 pm. FIG. 4L shows the quantification of proliferating OPCs in the hippocampus of 20-month-old mice following 1 week of infusion with aCSF or Fgfl7 (aCSF, n = 8; Fgfl 7, // = 6; two-sided /test; mean ± s.e.m.). FIG. 4M is representative images of the experiment in 1. Arrowheads point to proliferating OPCs. Scale bars, 50 pm. FIG. 4N is the percentage of freezing of 20-month-old mice in the remote recall contextual fear conditioning test (aCSF, n = 10; Fgfl7, n = 11; two-sided t test; mean ± s.e.m.). FIG. 40 is the percentage of freezing of 3 -month-old mice in the short-term contextual fear conditioning (CFC) test (// = 10; onesided t test; mean ± s.e.m.). FIG. 4P is the percentage of entries to the novel arm of the forced-alternation Y maze (n = 10; one-sided / test; mean ± s.e.m.). FIG. 4Q shows the average number of active c-Fos+ cells in the dentate gyrus (IgG, n = 9; anti-Fgfl7, n = 10; two-sided t test; mean ± s.e.m.). FIG. 4R is representative images of the experiment in FIG. 4Q. Scale bars, 100 pm. FIG. 4S shows the percentage of proliferating OPCs treated with aCSF, YH-CSF or Fgfl7 in combination with IgG or anti-Fgfl7 antibodies (H = 3; two-way ANOVA with Sidak’ s post hoc test; mean ± s.e.m.).
FIGS. 5A-5C show that Fgfl7 is predominantly expressed in the brain by a subset of neurons and choroid plexus epithelial cells. FIG. 5 A shows that Fgfl7 is predominantly expressed in the brain based on the human protein atlas. FIG. 5B shows that Fgfl7 is lowly expressed by neurons but not glial cells in the adult human cortex (Allen Brain Atlas). FIG. 5C shows that Fgfl7 is lowly expressed by neurons and choroid plexus epithelial cells in a human COVID19 brain and choroid plexus dataset.
FIGS. 6A-6I show the bulk RNAseq, infusion site details and overall overview of proliferating cells. FIG. 6A shows the relative proportions of cell types as predicted by deconvolution analysis of bulk RNAseq of aged mice infused with aCSF or YM-CSF (aCSF n = 8, YM-CSF n = 7). FIG. 6B shows the predicted number of DEGs per cell type by deconvolution analysis of bulk RNAseq of aged mice infused with aCSF or YM-CSF (aCSF n = 8, YM-CSF n = 7). FIG. 6C shows the effect size of the subset of oligodendrocyte genes in FIG. ID 16 h following acute injection of YM-CSF or aged mouse CSF (AM- CSF) calculated over aCSF as control (n = 4; Wilcoxon rank sum test). FIG. 6D shows the location of the infusion site. Image source: Allen Institute, Mouse brain atlas (coronal). FIG. 6E shows the location of the analysis site. Image source: Allen Institute, Mouse brain atlas (coronal). FIG. 6F is a hippocampal slice of 10-month-old mice given an EdU pulse prior to surgery showing low baseline proliferation, and three pulses of BrdU at day 5 and 6 of infusion showing an overall increase in proliferating cells following YM-CSF infusion (n = 4 per group; repeated measures two-way ANOVA followed by Sidak’ s post-hoc test; Means ± SEM). FIG. 6G is representative images of EdU (red) and BrdU (green) cells in mice with no surgery or infused with aCSF or YM-CSF. Scale bar, 500 pm. FIG. 6H is RNAscope of Pdgfra+EdU+ cells in hippocampus of 2-month-old (young) and 19-month-old (aged) mice (n = 3; two-sided t-test; mean ± s.e.m.). FIG. 61 is representative images of analysis in panel FIG. 6H. Arrows pointing to Pdgfra+EdU+ cells. Scale bar, 100 pm.
FIGS. 7A-7I show cortical Pdgfra+EdU+ cells and identity of Pdgfra- EDU+ cells. FIG. 7A shows hippocampal density of Pdgfra+ EdU+ cells per mm2 (aCSF n = 7, YM-CSF n = 8; two-sided t-test; mean ± s.e.m.). FIG. 7B shows hippocampal density of Pdgfra+ cells per mm2 (aCSF n = 7, YM-CSF n = 8; two-sided t-test; mean ± s.e.m.). FIG. 7C shows location of region of interest in the cortex. Scale bar, 100 pm. FIG. 7D shows the percentage of Pdgfra+ EdU+ 1 Pdgfra+ cells showing very low proliferation rates of OPCs in the cortex (n = 6; two-sided t-test; mean ± s.e.m.). FIG. 7E shows the cortical density of Pdgfra+ EdU+ cells per mm2 (n = 6; two-sided t-test; mean ± s.e.m.). FIG. 7F shows the cortical density of Pdgfra+ cells per mm2 (n = 6; two-sided t-test; mean ± s.e.m.). FIG. 7G shows the percentage of Pdgfra+ EdU+ / EdU+ in the hippocampus of aged mice infused with YM-CSF
(n = 3). FIG. 7H shows exemplary IBA+ EdU+ cells in the hippocampus (n = 3). Scale bar, 50 pm. Insert, 10 pm. FIG. 71 shows exemplary GFAP+ EdU+ cells in the hippocampus (n = 3). Scale bar, 50 pm. Insert, 10 pm.
FIGS. 8A-8E show young CSF increases number of myelinated axons in the molecular layer. FIG. 8A is a representative overview of 1mm diameter biopsy punch in the hippocampus. FIG. 8B is a representative overview of molecular layer (MoL, between dashed lines) before and after TEM imaging of three 10x10 montage squares (n=7). FIG. 8C is a representative montage of MoL of aged mouse infused with aCSF and YM-CSF (n = 7). Scale bar, 10 pm. FIG. 8D is a representative higher resolution image of aged mouse infused with aCSF and YM-CSF (n = 7). Scale bar, 1 pm. FIG. 8E is a g-ratio analysis of myelinated axons in molecular layer, (n = 3 mice per group, aCSF n = 321 axons, YM-CSF n = 291 axons).
FIGS. 9A-9D show young CSF boosts OPC differentiation in vitro and validation of OPC culture purity. FIG. 9A is images of differentiated cultures related to images in FIG. 10. Overview of MBP stain of OLs at day 4 of differentiation supplemented with 10% aCSF or YH-CSF (aCSF n = 3 coverslips, YH- CSF n = 2 coverslips). FIG. 9B shows the quantification of MBP intensity of day 4 differentiated OLs. Scale bar, 200 pm. (aCSF n = 3 coverslips, YH-CSF n = 2 coverslips; two-sided t-test; mean ± s.e.m.). FIG. 9C is primary rat OPC cultures supplemented with 10% aCSF or YH-CSF for 6 h and stained for NG2 (green), Olig2 (grey) and Acta2 (red), (n = 3 coverslips; Scale bar, 100 pm). FIG. 9D is higher magnification of primary rat OPC cultures supplemented with 10% aCSF or YH-CSF for 6 h and stained for NG2 (green), Olig2 (grey) and Acta2 (red), (n = 3 coverslips; Scale bar, 20 pm).
FIGS. 10A-10G show SLAMseq QC and principal component analysis. FIG. 10A shows the overall conversion rates in all SLAMseq samples, showing an enrichment for T>C mutation rate (orange bar) which increases with longer incubation time (6 h). FIGS. 10B and 10C is the distribution of T>C mutations across read position (FIG. 10B) and 3’UTR position indicating an equal distribution of s4U incorporation along the positive strand (FIG. 10C). FIGS. 10D and 10E are UMAP of aCSF and YH-CSF samples in both time points by all genes detected in the total (FIG. 10D) and nascent (FIG. 10E) mRNA counts, (young CSF 1 h n = 4, all the rest n = 5). FIG. 10F is gene set enrichment analysis (GSEA) of 6hr genes sorted by log2-transformed fold change (log2FC) showing an enrichment for SRF target genes by TRANSFAC. FIG. 10G shows the overall log2FC enrichment indicating upregulation of SRF target genes (TRANSFAC and curated list) and actin cytoskeleton genes in YH-CSF treated OPCs over aCSF. (SRF TRANSFAC - 423 genes), validated SRF targets from literature (74 genes) and actin genes (212 genes); Wilcoxon rank sum test; box show the median and the 25— 75th percentiles, and the whiskers indicate values up to 1.5-times the interquartile range).
FIGS. 11 A-l II show YH-CSF induces actin cytoskeleton alterations in vitro. FIGS. 11 A and 1 IB show actin filament content measured by live imaging using SiR-actin (red) throughout 4hr of aCSF and YH-CSF exposure. Average SiR-actin intensity (FIG. 11 A) and area (FIG. 1 IB) in rat OPC cultures exposed to aCSF or YH-CSF (n = 6 wells per condition; Means ± SEM). FIG. 11C is representative images of experiment quantified in FIGS. 11 A and 1 IB. Scale bar 200 pm. FIG. 1 ID shows OPC coverslips treated with YH-CSF for 6 h and stained for phalloidin. Histogram of the percentage of OPC with the indicated number of growth cones per cell. YH-CSF treated cells show a shift towards more growth cones per cell (n = 3 coverslips per condition, total of 200 cells analyzed per condition; two-way ANOVA followed by Sidak’s post-hoc test; Means ± SEM). Scale bar 20 pm. FIG. 1 IE is mouse OPC primary cultures from SRF-fl/fl pups infected with CRE-GFP and ACRE-GFP AAVs to induce recombination. Representative images of infected cells (green) 48 h after infection. Scale bar, 100 pm. FIG. 1 IF shows normalized SRF mRNA levels as measured by RT-PCR (n = 3 coverslips per condition; mean ± s.e.m.). FIG. 11G is representative images of data presented in FIG. 2H. Scale bar, 20 pm. FIG. 11H is quantification of GFP+ cells per image in SRF-WT and SRF-KO cells treated with 10% aCSF or YH-CSF. (n = 3; mean ± s.e.m.). FIG. 1 II is quantification of number of DAPI cells per image in SRF-WT and SRF-KO cells treated with 10% aCSF or YH-CSF. (n = 3; mean ± s.e.m.). Data was replicated in two independent experiments.
FIGS. 12A-12D show bulk RNAseq of hippocampal OPC and OL nuclei from young and aged mice. FIG. 12A shows the gating strategy for sorting of hippocampal OPC and OL nuclei. FIG. 12B is a heatmap of expression OPC and OL specific genes across young and aged OPC and OL samples (aged OL n = 3, rest n = 4). FIG. 12C is a volcano plot showing OL genes up and downregulated with age (n = 4; p. adjusted value by Wald test in DESeq2). FIG. 12D shows pathways enriched (red) or depleted (blue) in hippocampal OLs with age (unweighted Kolmogorov- Smimow test).
FIGS. 13A-13H show bulk RNAseq of hippocampal OPC and OL nuclei from aged mice following acute injection and Srf levels in neurons. FIG. 13A is a box plot of effect size of Srf targets (TRANSFAC database) in hippocampal OLs from aged vs. young, YM-CSF vs. aCSF at 1 h and 6 h timepoints (n = 4; genes pre-filtered by p < 0.05 cutoff; Wilcoxon rank sum test, box show the median and the 25— 75th percentiles, and the whiskers indicate values up to 1.5-times the interquartile range). FIG. 13B shows pathways enriched (red) or depleted (blue) in hippocampal OPCs Ihr following injection of aCSF or YM-CSF (n = 4; p. adjusted value by Wald test in DESeq2). FIG. 13C is a volcano plot showing OPC genes up and down regulated Ihr following CSF injection (n = 4; p. adjusted value by Wald test in DESeq2). FIG. 13D is a volcano plot showing OPC genes up and down regulated 6hr following CSF injection (n = 4; p. adjusted value by Wald test in DESeq2). FIG. 13E shows neuronal Srf intensity in CAI in young and aged mice, (n = 3; two-sided t-test; mean ± s.e.m.). FIG. 13F is representative images of FIG. 13E. Scale bar, 70 pm. FIG. 13G is neuronal Srf intensity in CAI in aged mice following YM- CSF infusion. (n = 4; two-sided t-test; mean ± s.e.m.). FIG. 13H is representative images of FIG. 13G. Scale bar, 70 pm.
FIGS. 14A-14F show that Fgf8 induces OPC proliferation and Fgfl7 induces SRF reporter activation mediated by actin dynamics and Fgfr3. FIG. 14A is dose-dependent activation of SRE-GFP reporter by increasing concentrations of Fgf8 and representative images of the experiment at 15.5 h. Scale bar, 400 pm. (n = 3; similar control as in FIG. 4C; one-way ANOVA followed by Sidak’s post-hoc test; mean ± s.e.m.). FIG. 14B shows the percentage of BRDU+/DAPI primary rat OPCs treated with 10, 20, 40 ng/ml Fgf8. (n = 4; one-way ANOVA followed by Tukey’s post-hoc test; mean ± s.e.m.). FIG. 14C is the quantification of OPC proliferating cells (Pdgfra+EDU+ / Pdgfra+ cells) in the CAI region of the hippocampus of 20-month-old mice following a week of aCSF or Fgf8 infusion. (aCSF n = 8 similar control as in FIG. 4L, Fgf8 n = 4; two-sided t-test; mean ± s.e.m.). FIG. 14D shows SRE-GFP activation with 200 ng/ml Fgfl7 following 30 min pre-treatment with Jasplakinolide (Jasp, 125 or 250 nM) or Latrunculin A (LatA, 250 or 500 nM). (n = 3; Two-way ANOVA with Tukey’s multiple comparisons test; mean ± s.e.m.). FIG. 14E shows SRE-GFP activation with 200 ng/ml Fgfl7 following 30 min pretreatment with blocking antibodies for FgfRl, FgfR2, FgfR3 (all 50 pg/ml) or FgfR3 alone (n = 3; Oneway ANOVA with Sidak’s multiple comparisons test; mean ± s.e.m.). FIG. 14F shows exemplary Pdgfra+ Fgfr3+ cells in the hippocampus of young mice, (n = 3). Scale bar, 5 pm.
FIGS. 15A-15H show Fgfl7 is predominantly expressed in the brain by a subset of neurons and is downregulated with age. FIG. 15A shows Fgfl7 is expressed by cortical glutamatergic neurons in the young adult mouse (Allen brain atlas). FIG. 15B shows sub-clustering of mouse cortical layer 4/5 neurons indicates expression by a subset of cortical neurons (Allen brain atlas). FIG. 15C is gene set enrichment analysis of genes mostly correlated with Fgfl7 in layer 4/5 neurons (Allen brain atlas). FIG. 15D shows Fgfl7 is expressed by cortical glutamatergic and GABAergic neurons in the human cortex (Allen brain atlas). FIG. 15E is a representative image of analysis in FIG. 15F. Scale bar, 100 pm. FIG. 15F shows Fgfl7 mRNA expression in cortical neurons drops dramatically in aged mice, (n = 3; two-way student t- test; mean ± s.e.m.). FIG. 15G shows Fgfl7 protein expression in cortical and hippocampal neurons drops dramatically in aged mice, (n = 3; mean ± s.e.m.). FIG. 15H is representative images of analysis in FIGS. 15G and 4F. Scale bar, 20 pm.
FIGS. 16A-16G show perfusion of labeled YH-CSF and mouse Fgfl7 to the brain parenchyma and working model. FIG. 16A shows deposition of labeled Fgfl7 on ventricular walls 3 h post ICV acute injection (n = 3). Scale bar, 300 pm. FIG. 16B shows deposition of labeled YH-CSF on lateral ventricle walls 2 h post ICV acute injection (n = 3). Scale bar, 100 pm. FIG. 16C shows labeled Fgfl7 in perivascular spaces in the molecular layer of the hippocampus (n = 3). Scale bar, 50 pm. FIG. 16D shows labeled YH-CSF in perivascular spaces in the molecular layer of the hippocampus (n = 3). Scale bar, 20 pm. FIG. 16E shows YH-CSF in the perivascular space in between the vessel (green) and astrocyte endfeet (white; n = 3). Scale bar, 20 pm. FIG. 16F is an orthogonal slice of YH-CSF (magenta) in perivascular space, in between the vessel (green) and astrocyte endfeet (white; n = 3). Scale bar, 20 pm. FIG. 16G is a schematic of a working model. OPC proliferation and differentiation (termed oligodendrogenesis) slow down with age. Re-exposure of the aged brain to young CSF or the brainspecific growth factor Fgfl 7, boost hippocampal oligodendorgenesis, concomitant with improvement in long term memory recall.
FIGS. 17A and 17B show a subset of Srf targets present in CSF proteomic datasets. FIG. 17A provides exemplary proteins tested in the SRE reported assay (related to FIGS. 4A and 4B). FIG. 17B provides exemplary Srf targets in CSF datasets that were not tested in the SRE reporter assay. DEFINITIONS
To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below:
As used herein, the term “subject” refers to any animal (e.g, a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
As used herein, the term “non-human animals” refers to all non-human animals including, but not limited to, vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.
As used herein, the term “cell culture” refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro. As used herein, the term “zzz vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell culture. The term “/// vivo” refers to the natural environment (e.g, an animal or a cell) and to processes or reaction that occur within a natural environment.
The terms “test compound” and “candidate compound” refer to any chemical entity, pharmaceutical, drug, and the like that is a candidate for use to treat or prevent a disease, illness, sickness, or disorder of bodily function (e.g., Alzheimer’s disease, Parkinson’s disease, atherosclerosis, cancer). Test compounds comprise both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by screening using the screening methods of the present disclosure.
As used herein, the term “sample” is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum, and the like. Environmental samples include environmental material such as surface matter, soil, water, and industrial samples. Such examples are not however to be construed as limiting the sample types applicable to the present disclosure.
As used herein, the term “effective amount” refers to the amount of a compound (e.g., a compound described herein) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not limited to or intended to be limited to a particular formulation or administration route.
As used herein, the term “co-administration” refers to the administration of at least two agent(s) or therapies to a subject. In some embodiments, the co-administration of two or more agents/therapies is concurrent. In other embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various agents/therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents/therapies are co-administered, the respective agents/therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents/therapies lowers the requisite dosage of a known potentially harmful (e.g, toxic) agent(s).
As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo, or ex vivo. As used herein, the term “antigen binding agent (e.g., “antigen-binding protein” or protein mimetic such as an aptamer) refers to proteins that bind to a specific antigen. “Antigen-binding proteins” include, but are not limited to, immunoglobulins, including polyclonal, monoclonal, chimeric, single chain, single domain, scFv, minibody, nanobody, and humanized antibodies, Fab fragments, F(ab’)2 fragments, and Fab expression libraries.
As used herein, the term “single-chain variable fragment” (scFv) refers to an antibody fragment that comprises a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin. In some embodiments, the VH and VL are connected with a short linker peptide.
As used herein, the term “minibody” refers to an antibody fragment that retains antigen binding activity. In some embodiments, minobodies comprise an scFv fused to an Fc region e.g., an IgG Fc region).
Various procedures known in the art are used for the production of polyclonal antibodies. For the production of antibody, various host animals can be immunized by injection with the peptide or protein containing the desired epitope including but not limited to rabbits, mice, rats, sheep, goats, llamas, alpacas, etc. In a specific embodiment, the peptide is conjugated to an immunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyhole limpet hemocyanin (KLH)). Various adjuvants are used to increase the immunological response, depending on the host species, including but not limited to Freund’s (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, Gerbu adjuvant and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.
For preparation of monoclonal antibodies, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used (See e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). These include, but are not limited to, the hybridoma technique originally developed by Kohler and Milstein (Kohler and Milstein, Nature, 256:495-497 [1975]), as well as the trioma technique, the human B-cell hybridoma technique (See e.g., Kozbor et al., Immunol. Today, 4:72 [1983]), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 [1985]). In other embodiments, suitable monoclonal antibodies, including recombinant chimeric monoclonal antibodies and chimeric monoclonal antibody fusion proteins are prepared as described herein. According to the invention, techniques described for the production of single chain antibodies (US 4,946,778; herein incorporated by reference) can be adapted to produce specific single chain antibodies as desired. An additional embodiment of the invention utilizes the techniques known in the art for the construction of Fab expression libraries (e.g., Huse et al., Science, 246:1275-1281 [1989]) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
In some embodiments, monoclonal antibodies are generated using the ABL-MYC method (See e.g., U.S. Patent 5,705,150 and 5,244,656, each of which is herein incorporated by reference) (Neoclone, Madison, WI). ABL-MYC is a recombinant retrovirus that constitutively expresses v-abl and c-myc oncogenes. When used to infect antigen-activated splenocytes, this retroviral system rapidly induces antigen-specific plasmacytomas. ABL-MYC targets antigen-stimulated (Ag-stimulated) B-cells for transformation.
In some embodiments, biopanning as described in Pardon etal., Nat Protoc. 2014 Mar;9(3):674- 93 is used to generate single domain antibodies. In some embodiments, to generate murine scFv units, phage-based biopanning strategies, of which there are several published protocols available, are used.
Antibody fragments that contain the idiotype (antigen binding region) of the antibody molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab’)2 fragment that can be produced by pepsin digestion of an antibody molecule; the Fab’ fragments that can be generated by reducing the disulfide bridges of an F(ab’)2 fragment, and the Fab fragments that can be generated by treating an antibody molecule with papain and a reducing agent.
Genes encoding antigen-binding proteins can be isolated by methods known in the art. In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art (e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), Western Blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein A assays, phage display biopanning, and immunoelectrophoresis assays, etc.).
As used herein, the term “toxic” refers to any detrimental or harmful effects on a cell or tissue as compared to the same cell or tissue prior to the administration of the toxicant.
DETAILED DESCRIPTION OF THE DISCLOSURE Provided herein are compositions and method for treating cells, tissues, and subjects to improve memory in the aging brain, and to study or to treat age-related diseases and conditions associated with memory loss. In particular, provided herein are compositions, methods, systems, kits and uses for delivery of Fibroblast growth factor 17 (Fgf 17) to restore memory and to rejuvenate oligodendrocyte progenitor cells.
Brain aging underlies dementia and neurodegenerative diseases, imposing an immense societal burden. Systemic interventions in model organisms have shown great promise in reversing aging related decline of various tissues, including the brain. For example, heterochronic parabiosis and young plasma transfer rejuvenates the aged brain and restores memory function. Nevertheless, the brain is protected with barriers which may limit access of these factors, presumably impeding their rejuvenation potential. Cerebrospinal fluid (CSF), which is in close association with brain cells, carries signals that instruct neuronal progenitor proliferation and specification during development. However, CSF protein composition changes with human aging, marked by an increase in inflammatory proteins and decrease in growth factors such as BDNF. Whether these CSF changes contribute to age-related cognitive decline is unknown. Testing this functionally by performing CSF transfers in vivo has been difficult due to technical limitations in CSF collection and direct CSF infusion to the brain. In experiments conducted in development of the present invention, intracerebroventricular (i.c.v.) administration of young CSF to aged mice has rejuvenating effects on the brain (FIG. 1 A).
First tested was if young CSF infusion to aged mice improves aging-related impairments in hippocampal dependent learning and memory tasks. Twenty-month-old mice received 3 foot shocks associated with a tone and a flashing light. Mice were then randomly split into 2 groups and infused with either artificial CSF (aCSF) or young mouse CSF (YM-CSF) for 1 week and remote memory recall was tested 3 weeks after memory acquisition. YM-CSF infusion resulted in higher average freezing rates following exposure to the tone and light, indicating improved preservation of remote fear memory (FIG. IB). This paradigm allowed testing of interventions that affect remote memory formation and consolidation, a process implicated in ageing-related cognitive decline. Aged mice infused with aCSF had comparable freezing rates to age-matched naive mice (aCSF, 17.82% ± 0.93; naive, 17.73% ± 0.93; mean + s.e.m.). Because the hippocampus is central to age-related cognitive decline and is in close apposition to CSF, the effect of young CSF infusion on its transcriptome was measured by RNA sequencing. Differential gene expression analysis between aCSF and YM-CSF identified 271 differentially-expressed genes (DEGs) significantly altered (FDRO. l) after young CSF treatment with 115 down- and 156 upregulated. Oligodendrocyte genes were highly upregulated identifying this cell type as a major cellular substrate for CSF (FIGS. 1C and 6A-6B). Specifically, young CSF promoted upregulation of transcription factors driving oligodendrocyte differentiation and maj or myelin protein components (for example, Oligl, Myrf, Mag, Mbp and Mobp) (FIG. ID).
Neuronal activity, induced by optogenetic tools or by learning tasks, promotes OPC proliferation and differentiation, and regulates myelin plasticity in mature oligodendrocytes (termed activitydependent myelination. To determine whether OPC proliferation underlies the transcriptomic signature observed following young CSF infusion, dividing cells were labeled in the last 2 days of young CSF infusion with the thymidine analogue EdU. While overall cell proliferation was very low in the aged brains, a surge in overall cell proliferation specifically in the hippocampus relatively distant from the infusion site was discovered (FIGS. 6D-6I). Young CSF induced a 2.35-fold increase in the percentage of proliferating OPCs (EdU+ PdgfraV Pdgfra+) in the CAI region of the hippocampus, but not in the cortex (FIGS. 1E-1F and 6D-6I). In an additional cohort of mice, aged mice were infused with human CSF pooled from healthy young donors (YH-CSF, mean age of 24.6 years) or aged donors (AH-CSF, mean age of 69 years). YH-CSF induced OPC proliferation at comparable levels to YM-CSF, whereas AH-CSF induced proliferation for only half as many cells (FIGS. 1G and 1H). CSF infusions also triggered EdU incorporation in astrocytes and microglia (FIGS. 7G-7I). Next, the proliferating cells were allowed to mature for 3 weeks to assess effects of young CSF on hippocampal myelination by MBP staining and transmission electron microscopy. Increases in MBP intensity in the molecular layer of the hippocampus (FIGS. II and 1 J) and in the number of myelinated axons (FIGS. IK and 8) in the molecular layer of the hippocampus were observed. These data indicate indicates that young CSF contains signals that promote cell growth for the oligodendrocyte lineage and/or substances that neutralize inhibitory factors.
To assess whether young CSF can stimulate OPC proliferation and differentiation directly, an established primary rat OPC culture system was used. Cells were grown in 90% full proliferation media supplemented with 10% human CSF pools (3 pools as replicates, each pool from 3 young adult healthy males (YH-CSF; mean age 24.6) or aCSF as control). Human instead of rodent CSF was used because larger volumes of human CSF are available. Similar to the in vivo infusions, BrdU pulsing confirmed a dose-dependent increase in OPC proliferation (FIGS. IL and IM). When OPCs were deprived of mitogens to promote cell differentiation over 4 days, YH-CSF not only induced a 2-fold increase in cell survival, a process in which a significant fraction of cells typically undergo apoptotic cell death, but also promoted a prominent expansion of the more differentiated mature cell morphology with an overall increase in MBP intensity per cell (FIGS. IN, 10, 9A and 9B). These data show that human CSF from young healthy donors induces neuronal viability while CSF from multiple sclerosis patients is toxic to neuronal and OPC cultures.
To test the cellular processes induced by young CSF in OPCs nascent mRNA was metabolically labeled with 4-thiouridine (s4U) using thiol(SH)-linked alkylation and sequenced RNA (SLAMseq) from cultured OPCs 1- or 6 hr after exposure to YH-CSF. The top gene induced after 1 hr was Serum Response Factor (Srf) (FIG. 2A), a transcription factor in skeletal muscle, heart and in neurons in the brain. Srf binds to serum response element (SRE) promoter sequences to induce cell motility, proliferation, and differentiation through modulation of immediate early genes (such as Egrf) and the actin cytoskeleton. A dramatic downregulation of the negative regulator of Wnt signaling and pro-apoptotic factor Bcl7b as well as the DNA repair protein Rpa3 was noted, in keeping with an overall pro-survival response following YH-CSF exposure. Nascent Srf mRNA transcripts peaked at 1 hr and returned to baseline by 6 hr (FIG. 2B). Many of the DEGs peaking at 6 hr are known Srf target genes (FIG. 2C, red). The most strongly increased genes were enriched as “target genes of SRF” based on gene set enrichment analysis (GSEA) and the TRANSFAC database (FIG. 10F), while the cells maintained OPC identity as determined by NG2 expression (FIGS. 9C and9D). The combined log2-transformed fold change(log2FC) in the TRANSFAC -predicted targets for SRF (423 genes), validated SRF targets (74 genes, curated list from literature) and actin genes (212 genes) in the dataset indicating overall activation of the SRF pathway and actin cytoskeleton transcripts (FIG. 10G). To test whether CSF- induced SRF expression regulates the OPC actin cytoskeleton, OPCs were exposed to YH-CSF in the presence of SiR-actin, a fluorescent probe used to label actin filaments for live imaging, or OPCs were fixed and stained with the actin filament dye phalloidin. SiR-actin intensity increased within hours of OPC stimulation with YH-CSF, without a change in total area, indicating an increase in cellular actin filament levels (FIGS. 11 A-l 1C). In fixed cells, OPCs exposed to YH-CSF for 6 h expressed twice as much phalloidin per cell as controls (FIGS. 2D and 2E). The increase in phalloidin intensity was confirmed in hippocampal OPCs in aged mice infused with YM-CSF for 6 d (FIGS. 2F and 2G).
Because SRF is necessary for the formation of actin filaments in axonal growth cones in neurons it was tested whether SRF has a similar role in OPCs and quantified the number of growth cones per OPC. YH-CSF induced significantly more growth cones per cell compared with aCSF (FIG. 1 ID). To test whether SRF mediates the effect of young CSF, cultured OPCs from mice with /oxP-flanked SrfwQXQ infected with adeno-associated viruses (AAVs) encoding Cre-GFP (to create SRF-knockout (SRF -KO) OPCs) or truncated Cre-GFP as a control (SRF-WT). The YH-CSF proliferation experiment was repeated and showed that CSF-induced proliferation is dependent on SRF (FIGS. 7H, 71, and 1 IE- 1 II). These results denote SRF and actin cytoskeleton regulation as potential mediators of the effects of young CSF in vivo.
Because deletion of SRF signaling in muscle cells leads to accelerated aging phenotypes in skeletal muscle of mice and worms, whether SRF signaling is downregulated in OPCs in the aging brain was tested. The fraction of SRF positive oligodendrocytes (SrE Pdgfra+/Pdgfra+) in the CAI region of the hippocampus detected by in situ hybridization decreased dramatically with age (FIGS. 3 A and 3B). To expand this analysis to other SRF targets and cellular processes, hippocampal OPC and OL nuclei were sorted by expression of the oligodendrocyte transcription factor Olig2 (Olig2high for OPCs and Olig2low for OLs) from young (3-month-old) and aged (25-month old) mice and performed bulk RNAseq (FIGS. 12A and 12B). The top pathways downregulated in OPCs with aging were related to oligodendrocyte cell markers, regulation of glial cell differentiation, cellular respiration and metabolism and protein folding. Conversely, immune related pathways and microglial specific genes were upregulated as previously reported for OPCs in aging and multiple sclerosis (FIGS. 3C, 3D, 12C, and 12D). A focused analysis of SRF TRANSFAC target genes in aging OPCs indicated overall downregulation with age (FIG. 3E, left box plot).
Next tested was if young CSF induces SRF pathway activation in vivo in the aged brain. Because of the transient nature of SRF induction in the SLAMseq experiment, an acute injection paradigm was designed wherein CSF was injected into the lateral ventricle of 18 month-old mice, and contralateral hippocampi were dissected 1 and 6 hours post injection for RNAseq of OPC and OL nuclei. Genes predicted to be targets of SRF (on the basis of TRANSFAC) were upregulated in POCs at both timepoints (FIGS. 3E and 13A). At the 6 hour time point, genes upregulated in OPCs were linked to SRF-related pathways such as “regulation of GTPase activity,” “chromatin organization,” “transcription factor binding,” “cell cycle,” and “regulation of cytoskeleton organization,” respectively (FIGS. 3F and 13B- 13D). SRF target genes were also downregulated in other published human and murine datasets of OPCs in aging and Alzheimer’s disease (AD) (FIG. 3G). Srf mRNA levels in neurons decreased with ageing and were not changed following acute injection with CSF (FIGS. 13E-13H). These experiments indicate that SRF signaling is downregulated with aging and induced following acute injection of young CSF in vivo. CSF contains hundreds of proteins that could potentially induce SRF signaling. Several SRF target genes are also upstream inducers of SRF itself such as BDNF and IGF1. Based on these data, 2 published CSF proteomic datasets were cross-referenced with the list of TRANSF AC-predicted SRF targets and a list of 35 potential SRF inducers was generated (FIGS. 17A-17B). To test the activity of these candidate, HEK293 cells were transfected with a SRE-GFP reporter and proteins were added at different concentrations (FIG. 4A). Fibroblast growth factor 8 (Fgf8) and Fgfl7 induced the strongest dosedependent responses (FIGS. 4B-4D and 14A-14B). Fgfl7 was explored because it is a brain enriched protein (FIG. 16A), and its levels decrease with age in human CSF (FIG. 4E) and in human plasma in males and females and in mouse neurons (FIGS. 4F, 4G, and 15A-H). Actin polymerization was enhanced or inhibited using jasplakinolide and latrunculin A, respectively, and Fgfl7 activated SRF signaling through actin modulation (FIG. 14D), indicating that Fgfl7 activates the SRF pathway through Rho GTPase activation and modulation of actin dynamics. The reporter experiment was performed in the presence of blocking antibodies for Fgfrl, Fgfr2 or Fgfr3 and found that Fgfl7 reporter activation was dependent on signaling through Fgfr3 (FIG. 14E). While Fgfr3 is very highly expressed by astrocytes, a fraction of Fgfr3 -positive hippocampal OPCs were also detected (FIG. 14F).
When added to primary rat OPCs, Fgfl7 (40 ng/ml) induced OPC proliferation (FIGS. 4H-4I) and differentiation (FIGS. 4J and 4K). Earlier work with cultured OPCs suggested that Fgfl7 slightly promoted proliferation, although it may inhibit OPC differentiation in some contexts. To determine the in vivo activity of Fgf8 and Fgfl7 recombinant proteins were infused over 7 days similar to administration of CSF (FIG. 1). Fgfl7 induced OPC proliferation in the aged hippocampus, but Fgf8 did not (FIGS. 4L, 4M and 14C). The effect of Fgfl7 infusion on cognition was tested and it was found that it improves long-term memory performance in the remote memory recall paradigm described in FIG. 1 (FIG. 4N). This evidence demonstrates that Fgfl7 is sufficient to mimic the effect of young CSF on OPCs in the hippocampus and memory consolidation in aged mice.
Young mice were infused with an anti-Fgfl7 blocking antibody ICV to test whether Fgfl7 is necessary for normal memory function. Mice infused with anti-Fgfl7, but not with control antibody, showed impaired performance in 2 hippocampal-dependent cognitive tests (Y maze and contextual fear conditioning; FIGS. 40 and 4P), and impaired neuronal plasticity measured by lower c-Fos levels in dentate gyrus granule cells following behavioral tests (FIGS. 4Q and 4R). In OPC cultures, the same concentration of anti-Fgfl7 antibody inhibited OPC proliferation induced by young CSF or Fgfl7. These data indicate that the boost in proliferation is in part mediated by Fgfl7 (FIG. 4S). Taken together, these experiments link Fgfl7 and cognitive function in young and aged mice.
In experiments conducted in the development of the present invention, it was shown that hippocampal OPCs proliferate and differentiate together with an increase in methylation in response to young CSF-derived cues in the aged brain, and generate beneficial effects on remote memory consolidation. Young CSF induces expression of the transcription factor SRF, and its actin cytoskeleton target genes to promote OPC proliferation. SRF is a versatile regulator of neuronal development, activity dependent plasticity and regeneration. SRF is widely expressed by OPCs wherein it is downregulated with aging. Fgfl7, a growth factor decreased in human CSF with aging, induces OPC proliferation in the aged brain. Fgfl7, whose levels decrease with age in mouse neurons and in human CSF, is sufficient and necessary to improve cognition in aged mice and promotes OPC proliferation in vivo and in vitro, suggesting that it constitutes a major component of the rejuvenating effects of young CSF (FIG. 16G).
The CSF proteome comprises proteins secreted by the choroid plexus or transferred through it from the blood plasma, as well as proteins secreted from parenchymal and immune cells. During aging, improper signaling cues derived from the aging choroid plexus led to neuronal stem cell quiescence with aging. OPCs, which account for the largest population of stem cells in the aged brain have been less extensively investigated. Studies in young rodents reveal that oligodendrogenesis, the formation of myelinating oligodendrocytes from OPCs, facilitates consolidation of newly formed memories, implicating their active role in cognitive function. Hippocampal oligodendrogenesis is inhibited with age and boosting it improves performance in learning and memory tasks in aged mice and in AD mouse models in keeping with data showing that aged OPCs in white matter regions, are slow to proliferate and to differentiate following demyelination in diseases such as multiple sclerosis, and that local or systemic environmental manipulations restored their myelination capacity.
A recent study showed that hippocampal oligodendrogenesis is markedly reduced with age and that increasing it was sufficient to improve performance in learning and memory tasks in aged mice and in AD mouse models.
In experiments conducted in the development of the present invention, Fgfl7 was infused into the CSF of aged mice and it recapitulates the effects of young CSF on OPC proliferation and long-term memory recall. Conversely, blocking Fgfl7 by infusing mice with an inhibitory antibody resulted in impaired function in hippocampal-dependent memory tests. In the young adult mouse Fgfl7 is abundantly expressed by cortical neurons and its expression drops markedly with ageing. Fgfr signaling is critical for oligodendrocyte development, with complex and diverse functions in disease processes such as demyelination and remyelination in multiple sclerosis. Specifically, studies using Fg/3r-null mice have shown a delay in the terminal differentiation of pro-oligodendrocytes and transient expression of Fgfr3 in subventricular zone progenitors drives oligodendrogenesis and promotes remyelination following a demyelinating injury. Young mice lacking Fgfl7 have a diversity of social behavior abnormalities coinciding with lower c-fos expressing cells in the prefrontal cortex following a novel social interaction test indicating that Srf, which regulates c-fos expression, may participate in these circuits and in neuropsychiatric disorders. Fgfl7 is critical for normal embryonic brain development but little is certain about its function in the adult nervous system. Searches in transcriptomic and proteomic datasets suggest that Fgfl7 is a brain-derived protein, that in the adult mouse and human brain is expressed by a small subset of cortical neurons and by the choroid plexus epithelial cells immediately accessible to the CSF (FIG. 5). Taken together, these data indicate that targeting hippocampal myelination through factors present in young CSF is a therapeutic strategy to prevent or to rescue cognitive decline associated with aging and neurodegenerative diseases.
In some embodiments, methods and compositions of the present invention comprise de novo peptide targeted therapeutics as described, for example, by Chevalier A. el al. Nature Publishing Group 2017:550;74-79 incorporated by reference herein in its entirety.
In some embodiments, the present disclosure provides peptides that directly or indirectly enhance Fgfl7 function. In certain embodiments, the peptide is an agonist to, for example, the receptor for Fgfl7. The peptide may comprise a fragment or portion of Fgfl7 which binds to the receptor for Fgfl7.
In some embodiments, compositions comprise oligomeric antisense compounds, particularly oligonucleotides used to modulate the function of nucleic acid molecules encoding Fgfl7, ultimately modulating the amount of Fgfl7 expressed. This is accomplished by providing antisense compounds that specifically hybridize with one or more nucleic acids encoding Fgfl7 or that hybridize to a nucleic acid that encodes a specific direct or indirect inhibitor of Fgfl7. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds that specifically hybridize to it is generally referred to as “antisense.” The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity that may be engaged in or facilitated by the RNA. The overall effect of such interference with a target nucleic acid function is modulation of Fgfl7. In the context of the present disclosure, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. For example, Fgfl7 expression may be stimulated to treat or prevent dementia, cognitive impairment, cognitive aging, or a white matter disorder, particularly in an aged subject.
In some embodiments, nucleic acids are small RNAs, for example, siRNAs. “RNA interference (RNAi)” is the process of sequence-specific, post-transcriptional gene silencing initiated by a small interfering RNA (siRNA). During RNAi, siRNA induces degradation of target mRNA with consequent sequence-specific inhibition of gene expression. An “RNA interference,” “RNAi,” “small interfering RNA” or “short interfering RNA” or “siRNA” or “short hairpin RNA” or “shRNA” molecule, or “miRNA” is an RNA duplex of nucleotides that is targeted to a nucleic acid sequence of interest, for example, Fgfl7. As used herein, the term “siRNA” is a generic term that encompasses all possible RNAi triggers. An “RNA duplex” refers to the structure formed by the complementary pairing between two regions of an RNA molecule. siRNA is “targeted” to a gene in that the nucleotide sequence of the duplex portion of the siRNA is complementary to a nucleotide sequence of the targeted gene. In certain embodiments, the siRNAs are targeted to the sequence encoding Fgfl7. In some embodiments, the length of the duplex of siRNAs is less than 30 base pairs. In some embodiments, the duplex can be 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 base pairs in length. In some embodiments, the length of the duplex is 19 to 32 base pairs in length. In certain embodiment, the length of the duplex is 19 or 21 base pairs in length. The RNA duplex portion of the siRNA can be part of a hairpin structure. In addition to the duplex portion, the hairpin structure may contain a loop portion positioned between the two sequences that form the duplex. The loop can vary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides in length. In certain embodiments, the loop is 18 nucleotides in length. The hairpin structure can also contain 3' and/or 5' overhang portions. In some embodiments, the overhang is a 3' and/or a 5' overhang 0, 1, 2, 3, 4 or 5 nucleotides in length.
As used herein, Dicer- substrate RNAs (DsiRNAs) are chemically synthesized asymmetric 25- mer/27-mer duplex RNAs that have increased potency in RNA interference compared to traditional siRNAs. Traditional 21-mer siRNAs are designed to mimic Dicer products and therefore bypass interaction with the enzyme Dicer. Dicer has been recently shown to be a component of RISC and involved with entry of the siRNA duplex into RISC. Dicer- substrate siRNAs are designed to be optimally processed by Dicer and show increased potency by engaging this natural processing pathway. Using this approach, sustained knockdown has been regularly achieved using sub-nanomolar concentrations. (U.S. Pat. No. 8,084,599; Kim et al., Nature Biotechnology 23:222 2005; Rose et al., Nucleic Acids Res., 33:4140 2005).
The transcriptional unit of a “shRNA” is comprised of sense and antisense sequences connected by a loop of unpaired nucleotides. shRNAs are exported from the nucleus by Exportin-5, and once in the cytoplasm, are processed by Dicer to generate functional siRNAs. “miRNAs” stem-loops are comprised of sense and antisense sequences connected by a loop of unpaired nucleotides typically expressed as part of larger primary transcripts (pri -miRNAs), which are excised by the Drosha-DGCR8 complex generating intermediates known as pre-miRNAs, which are subsequently exported from the nucleus by Exportin-5, and once in the cytoplasm, are processed by Dicer to generate functional miRNAs or siRNAs. “Artificial miRNA” or an “artificial miRNA shuttle vector”, as used herein interchangeably, refers to a primary miRNA transcript that has had a region of the duplex stem loop (at least about 9-20 nucleotides) which is excised via Drosha and Dicer processing replaced with the siRNA sequences for the target gene while retaining the structural elements within the stem loop necessary for effective Drosha processing. The term “artificial” arises from the fact the flanking sequences (~35 nucleotides upstream and ~ 40 nucleotides downstream) arise from restriction enzyme sites within the multiple cloning site of the siRNA. As used herein the term “miRNA” encompasses both the naturally occurring miRNA sequences as well as artificially generated miRNA shuttle vectors.
The siRNA can be encoded by a nucleic acid sequence, and the nucleic acid sequence can also include a promoter. The nucleic acid sequence can also include a polyadenylation signal. In some embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal or a sequence of six Ts.
The present disclosure contemplates the use of any genetic manipulation for use in modulating the expression of Fgfl7. Examples of genetic manipulation include, but are not limited to, gene knockout or knock-in (e.g, removing or adding the Fgfl7 gene from the chromosome using, for example, recombination), expression of antisense constructs with or without inducible promoters, and the like. Delivery of nucleic acid construct to cells in vitro or in vivo may be conducted using any suitable method. A suitable method is one that introduces the nucleic acid construct into the cell such that the desired event occurs (e.g, expression of an antisense construct or stimulation of Fgfl7 expression).
Introduction of molecules carrying genetic information into cells is achieved by any of various methods including, but not limited to, directed injection of naked DNA constructs, bombardment with gold particles loaded with said constructs, and macromolecule mediated gene transfer using, for example, liposomes, biopolymers, and the like. Exemplary methods use gene delivery vehicles derived from viruses, including, but not limited to, adenoviruses, retroviruses, vaccinia viruses, and adeno-associated viruses. Because of the higher efficiency as compared to retroviruses, vectors derived from adenoviruses are of use as gene delivery vehicles for transferring nucleic acid molecules into host cells in vivo. Adenoviral vectors have been shown to provide very efficient in vivo gene transfer into a variety of solid tumors in animal models and into human solid tumor xenografts in immune-deficient mice. Examples of adenoviral vectors and methods for gene transfer are described in PCT publications WO 00/12738 and WO 00/09675 and U.S. Pat. Appl. Nos. 6,033,908, 6,019,978, 6,001,557, 5,994,132, 5,994,128, 5,994,106, 5,981,225, 5,885,808, 5,872,154, 5,830,730, and 5,824,544, each of which is herein incorporated by reference in its entirety.
Vectors may be administered to the subject in a variety of ways. For example, in some embodiments of the present disclosure, vectors are administered into nervous system tissue or other tissue associated with aging using direct injection. In other embodiments, administration is via the blood or lymphatic circulation (See e.g., PCT publication 1999/02685 herein incorporated by reference in its entirety). Exemplary dose levels of adenoviral vector are preferably 108 to 1011 vector particles added to the perfusate.
In some embodiments, the present disclosure provides antibodies that directly or indirectly enhance Fgfl7 expression and or function. Any suitable antibody (e.g., monoclonal, polyclonal, or synthetic) may be utilized in the therapeutic methods disclosed herein. In some embodiments, the antibodies are humanized antibodies. Methods for humanizing antibodies are well known in the art (See e.g., U.S. Patents 6,180,370, 5,585,089, 6,054,297, and 5,565,332; each of which is herein incorporated by reference). In certain embodiments, the antibody is an agonist antibody to, for example, and the receptor for Fgfl7. Receptor agonism may be a critical step in the transmission of a signal from the outside to the inside of a cell. Agonist activity may occur when an antibody binds a receptor, for example the receptor for Fgfl7, such that it mimics binding of the natural ligand resulting in antibody-mediated downstream signaling or agonism. Antibody-mediated agonist activity may occur, for example, when 2 FAb arms of an IgG each bind to a half-receptor of a homo-dimeric receptor pair, causing the receptors to link and mimic the activity of a natural ligand.
The present invention is not limited to the use of any particular antibody configuration. In some embodiments, the targeting unit is an antigen binding protein. Antigen binding proteins include, but are not limited to an immunoglobulins, a Fab, F(ab')2, Fab' single chain antibody, Fv, single chain (scFv), mono-specific antibody, bi-specific antibody, tri-specific antibody, multivalent antibody, chimeric antibody, humanized antibody, human antibody, CDR-grafted antibody, shark antibody, an immunoglobulin single variable domain (e.g., a nanobody or a single variable domain antibody), minibody, camelid antibody (e.g., from the Camelidae family) microbody, intrabody (e.g., intracellular antibody), and /or de-fucosylated antibody and I or derivative thereof. Mimetics of binding agents and/or antibodies are also provided.
In some embodiments, scFv polypeptides described herein are fused to Fc regions to generate minibodies. As used herein, the term “fragment crystallizable region (Fc region)” refers to the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system. In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains; IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. The Fc regions of IgGs bear a highly conserved N-glycosylation site.
In some embodiments, the Fc region is derived from an IgG. In some embodiments, the IgG is human IgGl, although other suitable Fc regions derived from other organisms or antibody frameworks may be utilized.
In some embodiments, scFv polypeptides described herein are fused to chimeric antigen receptors. Chimeric antigen receptors (CARs), (also known as chimeric immunoreceptors, chimeric T cell receptors, artificial T cell receptors or CAR-T) are engineered receptors, which graft an arbitrary specificity onto an immune effector cell (T cell). Typically, these receptors are used to graft the specificity of an antibody (e.g., an scFv described herein) onto a T cell, with transfer of their coding sequence facilitated by retroviral vectors. The receptors are called chimeric because they are composed of parts from different sources.
Further, the present invention also envisages expression vectors comprising nucleic acid sequences encoding any of the above polypeptides or fusion proteins thereof or functional fragments thereof, as well as host cells expressing such expression vectors. Suitable expression systems include constitutive and inducible expression systems in bacteria or yeasts, virus expression systems, such as baculovirus, Semliki forest virus and lentiviruses, or transient transfection in insect or mammalian cells. Suitable host cells include E. co/i. Lactococcus lactis, Saccharomyces cerevisiae, Schizosaccharomyces pomhe. Pichia pastoris, and the like. Suitable animal host cells include HEK 293, COS, S2, CHO, NSO, DT40 and the like. The cloning, expression and/or purification of the antibodies can be done according to techniques known by the skilled person in the art.
It will be understood that polypeptides described herein may be identified with reference to the nucleotide and /or amino acid sequence corresponding to the variable and/or complementarity determining regions (“CDRs”) thereof.
Also within the scope of the invention are natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (herein collectively referred to as “variants”) of the immunoglobulin single variable domains of the invention as defined herein. Thus, according to one embodiment of the invention, the term “immunoglobulin single variable domain of the invention” or “nanobody” in their broadest sense also covers such variants, in particular variants of the antibodies described herein. Generally, in such variants, one or more amino acid residues may have been replaced, deleted and/or added compared to the antibodies of the invention as defined herein. Such substitutions, insertions or deletions may be made in one or more of the framework regions and/or in one or more of the CDRs. Variants, as used herein, are sequences wherein each or any framework region and each or any complementarity determining region shows at least 80% identity, preferably at least 85% identity, more preferably 90% identity, even more preferably 95% identity or, still even more preferably 99% identity with the corresponding region in the reference sequence (i.e., FRl variant versus FR1 reference, CDR1 variant versus CDRl reference, FR2_variant versus FR2_reference, CDR2_variant versus CDR2_reference, FR3_variant versus FR3_reference, CDR3_variant versus CDR3_reference, FR4_variant versus FR4_reference), as can be measured electronically by making use of algorithms such as PILEUP and BLAST. (See, e.g., Higgins & Sharp, CABIOS 5:151 (1989); Altschul S. F., W. Gish, W. Miller, E. W. Myers, D. I. Lipman. Basic local alignment search tool. J. Mol. Biol. 1990; 215:403-10.) Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the worldwide web at ncbi.nlm.nih.gov/). Such variants of immunoglobulin single variable domains may be of particular advantage since they may have improved potency or other desired properties.
A “deletion” is defined here as a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent as compared to an amino acid sequence or nucleotide sequence of a parental polypeptide or nucleic acid. Within the context of a protein, a deletion can involve deletion of about two, about five, about ten, up to about twenty, up to about thirty or up to about fifty or more amino acids. A protein or a fragment thereof may contain more than one deletion.
An “insertion” or “addition” is that change in an amino acid or nucleotide sequences which has resulted in the addition of one or more amino acid or nucleotide residues, respectively, as compared to an amino acid sequence or nucleotide sequence of a parental protein. “Insertion” generally refers to addition to one or more amino acid residues within an amino acid sequence of a polypeptide, while “addition” can be an insertion or refer to amino acid residues added at an N- or C-terminus, or both termini. Within the context of a protein or a fragment thereof, an insertion or addition is usually of about one, about three, about five, about ten, up to about twenty, up to about thirty or up to about fifty or more amino acids. A protein or fragment thereof may contain more than one insertion.
A “substitution,” as used herein, results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a parental protein or a fragment thereof. It is understood that a protein or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on the protein's activity. By conservative substitutions is intended combinations such as gly, ala; val, ile, leu, met; asp, glu; asn, gin; ser, thr; lys, arg; cys, met; and phe, tyr, trp.
By means of non-limiting examples, a substitution may, for example, be a conservative substitution (as described herein) and/or an amino acid residue may be replaced by another amino acid residue that naturally occurs at the same position in another variable domain. Thus, any one or more substitutions, deletions or insertions, or any combination thereof, that either improve the properties of the antibody of the invention or that at least do not detract too much from the desired properties or from the balance or combination of desired properties of the antibody of the invention (i.e., to the extent that the antibody is no longer suited for its intended use) are included within the scope of the invention. A skilled person will generally be able to determine and select suitable substitutions, deletions or insertions, or suitable combinations of thereof, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may, for example, involve introducing a limited number of possible substitutions and determining their influence on the properties of the antibodies thus obtained.
Further, depending on the host organism used to express the immunoglobulin single variable domain of the invention, such deletions and/or substitutions may be designed in such a way that one or more sites for post-translational modification (such as one or more glycosylation sites) are removed, as will be within the ability of the person skilled in the art. Alternatively, substitutions or insertions may be designed to introduce one or more sites for attachment of functional groups (as described herein), for example, to allow site-specific PEGylation.
Examples of modifications, as well as examples of amino acid residues within the immunoglobulin single variable domain, that can be modified (z.e., either on the protein backbone but preferably on a side chain), methods and techniques that can be used to introduce such modifications and the potential uses and advantages of such modifications will be clear to the skilled person. For example, such a modification may involve the introduction (e.g., by covalent linking or in another suitable manner) of one or more functional groups, residues or moieties into or onto the immunoglobulin single variable domain of the invention, and in particular of one or more functional groups, residues or moieties that confer one or more desired properties or functionalities to the immunoglobulin single variable domain of the invention. Examples of such functional groups and of techniques for introducing them will be clear to the skilled person, and can generally comprise all functional groups and techniques mentioned in the general background art cited hereinabove as well as the functional groups and techniques known per se for the modification of pharmaceutical proteins, and in particular for the modification of antibodies or antibody fragments (including ScFvs and single domain antibodies), for which reference is, for example, made to Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980). Such functional groups may, for example, be linked directly (for example, covalently) to an immunoglobulin single variable domain of the invention, or optionally via a suitable linker or spacer, as will again be clear to the skilled person. One of the most widely used techniques for increasing the half-life and/or reducing immunogenicity of pharmaceutical proteins comprises attachment of a suitable pharmacologically acceptable polymer, such as poly(ethyleneglycol) (PEG) or derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form of PEGylation can be used, such as the PEGylation used in the art for antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFvs); reference is made to, for example, Chapman, Nat. Biotechnol., 54, 531- 545 (2002); by Veronese and Harris, Adv. Drug Deliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug. Discov., 2, (2003) and in W004060965. Various reagents for PEGylation of proteins are also commercially available, for example, from Nektar Therapeutics, USA. Preferably, site-directed PEGylation is used, in particular via a cysteine-residue (see, for example, Yang et al., Protein Engineering, 16, 10, 761-770 (2003). For example, for this purpose, PEG may be attached to a cysteine residue that naturally occurs in an antibody of the invention, an antibody of the invention may be modified so as to suitably introduce one or more cysteine residues for attachment of PEG, or an amino acid sequence comprising one or more cysteine residues for attachment of PEG may be fused to the N- and/or C-terminus of an antibody of the invention, all using techniques of protein engineering known per se to the skilled person. Preferably, for the immunoglobulin single variable domains and proteins of the invention, a PEG is used with a molecular weight of more than 5000, such as more than 10,000 and less than 200,000, such as less than 100,000; for example, in the range of 20,000-80,000. Another, usually modification comprises N-linked or O-linked glycosylation, usually as part of co-translational and/or post-translational modification, depending on the host cell used for expressing the immunoglobulin single variable domain or polypeptide of the invention. Another technique for increasing the half-life of an immunoglobulin single variable domain may comprise the engineering into bifunctional constructs or into fusions of immunoglobulin single variable domains with peptides (for example, a peptide against a serum protein such as albumin).
Yet another modification may comprise the introduction of one or more detectable labels or other signal-generating groups or moieties, depending on the intended use of the labeled antibody. Suitable labels and techniques for attaching, using and detecting them will be clear to the skilled person and, for example, include, but are not limited to, fluorescent labels (such as fluorescein, isothiocyanate, 1 rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine and fluorescent metals such as Eu or others metals from the lanthanide series), phosphorescent labels, chemiluminescent labels or bioluminescent labels (such as luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogs), radio-isotopes, metals, metals chelates or metallic cations or other metals or metallic cations that are particularly suited for use in in vivo, in vitro or in situ diagnosis and imaging, as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alphaglycerophosphate dehydrogenase, triose phosphate isomerase, biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose- Vl-phosphate dehydrogenase, glucoamylase and acetylcholine esterase). Other suitable labels will be clear to the skilled person and, for example, include moieties that can be detected using NMR or ESR spectroscopy. Such labeled antibodies and polypeptides of the invention may, for example, be used for in vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA, RIA, EIA and other “sandwich assays,” etc.), as well as in vivo diagnostic and imaging purposes, depending on the choice of the specific label. As will be clear to the skilled person, another modification may involve the introduction of a chelating group, for example, to chelate one of the metals or metallic cations referred to above. Suitable chelating groups, for example, include, without limitation, diethylenetriaminepentaacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA). Yet another modification may comprise the introduction of a functional group that is one part of a specific binding pair, such as the biotin-(strept)avidin binding pair. Such a functional group may be used to link the antibody of the invention to another protein, polypeptide or chemical compound that is bound to the other half of the binding pair, i.e., through formation of the binding pair. For example, an agonist antibody of the invention may be conjugated to biotin, and linked to another protein, polypeptide, compound, or carrier conjugated to avidin or streptavidin. For example, such a conjugated antibody may be used as a reporter, for example, in a diagnostic system where a detectable signal -producing agent is conjugated to avidin or streptavidin. Such binding pairs may, for example, also be used to bind the antibody of the invention to a carrier, including carriers suitable for pharmaceutical purposes. One non-limiting example is the liposomal formulations described by Cao and Suresh, lournal of Drug Targeting, 8, 4, 257 (2000). Such binding pairs may also be used to link a therapeutically active agent to the antibody of the invention.
In some embodiments, the immunoglobulin single variable domain of the present invention is fused to a detectable label, either directly or through a linker. Preferably, the detectable label is a radioisotope or radioactive tracer, which is suitable for medical applications, such as in in vivo nuclear imaging. Examples include, without the purpose of being limitative, "mTc, 1231, 125I, inIn, 18F, 64Cu, 67Ga, 68Ga, and any other radio-isotope which can be used in animals, in particular mouse or human.
In still another embodiment, the immunoglobulin single variable domain of the present invention is fused to a moiety selected from the group consisting of a toxin, or to a cytotoxic drug, or to an enzyme capable of converting a prodrug into a cytotoxic drug, or to a radionuclide, or coupled to a cytotoxic cell, either directly or through a linker.
In some embodiments, the present invention provides an antibody-drug conjugate and/or an antibody-enzyme conjugate comprising, for example, a Fgfl7 agonist. In certain embodiments, the antibody drug conjugates are administered to cells expressing Fgfl7.
As used herein, “linkers” are peptides of 1 to 50 amino acids length and are typically chosen or designed to be unstructured and flexible. These include, but are not limited to, synthetic peptides rich in Gly, Ser, Thr, Gin, Glu or further amino acids that are frequently associated with unstructured regions in natural proteins. (See, e.g., Dosztanyi Z., V. Csizmok, P. Tompa, and I. Simon (2005). lUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics (Oxford, England), 21(16), 3433-4.)
In some embodiments, the therapeutic polypeptide is an immunoglobulin or fragment thereof. Examples include, but are not limited to, aptamers and immunoglobulins. Immunoglobulins (antibodies) are proteins generated by the immune system to provide a specific molecule capable of complexing with an invading molecule commonly referred to as an antigen. Natural antibodies have two identical antigenbinding sites, both of which are specific to a particular antigen. The antibody molecule recognizes the antigen by complexing its antigen-binding sites with areas of the antigen termed epitopes. The epitopes fit into the conformational architecture of the antigen-binding sites of the antibody, enabling the antibody to bind to the antigen.
The immunoglobulin molecule is composed of two identical heavy and two identical light polypeptide chains, held together by interchain disulfide bonds. Each individual light and heavy chain folds into regions of about 110 amino acids, assuming a conserved three-dimensional conformation. The light chain comprises one variable region (termed VL) and one constant region (CL), while the heavy chain comprises one variable region (VH) and three constant regions (CHI, CH2 and CH3). Pairs of regions associate to form discrete structures. In particular, the light and heavy chain variable regions, VL and VH, associate to form an “Fv “ area that contains the antigen-binding site.
The variable regions of both heavy and light chains show variability in structure and amino acid composition from one antibody molecule to another, whereas the constant regions show little variability. Each antibody recognizes and binds an antigen through the binding site defined by the association of the heavy and light chain, variable regions into an Fv area. The light-chain variable region VL and the heavychain variable region VH of a particular antibody molecule have specific amino acid sequences that allow the antigen-binding site to assume a conformation that binds to the antigen epitope recognized by that particular antibody.
Within the variable regions are found regions in which the amino acid sequence is extremely variable from one antibody to another. Three of these so-called “hypervariable” regions or “complementarity-determining regions” (CDR's) are found in each of the light and heavy chains. The three CDRs from a light chain and the three CDRs from a corresponding heavy chain form the antigenbinding site.
Cleavage of naturally occurring antibody molecules with the proteolytic enzyme papain generates fragments that retain their antigen-binding site. These fragments, commonly known as Fab's (for Fragment, antigen binding site) are composed of the CL, VL, CHI and VH regions of the antibody. In the Fab the light chain and the fragment of the heavy chain are covalently linked by a disulfide linkage.
Monoclonal antibodies against target antigens (e.g., Fgfl7 inhibitors) are produced by a variety of techniques including conventional monoclonal antibody methodologies such as the somatic cell hybridization techniques of Kohler and Milstein, Nature, 256:495 (1975). Although in some embodiments, somatic cell hybridization procedures are of use, other techniques for producing monoclonal antibodies are contemplated as well (e.g., viral or oncogenic transformation of B lymphocytes).
An animal system for preparing hybridomas is the murine system. Hybridoma production in the mouse is a well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.
Human monoclonal antibodies (mAbs) directed against human proteins can be generated using transgenic mice carrying the complete human immune system rather than-the mouse system. Splenocytes from the transgenic mice are immunized with the antigen of interest, which are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein. (See e.g., Wood et al., WO 91/00906, Kucherlapati etal., WO 91/10741; Lonberg etal., WO 92/03918; Kay et al., WO 92/03917 (each of which is herein incorporated by reference in its entirety); N. Lonberg et al., Nature, 368:856-859 [1994]; L.L. Green et al., Nature Genet., 7: 13-21 [1994]; S.L. Morrison et al., Proc. Nat. Acad. Sci. USA, 81 :6851-6855 [1994]; Bruggeman e/ u'/., Immunol., 7:33-40 [1993]; Tuaillon et al., Proc. Nat. Acad. Sci. USA, 90:3720-3724 [1993]; and Bruggernan etal. Eur. J. Immunol., 21: 1323-1326 [1991]).
Monoclonal antibodies can also be generated by other methods known to those skilled in the art of recombinant DNA technology. An alternative method, referred to as the “combinatorial antibody display” method, has been developed to identify and isolate antibody fragments having a particular antigen specificity, and can be utilized to produce monoclonal antibodies. (See e.g., Sastry et al., Proc. Nat. Acad. Sci. USA, 86:5728 [1989]; Huse et al., Science, 246:1275 [1989]; and Orlandi et al., Proc. Nat. Acad. Sci. USA, 86:3833 [1989]). After immunizing an animal with an immunogen as described above, the antibody repertoire of the resulting B-cell pool is cloned. Methods are generally known for obtaining the DNA sequence of the variable regions of a diverse population of immunoglobulin molecules by using a mixture of oligomer primers and PCR. For instance, mixed oligonucleotide primers corresponding to the 5' leader (signal peptide) sequences and/or framework 1 (FR1) sequences, as well as primer to a conserved 3' constant region primer can be used for PCR amplification of the heavy and light chain variable regions from a number of murine antibodies. (See e.g., Larrick et al., Biotechniques, 11 :152-156 [1991]). A similar strategy can also be used to amplify human heavy and light chain variable regions from human antibodies (See e.g., Larrick et al., Methods: Companion to Methods in Enzymology, 2:106-110 [1991]).
The term modified antibody is also intended to include antibodies, such as monoclonal antibodies, chimeric antibodies, and humanized antibodies which have been modified by, for example, deleting, adding, or substituting portions of the antibody. For example, an antibody can be modified by deleting the hinge region, thus generating a monovalent antibody. Any modification is within the scope of the invention so long as the antibody has at least one antigen binding region specific.
Chimeric mouse-human monoclonal antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted. (See e.g., Robinson et al., PCT/US86/02269; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; WO 86/01533; US 4,816,567; European Patent Application 125,023 [each of which is herein incorporated by reference in its entirety]; Better et al., Science, 240: 1041-1043 [1988]; Liu et al., Proc. Nat. Acad. Sci. USA, 84:3439-3443 [1987]; Liu et al., J. Immunol., 139:3521-3526 [1987]; Sun etal., Proc. Nat. Acad. Sci. USA, 84:214-218 [1987]; Nishimura etal., Cane. Res., 47:999-1005 [1987]; Wood et al., Nature, 314:446-449 [1985]; and Shaw et al., J. Natl. Cancer Inst., 80:1553-1559 [1988]). The chimeric antibody can be further humanized by replacing sequences of the Fv variable region that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General reviews of humanized chimeric antibodies are provided by S.L. Morrison, Science, 229: 1202-1207 (1985) and by Oi et al., Bio. Techniques, 4:214 (1986). Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain.
Suitable humanized antibodies can alternatively be produced by CDR substitution (e.g, US 5,225,539 (incorporated herein by reference in its entirety); Jones et al., Nature, 321:552-525 [1986]; Verhoeyan et al., Science, 239:1534 [1988]; and Beidler et rz/., J. Immunol., 141:4053 [1988]). All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to the Fc receptor.
An antibody can be humanized by any method that is capable of replacing at least a portion of a CDR of a human antibody with a CDR derived from a non-human antibody. The human CDRs may be replaced with non-human CDRs; using oligonucleotide site-directed mutagenesis.
Also within the scope of the invention are chimeric and humanized antibodies in which specific amino acids have been substituted, deleted, or added. In particular, humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen. For example, in a humanized antibody having mouse CDRs, amino acids located in the human framework region can be replaced with the amino acids located at the corresponding positions in the mouse antibody. Such substitutions are known to improve binding of humanized antibodies to the antigen in some instances.
The antibodies can be of various isotypes, including, but not limited to: IgG (e.g., IgGl, IgG2, IgG2a, IgG2b, IgG2c, IgG3, IgG4); IgM; IgAl; IgA2; IgAsec; IgD; and IgE. In some embodiments, the antibody is an IgG isotype. In other embodiments, the antibody is an IgM isotype. The antibodies can be full-length (e.g., an IgGl, IgG2, IgG3, or IgG4 antibody) or can include only an antigen-binding portion (e.g., a Fab, F(ab')2, Fv or a single chain Fv fragment).
In certain embodiments, the immunoglobulin is a recombinant antibody (e.g., a chimeric or a humanized antibody), a subunit, or an antigen binding fragment thereof (e.g, has a variable region, or at least a complementarity determining region (CDR)).
In some embodiments, the immunoglobulin is monovalent (e.g, includes one pair of heavy and light chains, or antigen binding portions thereof). In other embodiments, the immunoglobulin is a divalent (e.g., includes two pairs of heavy and light chains, or antigen binding portions thereof). In some embodiments, recombinant Fgfl7 fusion proteins that agonize the Fgfl7 receptor are provided. Where clinical applications are contemplated, in some embodiments of the present invention, the fusion proteins are prepared as part of a pharmaceutical composition in a form appropriate for the intended application. Generally, this entails preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals. However, in some embodiments of the present invention, a fusion protein composition formulation may be administered using one or more of the routes described herein.
In some embodiments, the fusion protein compositions are used in conjunction with appropriate salts and buffers to render delivery of the compositions in a stable manner to allow for uptake by target cells. Buffers also are employed when the compositions are introduced into a patient. Aqueous compositions comprise an effective amount of composition dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula. In some embodiments, candidate Fgfl7 agonists are screened for activity (e.g, using the methods described in the experimental methods or another suitable assay). In particular embodiments, cell permeation is enhanced by administration of a high affinity and selectivity glycan ligand as a prodrug, by replacing, for example, the carboxylate with a bioisostere, or by administering high affinity Fgfl7 receptor ligands on the surface of liposomal nanoparticles.
In some embodiments, the Fgfl7 agonists and agents delivered to the CNS by methods and compositions that promote transfer across the blood brain barrier (BBB). In certain embodiments, the methods and compositions comprise one or more bi-specific antibodies comprising, for example, antibodies to highly expressed proteins, including basigin, Glutl, and CD98hc. Antibodies to these targets are significantly enriched in the brain after administration in vivo. In particular, antibodies against CD98hc show robust accumulation in the brain after systemic dosing. Accordingly, in specific embodiments, methods and compositions of the present invention comprise, for example, use of CD98hc as a robust receptor-mediated transcytosis pathway for antibody delivery to the brain. (Zuchero et al. Neuron 89;70-82, 2016.) In further embodiments, transfer across the BBB is enhanced by transient disruption, for example, osmotic or pharmacologic disruption, and/or by other membrane protein pathways using receptor-mediate transcytosis comprising, for example, antibodies against the transferrin receptor.
In some embodiments, the present invention provides methods and compositions for increasing Fgfl7 activity comprising, for example, methods and compositions that enhance Fgfl7 transcription, translation, and expression, which inhibit Fgfl7 degradation, and/or that agonize Fgfl7 activity comprising, for example, a nucleic acid, an antibody, a small molecule, or a combination thereof. In particular embodiments, two or more methods and compositions that increase Fgfl7 provided in combination.
The present disclosure further provides pharmaceutical compositions (e.g, comprising the compounds described above). The pharmaceutical compositions of the present disclosure may be administered in a number of ways depending upon whether local or systemic treatment, for example, treatment to the central nervous system (CNS), the autonomic nervous system and/or the peripheral nervous system is desired, and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including oral and nasal delivery), pulmonary (e.g, by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, epidermal and transdermal), or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intracerebroventricular, administration. In certain embodiments, Fgfl7 agonists and agents that increase Fgfl7 activity are administered by methods that bypass the BBB including, for example, direct application to the surface of the CNS, to the parenchyma of the CNS, to the ventricles of the CNS, and to the cerebrospinal fluid (CSF) of the CNS. In particular, intrathecal and epidural administration may be achieved by single shot, a series of single shots, and/or by continuous administration to the CSF. In certain embodiments, continuous administration to the CSF is provided by a programmable external pump, for example, an osmotic pump. In other embodiments, continuous administration is provided by a programmable implantable pump.
Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, or binders may be desirable.
Compositions and formulations for parenteral, intrathecal or intracerebroventricular administration may include sterile aqueous solutions that may also contain buffers, diluents, and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients. Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids.
The pharmaceutical formulations of the present disclosure, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
The compositions of the present disclosure may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present disclosure may also be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present disclosure. For example, cationic lipids, such as lipofectin (U.S. Pat. No. 5,705,188), cationic glycerol derivatives, nanoparticles and polycationic molecules, such as polylysine (WO 97/30731), also enhance the cellular uptake of oligonucleotides.
The compositions of the present disclosure may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until prevention or a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering physician can determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of, for example, individual peptides, antibodies, oligonucleotides, and the like, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models or based on the examples described herein. In general, dosage is from 0.01 pg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly, or yearly. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease state, wherein the Fgfl7 agonist is administered in maintenance doses, ranging from 0.01 pg to 100 g per kg of body weight, once or more daily, to once every 20 years.
EXPERIMENTAL METHODS
Animals
Aged C57BL/6 mice (18-22 months old) were obtained from the National Institute on Aging rodent colony. Young male C57BL/6 mice (2 months old) were obtained from Charles River Laboratories. All experiments used male mice. All mice were housed at the Palo Alto VA animal facility under a 12-h light/12-h dark cycle with dark hours from 18:30-06:30 and housed at 68-73 °F under 40- 60% humidity. Published data (See, Castellano JM et al. Nature 544, 488-492, doi: 10.1038/nature22067 (2017), Villeda SA et al. NatMed , 659-663, doi: 10.1038/nm.3569 (2014), and De Miguel Z. et al. Nature 600, 494-499, doi: 10.1038/s41586-021-04183-x (2021)) to determine an optimal n for the studies on the effects of young CSF on behavior. For bulk RNA-seq of sorted oligodendrocyte nuclei, preliminary studies were performed to ensure capture of enough nuclei for downstream library preparation and statistical analysis. Age-matched mice were allocated into groups to achieve an equal average weight. All other criteria were not considered and, as such, were randomized. All animal care and procedures complied with the Animal Welfare Act and were in accordance with institutional guidelines and approved by the V.A. Palo Alto Committee on Animal Research and the institutional administrative panel of laboratory animal care at Stanford University.
Young mouse CSF collection
CSF was collected as previously described with several adaptations (Liu L. & Duff K. J Vis Exp, doi: 10.3791/960 (2008)). Ten-week-old mice were anesthetized intraperitoneally with Ketamine (120mg/kg) and xylazine (8mg/kg), and then placed in a stereotactic instrument (KOPF) with the head secured at a 45-degree angle facing downwards. An incision was made above the neck and muscles were held separated with microretractors allowing exposure of the cisterna magna by blunt forceps without any bleeding. CSF was pulled out of the cisterna magna cavity with a 20pl pipettor connected through an aspirator tube assembly (Sigma, A5177) to a pooled glass capillary (Borosilicate glass tubes, ID 1.30 mm, OD 1.70 mm, Length 4.00 in, type 8250, King precision glass) held secure by Model 1769 90° Electrode Holder (KOPF). CSF was kept in a low-protein bind tube on ice and spun in a cold centrifuge for 10 min at 1500 rpm to exclude CSF immune cells. Supernatant was collected and kept in -80 until use. The pellet was resuspended in 6ul of milli-q ultra-pure water for blood contamination quality control using the Nanodrop UV-vis setting with a 415nm wavelength for detection of oxyhemoglobin. A cutoff of below than 0.02 AU was used for CSF infusions.
Human CSF CSF samples of nine young heathy individuals (ages 24-26) were obtained through a collaboration with Dr. Henrik Zetterberg, University of Gothenburg, Sweden. The samples were baseline (normal sleep) lumbar CSF samples, collected in the morning, from healthy volunteers who took part in a study on sleep restriction-induced changes of CSF composition. For in vitro experiments, three pools consisting of 3 individuals each were made for each experiment, 2 pools from 6 male samples and 1 of female samples were each used in 3-4 technical triplicates.
Osmotic pump intracerebroventricular infusion
To minimize the volume of mouse CSF infused per mouse, young CSF or artificial CSF were loaded to a coiled polyethylene (PE-60) catheter prepared in house following the lynch coil technique. In brief, the total length of the coil needed was calculated at 4.56ul internal volume per cm tube. Usually, 20cm were wound around a syringe of the same outside diameter as the pump and secured with tape. The syringe was submerged in boiling water for 1 min and immediately immersed in ice cold water for 1 min. Coils were disassembled and left to dry overnight. 90 pl of pooled young CSF or artificial CSF (Tocris) were loaded to the coiled catheter connected to lOOul osmotic pumps (Alzet, 1007D) with a 7-day infusion at a rate of 0.5 pl/hr. Osmotic pumps were connected to a cannula (Brain infusion kit III, Alzet) and incubated overnight in a 37°C water bath. A cannula was inserted at +1 mm medio-lateral, 0 mm anterior-posterior, and -3mm dorso-ventral relative to bregma in order to target the right lateral ventricle. The pump was placed subcutaneously and mice received post-surgical buprenorphine and Baytril. Mice were split into groups to achieve an equal average body weight in all groups. In the human CSF infusion experiments, a pool of 3 young or 3 aged human CSF samples, pooled YM-CSF or aCSF was loaded into an osmotic pump (without a coil) and surgery was performed as described above. Recombinant carrier free human /mouse Fgf8b (423-F8/CF, R&D) and mouse Fgfl7 (7400-FG-025/CF, R&D) were resuspended in aCSF (Tocris) to a concentration of 25 pg/ml and loaded to an osmotic pump (Alzet, 1007D) with a 7-day infusion at a rate of 0.5ul I hr. In experiments with anti-Fgfl7 blocking antibody, polyclonal rabbit anti-Fgfl7 (PA5-109722, Thermo) and a rabbit IgG isotype control (31235, Thermo) were diluted in aCSF, loaded on a 3-kDa MWCO Amicon Ultra centrifugal filter (UFC500396, ThermoFisher) for buffer exchange and spun at 14,000g for 30 min at 4 °C. This step was repeated twice. Osmotic pumps (Alzet, 1004) were loaded with 0.68 mg ml-1 antibody to achieve a final CSF steady-state concentration of 5 pg ml-1. The surgery was performed as described above. In all experiments, mice were split into groups to achieve an equal average body weight in all groups.
Acute intracerebroventricular infusion Eighteen-month old mice were anesthetized with 2.5% isoflurane and then placed in a stereotactic instrument (KOPF). 3 pl of a pool of young mouse CSF (YM-CSF) (as described above) or aCSF were injected to the right lateral ventricle using a digital pump (WPI syringe pump with Micro4t controller model UMP3T-1) at a rate of I pl/min. Mice received post-surgical buprenorphine and Baytril. At Ihr or 6hr following the injection, mice were perfused and the contralateral hippocampus was dissected and used for RNAseq to avoid gene signatures induced by local immune response to the surgery. In labelled Fgfl7 experiments, 10 pg of mouse Fgfl7 (7400-FG-025/CF, R&D) was conjugated to Alexa Fluor 647 using the lightning-link conjugation kit (ab269823, ThermoFisher) following the manufacturer’s instructions and then loaded twice on a 3-kDa MWCO Amicon Ultra centrifugal filter (UFC500396, ThermoFisher) and spun at 14,000g for 30 min at 4 °C for free-dye clean-up and buffer exchange with aCSF. As control, a parallel labelling reaction (dye-only control) was carried out with a similar volume of aCSF as input. In CSF labelling experiments, 1 ml of human CSF was conjugated to Alexa Fluor 647 (ThermoFisher) using NHS chemistry and then loaded on a 3-kDa MWCO Amicon Ultra centrifugal filter (UFC500396, ThermoFisher) and spun at 14,000g for 30 min at 4 °C for free-dye clean-up and buffer exchange with aCSF and for protein concentration. Then, 0.5 pg of labelled Fgfl7 in 2 pl, dye-only control or YH-CSF was injected at a rate of 1 pl min-1 into young (2-3 months) mice and mice were killed 2 or 3 h after injection. Mice received postsurgical buprenorphine and Baytril.
Behavioral assays
The remote fear-conditioning recall test was performed as previously described with modifications. Mice were trained to associate cage context or an audiovisual cue with an aversive stimulus (foot shock). On day 1, mice were placed in a cage and exposed to 3 periods of 30 s of paired cue light and 1,000-Hz tone followed by a 2s foot shock (0.6 mA), with a 60s interval. On day 2 and day 22, mice were subjected to 2 trials. In the first trial assessing contextual memory, mice were re-exposed to the same cage context, and freezing behavior was measured during minute 390s using a FreezeScan tracking system (Cleversys). In the second trial measuring cued memory, mice were placed in a novel context and exposed to the same cue light and tone from day 1 on min 2, 3 and 4 of the trial. Freezing behavior was averaged across min 3-5. No significant differences in contextual fear conditioning were observed between groups at day 22. All experiments were performed by a blinded researcher
In the experiment presented in FIG. IB, initial group numbers were n = 11 for aCSF and n = 9 for YM-CSF (owing to limited amounts of mouse CSF) and 1 mouse from each group died during the experiment. In the experiment presented in FIG. 4N, initial group numbers were n = 11 for aCSF (one pump was not implanted due to an air bubble) and n = 12 for Fgfl7 and one mouse from each group died during the experiment.
In the anti-Fgfl7 blocking antibody experiments, the forced-alternation Y-maze and contextual fear conditioning tests were performed as previously described (Pluvinage JV et al. CD22 blockade restores homeostatic microglial phagocytosis in ageing brains. Nature 568, 187-192, doi: 10.1038/s41586-019- 1088-4 (2019)). The forced-alteration Y-maze test consisted of a 5-min training trial followed by a 5-min retrieval trail, with a 1-h intertrial interval. For the training trial, one arm of the Y maze was blocked off and mice were allowed to explore the 2 open arms. One hour later, the mouse was again placed in the Y maze with all 3 arms open and a black-and-white pattern placed at the end of the novel arm. Between mice and trials, the maze was wiped with ethanol to remove odor cues. For analysis, video was analyzed by a blinded observer and both the number of arm entries and the time spent in each arm were quantified. In the fear conditioning paradigm, mice were trained to associate cage context or an audiovisual cue with an aversive stimulus (foot shock). The test was administered over 2 d. On day 1, mice were placed in a cage and exposed to two periods of 30 s of paired cue light and 1,000-Hz tone followed by a 2-s foot shock (0.6 mA), with a 180-s interval. On day 2, mice were subjected to 2 trials. In the first trial assessing contextual memory, mice were re-exposed to the same cage context and freezing behavior was measured during minutes 1-3 using a FreezeScan tracking system (Cleversys). In the second trial measuring cued memory, mice were placed in a novel context and exposed to the same cue light and tone from day 1 after 2 min of exploration. Freezing behavior was measured for 1-3 min following the cue. No significant differences in cued fear conditioning were observed between groups. Experimental groups consisted of n = 10 mice, and no mice were excluded during the experiment.
In vivo BRDU and EDU pulses
To assess baseline proliferation, mice received one EdU injection 15 mins before the stereotactic surgery (Invitrogen, E10415, 100 mg I kg intraperitoneally). To assess cell proliferation post infusion, mice were pulsed three times with BrdU to label proliferating cells; twice on day 5 of the infusion 10 hrs apart and once on day 6 two hours before perfusion (100 mg /kg intraperitoneally; B5002-5G, Sigma- Aldrich). In a subsequent cohort, in which young mouse CSF or Fgfs were infused to aged mice, mice received post-infusion pulses of EDU instead of BRDU in the same paradigm described above, with no baseline labeling prior to surgery.
Tissue processing prior to immunostaining
Mice were anesthetized with Avertin (2,2,2-tribromoethanol: T48402, Sigma-Aldrich; 2-methyl-2- butanol: 240486, Sigma-Aldrich) (0.018 ml (2.5%) per gram of body weight) and perfused with 20 ml cold PBS. Brains were collected and divided sagittally. One hemisphere was used to dissect the hippocampus for RNA sequencing, which was snap frozen and stored at -80 °C. The second hemisphere was fixed in phosphate-buffered 4% paraformaldehyde overnight at 4 °C before transfer to 30% sucrose in PBS at 4 °C until sectioning. Brains were frozen at -30 °C and cryosectioned coronally at 40 pm with a microtome (Leica, SM2010R). Brain sections were stored in cryoprotectant (40% PBS, 30% glycerol, 30% ethylene glycol) and kept at -20 °C until staining.
Immunostaining
Brain slice immunofluorescent staining
Brain sections were washed 3 times for 10 min in TBST and then blocked in TBS++ (TBS + 3% donkey serum (130787, Jackson ImmunoResearch) + 0.25% Triton X-100 (T8787, Sigma-Aldrich)) for 1 hr, followed by primary antibody incubation overnight on a rocking platform at 4 °C. The following primary antibodies were used in this study; PDGF receptor a (D1E1E) XP rabbit mAb (1 :500; Cell Signaling, 3174), rabbit anti-MBP (1: 100; Millipore, MAB386), rabbit anti-Fgfl7 (1 :500; Thermo, PAS- 109722), rabbit anti-c-Fos (1 :500; Cell Signaling, 9F6), rabbit anti-GFAP (1 :500; Dako, Z0334), goat anti-IBAl (1 :500; Abeam, ab5076), rabbit anti-NG2-Alexa488 (1 :200; Millipore, AB5230A4), rabbit anti-Olig2 (1:500; Millipore, AB9610), anti-Acta2-Cy3 (1 :500; Sigma, C6198) and mouse anti-NeuN (1:500; Sigma, MAB377). For secondary staining, brain sections were washed 3 times for 10 min in TBST, followed by incubation for 1.5 hrs in Alexa Fluor-conjugated secondary antibodies (1 :500). For in vivo phalloidin staining, phallidin-Alexa488 (Thermo, A12379) was added at a 1 :50 dilution to the secondary antibody mix. Brain sections were washed and mounted on Superfrost microscope slides (12- 550-15, Fisher Scientific) with Vectashield Hardset Antifade Mounting Medium with DAPI (Vector labs, H-1500/ NC9029229). For MBP stains an additional step of tissue de-lipidation was added before blocking: tissues were incubated in 100% EtOH for lOmin in room temperature and then washed twice with PBS. For MBP staining, an additional step of tissue delipidation was performed before blocking: tissues were incubated in 100% ethanol for 10 min at room temperature and then washed twice with PBS. EdU staining
Brain sections were washed 3 times for 10 min in PBS, then permeabilized for 20 min in 0.1% Triton X-100 (T8787, Sigma-Aldrich), washed again 3 times and then blocked in TBS++ for Ihr. EdU staining was performed following the Click-iT® Plus EdU Alexa Fluor® 555 Imaging Kit instructions (Cl 0638, Life Technologies). Sections were washed and stained with primary and secondary antibodies as described above.
BrdU staining- brain slices and 384-well plates Following staining with other primary and secondary antibodies, sections were incubated in 2N HC1 for 30 min at 37 °C and then washed 3times for 10 min in TBST. Sections were blocked for 1.5 hr in TBS++ and then transferred to primary antibody with Rat anti-BRDU antibody (1 :500, ab6326, Abeam) overnight at 4 °C. Secondary staining started with 3 washes for 10 min in TBST, followed by incubation with secondary antibody mix for 1.5 h. After 3 10-min washes in TBST, sections were mounted as described above. For 384-well plates, nuclei were stained and with Hoechst 33342 (1 :2000, H3570, Thermo) and immediately imaged on a Keyence microscope (BZ-X800). In cases where BRDU and EDU staining was performed on the same sections, the sequence was; permeabilization, HCL antigen retrieval, EDU Click-it reaction, blocking, primary and secondary antibody stain as described above in detail.
In situ RNA hybridization (RNAScope)
RNAScope was performed on fresh frozen coronal brain sections (10pm thick) using the Multiplex Fluorescence v.2 kit (Advanced Cell Diagnostics) according to the manufacturer’s protocol with minor modifications. Tissue fixation with 4% PFA was extended to 60 min at RT, and Protease IV treatment was shortened to 20 min to better preserve the hippocampal tissue. Probes for mouse Pdgfira and SRF were commercially available from the manufacturer and secondary Opal 690 and 520 reagents (FP1497001KT and FP1487001KT, Akoya Biosciences) were diluted at 1 :1500 in TSA buffer.
Image analysis ( Imaris)
Brain slices
Confocal z stacks of four coronal brain sections spanning the dorsal hippocampus were captured on a Zeiss confocal LSM880 microscope for each brain sample using a 20x magnification. Maximumprojection files (at least 4 hippocampal coronal slices per mouse, 400pm apart) were analyzed in Imaris by generating masks of 2 main regions of interest: (1) CAI : - stratum oriens, stratum pyramidale and stratum lacunosum-moleculare (SLM) combined, and (2) cortex. The percentage of newly proliferated OPCs was analyzed by dividing cell counts of BRDU+ PDGFRoT or EDU+ PDGFRa+ cells by that for PDGFRoC cells. For cell density, BRDU+ or PDGFRa+ cell counts were divided by the corresponding area of the mask per slice.
For RNAscope analysis, similar hippocampal tiled z-stacks were acquired (at least 4 hippocampal coronal slices per mouse, 100pm apart). Percentage of SRF^ OPCs was calculated by dividing the number of SRF* PDGFRoC nuclei by PDGFRcC nuclei in the CAI region of the hippocampus. For MBP analysis, confocal images of the molecular layer were acquired using the 20x magnification (Keyence microscope model BZ-X800),MBP intensity was measured using batch analysis in ImageJ. For in vivo phalloidin analysis, z stacks at x63 magnification of individual OPCs were obtained by staining for Pdgfra. Using Imaris, three-dimensional surface rendering was reconstructed by Pdgfra signal and phalloidin intensity was measured only inside the surface. All analyses were performed by a blinded observer.
In vitro cell culture
Three random 20x images of each well were analyzed using Imaris batch by setting similar surfaces to automatically count BRDU+ and Hoechst nuclei. For each image percentage of proliferating cells was calculated by dividing BRDU+ counts by total Hoechst4 counts. In differentiation experiments, cell morphology state was assessed manually as previously described (Zuchero JB et al. Dev Cell 34, 152-167, doi: 10.1016/j.devcel.2015.06.011 (2015)). Cellular phalloidin intensity and MBP intensity were measured by manually delineating cell borders and measuring intensity and cell area. All cell measurements were then averaged per coverslip. Representative images of phalloidin intensity were made with Fire LUT in IMAGEJ. All other quantifications were done using IMAGEJ and manual cell counts. In differentiation experiments, cell morphology state was assessed manually as previously described (Mathur D. et al. Front Cell Neurosci 11, 209, doi: 10.3389/fncel.2017.00209 (2017)). All analyses were performed by a blinded observer.
Transmission electron microscopy
Perfusion and sectioning
Mice were perfused with 20 ml cold EM fixation buffer consisting of EM-grade 2% glutaraldehyde (EMS/Fisher, 50-262-08) and 4% PFA (EMS/Fisher, 50-980-486) in 0.2 M sodium cacodylate (EMS/Fisher, 50-980-279) and kept in fixation buffer until sectioning. Brains were sectioned coronally to 100-pm sections using a Leica VT1200S vibratome and kept in EM fixative until TEM processing.
High-pressure freezing with freeze substitution
Vibratome sections of 100 pm were stained using an osmium-thiocarbohydrazide-osmium (OTO) method in combination with microwave-assisted processing, followed by high-pressure freezing and freeze substitution (HPF-FS), as previously described (Ewald AJ et al. JCellSci 125, 2638-2654, doi: 10.1242/jcs.096875 (2012)). Samples were stained with OTO, incubated with 2% aqueous uranyl acetate overnight and then subjected to HPF, followed by super-rapid FS with 4% osmium tetroxide, 0.1% uranyl acetate and 5% ddH2O in acetone. They were then thin-layer embedded and polymerized in hard epon resin. Resin-embedded samples were precision cut off the glass slide and glued with cyanoacrylate onto a blank resin block for sectioning or glued with silver paint onto a stub for focused ion beam imaging. Transmission electron microscopy Ultrathin sections of 90 nm were cut using a Leica UC6 ultramicrotome (Leica Microsystems) and collected onto formvar-coated 50-mesh copper grids or copper-rhodium slot grids. Because of native contrast from volume EM processing, no poststain was necessary. Sections were imaged using a Tecnai 12 120-kV transmission electron microscope (FEI), data were recorded using an UltraScan 1000 with Digital Micrograph 3 software (Gatan) and SerialEM was used to collect montages covering an area of 143 x 143 pm.
SRF reporter assay
HEK293 cells were plated at 50K cells/ well in a 96-well plate in full media (DMEM, 10% FCS and 1% P/S) and transfected on day 2 with Cignal SRE Reporter Assay Kit (GFP) (CCS-010G, Qiagen) using lipofectamine P3000 in experimental media (DMEM, 0.5% FCS, 1% non-essential amino acids (M7145, Sigma)), following the manufacturer’s instructions. On day 3 media was changed to 90pm fresh experimental media and supplemented with 10pm of lOx concentration of indicated concentrations of recombinant carrier free recombinant human kGF/FGF-7 (251-KG-010, R&D Systems), human /mouse Fgf8b (423-F8/CF, R R&D Systems), mouse Fgfl7 (7400-FG-025/CF, R&D Systems), human/murine/rat BDNF (450-02, Peprotech), human CX3CLl/fractalkine (365-FR-025, R&D Systems), human Dhh protein (ab78682, Abeam) or Ckm (9070-CK-050, R&D Systems) at the indicated concentration. Plates were incubated in the IncuCyte (Essen BioScience) and imaged every hour for 24 hours. In actin inhibitors experiments, on day 3 media was changed to fresh experimental media and pre-incubated for 30 min at 37°C with jasplakinolide (Jasp, 125 or 250nM, J7473, Fisher Scientific) or latrunculin A (LatA, 250 or 500nM, L12370, Thermo), before adding Fgfl7 at a final concentration of 200 ng/ml. Plates were incubated in the IncuCyte and imaged every hour for 24 hours. In anti-FgfR blocking antibody experiments, on day 3, the medium was changed to fresh experimental medium and cells were preincubated at 37 °C with anti-FgfRl (NBP2-12308), anti-FGFR2 (MAB684-100) or anti-FGFR3 (MAB7661-100; all from Novus Biologicals) at a final concentration of 50 pg ml-1, before adding Fgfl7 at a final concentration of 200 ng ml-1. Plates were incubated in the IncuCyte and imaged every hour for 24 h.
Western blot of Fgfl7 in mouse cortex.
Tissue was lysed in a RIPA lysis buffer (ThermoFisher, 89901) cocktail solution containing protease inhibitor (Roche, 11836153001) and Halt phosphatase inhibitor (ThermoFisher, 78420). The protein concentration of each sample was then measured using the Pierce BCA Protein Assay kit (ThermoFisher, 23225). 30ug of protein per sample were then heated at 95°C for lOmin before loading on a 4-12% 10-well gel (ThermoFisher, NP0321BOX). The gel was run at 80V for lOmin and then 150V until samples ran through gel. Transfer was completed at 100V for 1.5hrs. Membranes were blocked in 5% BSA for Ihr and then stained overnight at 4°C with 1 : 1000 dilutions of mouse a-GAPDH (Origene, TA802519) and rabbit a-FGF17 (ThermoFisher, PA5-109722). Then, membranes were washed and stained with 1 :20,000 Li-Cor a-Mouse (Li-Cor, 926-68072) and a-Rabbit (Li-Cor, 926-32213) secondary antibodies for Ihr covered with aluminum foil. Finally, they were washed 3 times with TBST for 5 mins and 2 times with TBS for 5 mins before being imaged using a Li-Cor Odyssey CLx imager.
OPC primary cultures.
Rat OPC cultures
OPCs were isolated from P7-P8 brains by immunopanning and grown in serum-free defined medium, as previously described (Emery B. & Dugas JC Cold Spring Harb Protoc 2013, 854-868, doi: 10.1101/pdb.prot073973 (2013)). Cell culture for proliferation and differentiation experiments was done following the protocol with several modifications. To use the least possible CSF of young healthy human subjects, the culture conditions were minimized to 384 well plates. In addition, to account for inter-subject variability, CSF of 3 subjects with similar ages were pooled, and 3 such pools were used in each experiment in triplicates. Following initial growth of 4 days in 10cm dishes, cells were trypsinized and split to 384-well (Falcon® 384 Well Optilux, 353962), PDL-covered (PDL; Sigma-Aldrich P6407; molecular weight 70-150 kDa) plates. For proliferation experiments, 2,500 cells were plated in a total volume of 50pl of full proliferation medium supplemented with 10 ng/ml PDGF (Peprotech 100- 13 A), 10 ng/ml CNTF Peprotech 450-02, 4.2 pg/ml forskolin (Sigma-Aldrich F6886) and Ing/ml NT3 (Peprotech 450-03) with 10% of YH-CSF or aCSF. Actin filaments were visualized by live imaging by addition of 500nM of SiR-Actin (Cytoskeleton, CY-SC002) added with 10% YH-CSF. Wells were imaged every hour for the remaining 6 hours of the experiment. In BRDU experiments, 18hrs after plating in 384-well plates (with 10% CSF), 5ul of 200pM BRDU (20pM final concentration) was added for a pulse of 6 hrs followed by fixation with 4% PFA for 20 min. BrdU experiments were performed with the indicated concentrations of YH-CSF or Fgfl 7 and in FIG. 4S in combination with rabbit anti-Fgf!7 (Thermo, PA5- 109722) or IgG isotype control (Thermo, 31235) to achieve a final concentration of 5 pg ml-1 antibody, 40 ng ml-1 Fgfl7 and 10% YH-CSF in full proliferation medium, as indicated above.
In experiments that required phalloidin staining, 10,000 OPCs were plated on PDL-covered 12mm coverslips in a 24-well dish in 90% full proliferation medium (315 pl) and let to adhere overnight. The medium was then supplemented with 10% CSF (35pl) for 6 h. Coverslips were fixed with 4% PFA for 20 mins, washed and stained with 555-phalloidin in PBS (1 :143, Invitrogen) for 15 min. Coverslips were washed and mounted with Vectashield Hardset Antifade Mounting Medium with DAPI (Vector labs, H- 1500/NC9029229). For differentiation experiments, 10,000 OPCs were plated on PDL-covered 12mm coverslips in a 24-well dish in full proliferation medium overnight. Proliferation medium was completely changed to differentiation media (basal growth media supplemented with 40 ng/ml T3 (Sigma-Aldrich T6397)) with 10% CSF with a 50% media change (with 10% CSF) on day 2 of differentiation. At day 4 of differentiation coverslips were fixed with 4% PFA for 20 min, washed with PBS, permeabilized with 0.1% Triton X-100 for 3 mins followed by wash and blocking in 3% BSA for 1 hr. Primary antibodies were as follows; rabbit-anti-MBP (1 :100, abeam, ab7349, knock-out validated 19) and mouse anti-GFAP (1:500, Chemicon, MAB360). Primary antibodies were incubated overnight at 4 °C. Coverslips were washed, stained with Alexa Fluor-conjugated secondary antibodies (1:500) followed by a 15 min stain with Cell mask (1 :1000, Invitrogen, Cl 0046) mounted and set on a coverslip before imaging on a Keyence microscope (BZ-X800) or confocal laser-scanning microscope (Zeiss LSM880).
Mouse OPC cultures
Mouse OPCs were purified from brains of mice with loxP -flanked SRF (generated by David Ginty and kindly provided by Eric Small) by immunopanning as described above for rat OPCs (Emery B. & Dugas JC Cold Spring Harb Protoc 2013, 854-868, doi: 10.1101/pdb.prot073973 (2013)).
On day 3 of culture, SRPilf OPCs were split and plated in 384-well plates for proliferation experiments. When the cells were in suspension in proliferation media before plating, P IO10 viral genomes of AAV DJ-CMV eGFP-deleted ere (GVVC-AAV-62) or AAV DJ-CMV eGFP-cre (GVVC- AAV-63) (both generated by the Stanford Gene Vector and Virus Core) were added. The following day, the medium was fully replaced and 48h after infection 10% aCSF or YH-CSF was added to the proliferation media with BRDU (20pM final concentration) for 16 h. Cells were fixed and cell proliferation was assessed as indicated above for BRDU experiments.
SLAMseq experiment and data analysis
The optimal s4U concentration was assessed using the SLAMseq Explorer Kit - Cell Viability Titration Module (061, Lexogen) following manufacturer’s recommendations. OPCs were incubated with increasing doubling concentrations of s4U (1.95-2000pM), and viability was assessed with live-dead ratio as described above and with ATP incorporation following manufacturer’s recommendations (Promega,G7571). A final concentration of 62.5uM was found to be the highest concentration that did not compromise cell viability within 12 h (twice the duration of the intended experiment). The SLAMseq experiment was conducted following SLAMseq Kinetics Kit - Anabolic Kinetics Module protocol (061, Lexogen). Following initial growth for 4 days in 10cm dishes, 30,000 OPCs were plated in 315pl full proliferation medium on poly(D-lysine)-covered 24-well plates (one plate per timepoint) overnight. The next day 35 pl of pooled YH-CSF or aCSF with 625pM S4U (lOx concentration) was added gently to minimize confounding induction of gene expression. After 1 or 6 h, the medium was removed, and cells were scraped with 1ml Trizol (Thremo, 15596018), transferred to foil-covered tubes and frozen until RNA extraction. RNA extraction was performed following the protocol for the anabolic kit. All cell culture and RNA extraction steps were done in the dark under red-light, following manufacturer’s recommendations. Library preparation was done with QuantSeq 3' mRNA-Seq Library Prep Kit for Illumina (FWD) (015, Lexogen) with the indicated modifications to adjust to low-RNA input. After normalization and pooling, libraries were sequenced on a Nextseq550 (Illumina) using single-end 75-bp reads. Libraries were sequenced to a mean depth of ~30 million reads per sample. Raw sequencing files were demultiplexed with bcl2fastq and resulting FASTQ files combined across lanes and per sample. Sequencing quality control was performed using FastQC vO.l 1.8 and summary reports were generated with MultiQC vl.7. FASTQ files were then analyzed using the SLAM-DUNK pipeline vO.3.4 and the related alleyoop toolchain. The pipeline comprises 1) read mapping, 2) alignment filtering, 3) SNP calling and correction, and 4) 3'-UTR sequence counting. Using NextGenMap, reads were aligned against the genome of Rattus norvegicus (release 6.0), which was downloaded in FASTA format from Ensembl release v97. For mapping the following parameters were set; '-5 12, -a 4, -n 1, -ss' leaving the rest at default. Resulting Binary sequence Alignment/Map (BAM) format files were filtered to remove low- quality alignments using the parameters '-mq 2, -mi 0.95, -nm -1’ with the rest at default. Next, SNPs in alignments, in particular the T to C (T>C) conversions, were called with parameters '-c 10, -f 0.8' and other parameters remaining at default. The statistical independence of distributions of true SNP-callings identified by VarScan2 in relation to the number of T>C reads was assessed with a Mann-Whitney-U test for each sample as initially described in the alleyoop snpeval toolchain module. For the reference set of 3'-UTRs, a genome feature file (GFF3) for the Rattus norvegicus genome (release 6.0) was downloaded from Ensembl release v97, filtered to retain only three_prime_utr features and converted to Browser Extensible Data (BED) format using bedops v2.4.36. The BED file was used in combination with parameters '-c 1, -q 27' for the SLAMDUNK count command, leaving other parameters at default. Resulting 3'-UTR T>C and total read counts were added up on gene-level along all transcripts and alternative 3'-UTRs available per transcript. The entire down-stream analysis was performed using R v3.6.1, data.table vl.12.2, and common tidyverse packages (tidyr, dplyr, purrr, stringr, ggplot2). Differential gene expression analysis was carried out using DESeq2 vl.26.0 on the T>C count and total count matrices separately with standard settings with alpha level set to 0.05. In total, 6 DESeq2 experiments were performed, 2 with pooled timepoints (Ihr + 6hr) per read-type (design variables of treatment and time), and 4 with separate timepoint per read-type (design variable treatment only). Effect size was calculated using implementation of Cohen’s d from the effsize vO.7.6 package. Gene set enrichment and over-representation analyses were performed using GeneTrail 3.0. Category source databases were adjusted independently, and P-values were corrected using the false discovery rate controlling procedure by Benjamini -Hochberg and considered significant if smaller than 0.05.
Isolation and sorting of oligodendrocyte nuclei
Isolation of nuclei from frozen dissected hippocampi was done as previously described (Hahn 0. et al. Nucleic Acids Res 49, el l, doi: 10.1093/nar/gkaal 127 (2021)) using the Nuclei EZ Prep Kit (Sigma- Aldrich, St. Louis, USA). Following the final PBS wash, nuclei were pelleted and resuspended in 100 pl of antibody mix (1:100, Anti-NeuN antibody -Alexa Fluor® 647, EPR12763, and 1 : 100, Anti-Olig2 antibody-Alexa Fluor® 488) with 0.2U/pl RNAse Inhibitor (Takara, 23138) in FACS buffer (0.5% BSA in PBS) and were incubated on ice with intermittent shaking for 30 min. Nuclei were washed with 1ml FACS buffer and pelleted by centrifugation at 500 RCF. for 5 min, resuspended in FACS buffer with 0.2U/pl RNAse inhibitor and Hoechst 33342 (1 :2000, H3570, Thermo), sorted on a Sony Sorter (SH800) based on Houechst+NeuN" Olig2+high(OPC) and Houechst+NeuN'Olig2+low (OL) gating to 350pl of RTL buffer and stored in -80°C until RNA extraction. Data were analyzed using FlowJo software (TreeStar).
RT-qPCR
Oligodendrocyte nuclei were isolated by FACS and RNA was extracted with the RNeasy Plus Micro kit (Qiagen, 74034). cDNA was generated with qScript™ cDNA SuperMix (QuantaBio, 95048). Samples were diluted and mixed with SYBR green master mix before loading as technical triplicates for qPCR on a LightCycler 480 (Roche). AAC T values normalized to Gapdh were used to assess relative gene expression between samples. The following validated primer pairs for mouse Sr/were used: 5'- GGC CGC GTG AAG ATC AAG AT-3' (forward; SEQ ID NO: 1) and 5'- CAC ATG GCC TGT CTC ACT GG-3' (reverse; SEQ ID NO: 2).
Bulk RNA-seq
Hippocampal RNAseq
Frozen dissected hippocampi were thawed on ice and homogenized in 350pl RLT buffer by 20 strokes using a manual homogenizer, and total RNA was isolated with the RNeasy Plus Micro kit (Qiagen, 74034). RNA quantity and quality were assessed by Agilent 2100 Bioanalyzer (Agilent Technologies). All samples passed a quality-control threshold (RIN > 9) to proceed to library preparation and RNA-seq on HiSeq 4000 (Illumina) using paired-end 100-bp reads. Libraries were sequenced to a depth of >20 million reads per sample. Raw sequencing files were demultiplexed with bcl2fastq, reads were aligned using STAR, and counts of technical replicates were summed up using DESeq2 before performing normalization and differential expression analysis with standard settings.
For deconvolution analysis of bulk RNA-seq data, the CIBERSORTx algorithm (Newman AM et a . Nat Biotechnol Nl , 773-782, doi: 10.1038/s41587-019-0114-2 (2019)) was used to deconvolve the bulk RNA-seq data. First, single-nucleus RNA-seq data describing the brain of 3-month-old young mice (Hahn 0. et al. Nucleic Acids Res 49, el l, doi: 10.1093/nar/gkaal 127 (2021)) was used to construct a cell-type-specific signature matrix with CIBERSORTx. One hundred cells were equally sampled across the following cell types: astrocytes, choroid plexus, endothelial cells (BEC), interneurons, microglia, neurons of the trisynaptic loop (neuron CA), neurons of the dentate gyrus (neuron DG), oligodendrocytes, oligodendrocyte precursors and pericytes. Sampling was done over the annotated, quality-controlled data to ensure efficiency for CIBERSORTx. Next, CIBERSORTx was ran on the sampled and CPM- normalized dataset with default parameters and inferred a ‘signature matrix’ that provided gene signatures for each noted cell type. By following Steen CB, et al., (Methods Mol Biol 2117, 135-157, doi:
10.1007/978- 1-0716-0301-7_7 (2020)), CPM-normalized YM-CSF and aCSF samples were deconvolved separately in S-mode owing to the possibility of high technical variance. This step was conducted first to infer cell type fractions per sample. Next, cellular expression levels were estimated in ‘group mode’ to identify cell-type-specific gene expression profiles per condition group. Finally, the differential expression code was ran comparing the cell-type-specific expression profiles estimated for YM-CSF and aCSF. P values were corrected with the Benjamini-Hochberg procedure (FDR = 0.05), and log2- transformed fold change (log2FC) values reflect changes in the estimated expression levels of each gene between YM-CSF and aCSF. Single-cell subsampling and single-cell data normalization were conducted with Python Scanpy 1.6.0. All CIBERSORTx-specific analyses were done with the web service of the CIBERSORTx team (cibersortx-stanford-edu.ezproxy.library.wisc.edu/). Differential gene expression analysis was conducted in R 4.0.5.
RNA-seq of sorted OPCd
RNA was extracted with the RNeasy Plus Micro kit (Qiagen, 74034). cDNA and library synthesis were performed inhouse using the Smart-seq2 protocol (detailed protocol at doi- org.laneproxy.stanford.edu/10.17504/protocols.io.2uvgew6) with several modifications. Because of the low input RNA content, 2pl of RNA extracted from sorted nuclei was reversed transcribed using 16 cycles for OL samples and 18 cycles for OPC samples. Following bead cleanup using 0.7x ratio with AMPure beads (A63881, Fisher), the cDNA concentration was measured using the Qubit lx dsDNA HS kit (Q33231) and normalized to 0.4 ng/pl as input for library prep. 0.4 pl of each normalized sample was mixed with 1.2 pl of Tn5 Tagmentation mix (0.64 pl TAPS -PEG buffer (PEG 8000) ( Promega V3011), and TAPS-NaOH pH8.5)(Boston Bioproducts BB-2375), 0.46 pl H2O and 0.1 pl Tn5 enzyme (Illumina 20034198,), followed by incubation at 55 °C for 10 min. The reaction was stopped by adding 0.4 pl 0.1% sodium dodecyle sulfate (Fisher Scientific, BP166-500). Indexing PCR reactions were performed by adding 0.4 pl of 5 pM i5 indexing primer (IDT), 0.4 pl of 5 pM i7 indexing primer (IDT), and 1.2 pl of KAPA HiFi Non-Hot Start Master Mix (Kapa Biosystems) using 12 amplification cycles. Libraries were purified using 2 purification rounds with a ratio of 0.8x and 0.7x AMPure beads. Library quantity and quality was assessed using a Bioanalyzer (Agilent) and Qubit. All steps were done manually using 8-strip PCR tubes, and PCR reactions were carried out on a 96-plate thermal cycler (Biorad). Libraries were pooled and sequenced on a Nextseq550 sequencer (Illumina) using single end 63bp for Read 1 and 12bp for index 1 with a high output 75bp kit (Illumina 20024906,).
Libraries were sequenced to a depth of at least >10 million reads per sample. Raw sequencing files were demultiplexed and known adaptor sequences were trimmed with bcl2fastq. Data analysis of raw sequencing data was performed using the nextflow-core RNA-seq pipeline v3.0. The core workflow of the pipeline maps filtered reads against the species reference genome using STAR and computes transcript counts using RSEM. For nuclear RNA-seq data, a custom reference genome in which exon sequences in GTF files were modified to include all introns per transcript was created and used for the mapping. For mouse and rat sequencing data the reference genomes GRCm38 and Rnor 6.0 provided by Illumina igenomes were used, respectively. All gene annotations were based on the Ensembl database. Obtained raw gene transcript counts for each sample were loaded into DESeq2, performing normalization for transcript length and sequencing depth, and differential expression analysis was performed standard settings. Effect sizes for each gene were computed based on normalized counts computed by DESeq2 using the function cohen. d of the R package effsize. Gene set enrichment analysis (GSEA) was performed using GeneTrail 3 using BH-FDR p-value adjustment with all remaining parameters kept at default.
Meta-analysis of SRF targets in single-cell datasets from the literature
For each data set, gene counts, fold-changes and adjusted P-values calculated when comparing either case and control or old and young age groups were acquired from the publicly available supplement tables of the corresponding publications (Spitzer SO et al. Neuron 101, 459-471 e455, doi: 10.1016/j. neuron.2018.12.020 (2019), Mathys H. et al. Nature 570, 332-337, doi: 10.1038/s41586-019- 1195-2 (2019) and Zhou Y. et al. Nat Med 26, 131-142, doi: 10.1038/s41591-019-0695-9 (2020)). Next, the list of SRF targets known in Homo sapiens (by TRANSFAC dataset, See, Gerstner N. et al. Nucleic Acids Res, doi: 10.1093/nar/gkaa306 (2020)) was mapped to orthologous gene identifiers in Mus musculus using the Ensembl database at release 100. The process described in the following was performed independently for OPC and mature oligodendrocyte clusters. For each data set, the list of genes was intersected with the organism matched list of SRF targets. Next, genes that did not pass the significance threshold for the adjusted p-values at cut-off of 0.05 were discarded from subsequent analysis. Fold-changes were normalized for each data set to obtain a comparable scale and to mitigate project-dependent fluctuations of fold-changes due to varying sample counts.
Allen Brain Atlas analysis
For secondary analysis of Fgfl 7-positive cells and corresponding expression levels, raw gene expression counts were downloaded for 2 datasets from the Allen Brain Atlas data portal (portal. brainmap. org/). The first comprised human Ml cortex samples profiled with lOx 3' gene expression yielding -77,000 single-nucleus transcriptomes, and the second comprised mouse hippocampus and cortex samples profiled with Smart-seq2 yielding -77,000 single-cell transcriptomes. Each was loaded and analyzed separately using the R programming language (v4.0.5) together with the packages Seurat (v4.0.6), SeuratWrappers (v0.3.0), bioconductor-mast (vl .16.0), monocle3 (vl.0.0), data.table (vl .14.2), Isa (vO.73.2), umap (0.2.7.0) and ggplot2 (v3.3.5). The official metadata and annotations provided were used to construct Seurat objects and dissect the data. A minimal number of 250 expressed features per cell and at least 100 cells detected per feature were enforced to filter the raw matrices. The official Seurat analysis workflow was followed: normalization (step 1), selection of variable features using VST normalization and setting of the number of features to 2,000 (step 2), scaling and centering of informative features (step 3), principal-component analysis with 50 principal components (step 4), computation of the nearest-neighbor graph using 20 dimensions (step 5), identification of clusters in the graph when setting the resolution to 0.8 and the number of starts and iterations to 10 and 15, respectively (step 6), and UMAP-based dimension reduction with 20 input dimensions, the number of neighbors set to 30, the minimum distance set to 0.3 and the spread set to 1 (step 7). Fgfl 7-positive cells were defined by a normalized expression count larger than zero. For cell type cluster or region-specific analyses, the respective cells were extracted and analysis steps 2-7 from above were repeated. To compute enriched markers for the different subclusters, the FindAllMarkers function from Seurat was used together with the MAST package, requiring an absolute log-transformed fold change of at least 0.25 and a minimum percentage expressed of 10%. To find a global transcriptome signature for the Fgfl 7-positive cells, the expression of Fgfl 7 was correlated to that of all other expressed genes in selected clusters using the cosine similarity. Lists of genes ranked by decreasing correlation were then used to perform GSEA using GeneTrail 3.0. Statistics and reproducibility
All non-RNAseq analysis was done using GraphPad Prism 8 and 9 with the indicated statistical tests. Gene set enrichment and over-representation analyses were performed using GeneTrail 3. Category source databases were adjusted independently, and C- values were corrected using the FDR- controlling procedure by Benjamini -Hochberg and considered significant if smaller than 0.05 Box plots show the median and 25th— 75th percentile range, and the whiskers indicate values up to 1.5 times the interquartile range. In in vivo experiments with mice of the same age, mice were divided into groups to achieve a similar average weight across groups. No other criteria were considered and, as such, were randomized. Data in FIGS. IB, IE, II, IK, 4L and 6A-6F are combined raw data from 2 independent cohorts of mice. Data in FIGS. 1L-1O, 2E, 21, 4B, 4C, 4F, 4H, 41, 4S, and 7A, 12A, 12B, 12D-12F, 12H, 121, 14A, 14B, 14D, and 14E were successfully replicated in 2 independent experiments.
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All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled relevant fields are intended to be within the scope of the following claims.

Claims

1. A method of treating an age-related disease or condition, comprising: exposing one or more of a subject’s central nervous system cells to Fgfl7 and/or to a Fgfl7 agonist wherein said exposing prevents and or treats said age-related disease or condition.
2. The method of claim 1, wherein said age-related disease or condition is selected from the group of cognitive aging, neurodegeneration, or demyelination.
3. The method of claim 1 or 2, wherein said subject is a human subject.
4. The method of any of claims 1-3, wherein said Fgfl7 agonist is a Fgfl7 peptide or fragment thereof.
5. The method of any of claims 1-3, wherein said Fgfl7 agonist is an agonist antibody.
6. The method of any of claims 1-3, wherein said Fgfl7 agonist is a nucleic acid.
7. The method of any of claims 1-6, wherein said Fgfl7 agonist increases Fgfl7 expression.
8. The method of any of claims 1-7, wherein said exposing is in vivo exposing, ex vivo exposing or in vitro exposing.
9. The method of any of claims 1-8, wherein said exposing is selected from the group consisting of local administration, topical administration, intrathecal administration, intraparenchymal administration, intracerebroventrical administration, intravenous administration, intraarterial administration, intrapulmonary administration, and oral administration.
10. The method of any of claims 1-9, wherein said exposing comprises combination therapy with an agent that increases Fgfl7 function.
11. A composition, comprising: a. a Fgfl7 peptide and/or a Fgfl 7 agonist; and b. a pharmaceutically acceptable carrier.
12. Fgfl 7 and/or a Fgfl 7 agonist for use in treating an age-related disease or condition, comprising: exposing one or more of a subject’s central nervous system cells wherein said exposing prevents and or treats said age-related disease or condition.
13. A composition comprising Fgfl 7 and/or a Fgfl 7 agonist for use in treating an age-related disease or condition, comprising: exposing one or more of a subject’s central nervous system cells wherein said exposing prevents and or treats said age-related disease or condition.
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