WO2025010238A2 - Méthodes et aspects associés de traitement et de diagnostic de protéinopathies - Google Patents

Méthodes et aspects associés de traitement et de diagnostic de protéinopathies Download PDF

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WO2025010238A2
WO2025010238A2 PCT/US2024/036483 US2024036483W WO2025010238A2 WO 2025010238 A2 WO2025010238 A2 WO 2025010238A2 US 2024036483 W US2024036483 W US 2024036483W WO 2025010238 A2 WO2025010238 A2 WO 2025010238A2
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fgl1
variant
functional portion
protein
subject
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WO2025010238A3 (fr
Inventor
Xiaobo Mao
Ted M. Dawson
Valina L. Dawson
Wenjing Wang
Junkai HU
Mingyao YING
Jun Wang
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Johns Hopkins University
University of Michigan System
New York University NYU
University of Michigan Ann Arbor
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Johns Hopkins University
University of Michigan System
New York University NYU
University of Michigan Ann Arbor
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    • 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
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/761—Adenovirus
    • 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
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/363—Fibrinogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/75—Fibrinogen

Definitions

  • AD Alzheimer’s disease
  • LBD Lewy body dementia
  • PD Parkinson’s disease
  • AD is the most common neurodegenerative disease and dementia, which is characterized with widely distributed neurofibrillary tangles.
  • Misfolded microtubule-associated protein tau is the major component of these tangles.
  • tau-pathology-related diseases such as Pick’s disease (PiD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Argyrophilic gain disease (AGD) and chronic traumatic encephalopathy (CTE).
  • LBD is the second most common dementia, including dementia with Lewy body (DLB) and PD with dementia (PDD), with substantial ⁇ - amyloid plaques, tau tangles, and ⁇ -synuclein ( ⁇ -syn) inclusions.
  • PD is the second most common neurodegenerative disease with motor and non-motor dysfunction, which is correlated to ⁇ -syn pathology distribution.
  • ⁇ -syn and tau are prion-like proteins from tissue-to-tissue, region-to-region, and cell-to-cell, that significantly drive disease progression. To date, however, therapies to treat Atty Dkt.
  • the present disclosure provides therapeutic proteins, namely, fibrinogen- like protein 1 (FGL1) for inhibiting prion-like proteinopathies, including Alzheimer’s disease (AD), Lewy body dementia (LBD) and Parkinson’s disease (PD), among other aging-related neurodegenerative disorders.
  • FGL1 fibrinogen-like protein 1
  • the therapeutic proteins elevate FGL1 protein levels in the brains of patients to inhibit the prion-like proteinopathies, an important step in the pathogenesis of PD, LBD, AD, and other neurological disorders via various routes of delivery, including AAV-mediated FGL1 gene delivery, exosome-mediated FGL1 protein delivery, and blood exchange.
  • FGL1 is also used as a blood biomarker to predict the risk of proteinopathy in, for example, PD, LBD and AD patients, as well as the normal aging population.
  • the method includes administering an effective amount of a fibrinogen-like protein 1 (FGL1), or a functional portion or variant thereof, to the subject having the proteinopathy, which effective amount elevates a level of the FGL1, or the functional portion or variant thereof, in a brain of the subject sufficient to inhibit a binding interaction between a pathogenic protein associated with the proteinopathy and a lymphocyte-activation protein 3 (LAG3) present in the brain of the subject, thereby treating the proteinopathy in the subject.
  • a method of inhibiting a binding interaction is presented. The method includes contacting an effective amount of a fibrinogen-like protein 1 (FGL1), or a functional portion or variant thereof, with a Atty Dkt.
  • No.0184.0261-PCT (C17635_P17635-02) composition that comprises a pathogenic protein and a lymphocyte-activation protein 3 (LAG3), wherein the effective amount of the FGL1, or the functional portion or variant thereof, reduces binding of the pathogenic protein to the LAG3 in the composition relative to the composition in the absence of the FGL1, or the functional portion or variant thereof, thereby inhibiting the binding interaction.
  • LAG3 lymphocyte-activation protein 3
  • the method includes contacting an effective amount of an age-related rejuvenation factor, or a functional portion or variant thereof, with a composition that comprises a pathogenic protein and a lymphocyte-activation protein 3 (LAG3), wherein the effective amount of the age-related rejuvenation factor, or the functional portion or variant thereof, reduces binding of the pathogenic protein to the LAG3 in the composition relative to the composition in the absence of the age- related rejuvenation factor, or the functional portion or variant thereof, thereby inhibiting the binding interaction.
  • a composition that comprises a pathogenic protein and a lymphocyte-activation protein 3 (LAG3)
  • the present disclosure provides a use of a fibrinogen-like protein 1 (FGL1), or a functional portion or variant thereof, or a synthetic polynucleotide encoding the FGL1, or the functional portion or variant thereof, for the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a proteinopathy.
  • FGL1 fibrinogen-like protein 1
  • a synthetic polynucleotide encoding the FGL1, or the functional portion or variant thereof for the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a proteinopathy.
  • EVs exosome vesicles
  • the method includes transfecting one or more induced pluripotent stem (iPS) cells with a synthetic polynucleotide encoding a fibrinogen-like protein 1 (FGL1), or a functional portion or variant thereof, such that the iPS cells express the FGL1, or the functional portion or variant thereof, to produce transfected iPS cells; and purifying one or more EVs that comprise the FGL1, or the functional portion or variant thereof, from the transfected iPS cells to produce purified EVs that comprise the FGL1, or the functional portion or variant thereof, thereby forming the EVs.
  • the iPS cells comprise human iPS cells.
  • a method of generating a recombinant adeno-associated virus (AAV) comprising an AAV capsid includes culturing a host cell containing: (a) a nucleic acid sequence encoding an Atty Dkt.
  • No.0184.0261-PCT (C17635_P17635-02) AAV capsid protein; (b) a functional rep gene; (c) a minigene comprising AAV inverted terminal repeats (ITRs) and a transgene encoding a fibrinogen-like protein 1 (FGL1), or a functional portion or variant thereof; and (d) sufficient helper functions to permit packaging of the minigene into the AAV capsid; and purifying the AAV capsid from the host cell, thereby generating the recombinant AAV comprising the AAV capsid.
  • ITRs AAV inverted terminal repeats
  • FGL1 fibrinogen-like protein 1
  • the method includes determining a fibrinogen-like protein 1 (FGL1) expression level in a sample obtained from a subject, which sample is indicative of the FGL1 expression level in a brain of the subject; and identifying the subject as likely to develop, or having, the proteinopathy when the FGL1 expression level in the brain of the subject is below a control expression level, or identifying the subject as unlikely to develop, or not having, the proteinopathy when the FGL1 expression level in the brain of the subject is above the control expression level, thereby the predicting the risk of the proteinopathy in the subject.
  • FGL1 fibrinogen-like protein 1
  • the method includes administering an effective amount of an exogenous FGL1, or a functional portion or variant thereof, to the subject when the FGL1 expression level in the brain of the subject is below the control expression level, which effective amount elevates a level of the exogenous FGL1, or the functional portion or variant thereof, in the brain of the subject sufficient to inhibit a binding interaction between a pathogenic protein associated with the proteinopathy and a lymphocyte-activation protein 3 (LAG3) present in the brain of the subject.
  • a pharmaceutical composition comprises a fibrinogen-like protein 1 (FGL1), or a functional portion or variant thereof, of use in the treatment of a proteinopathy in a subject.
  • the pharmaceutical composition comprises exosome vesicles (EVs) that comprise the FGL1, or the functional portion or variant thereof.
  • the pharmaceutical composition comprises adeno-associated virus (AAV) capsids that comprise a transgene encoding the FGL1, or the functional portion or variant thereof.
  • the Atty Dkt. No.0184.0261-PCT (C17635_P17635-02) pharmaceutical composition comprises one or more blood components suitable for administering the pharmaceutical composition to the subject via blood exchange.
  • the proteinopathy comprises Alzheimer's disease (AD), Parkinson's disease (PD), and/or Lewy body dementia (LBD).
  • a kit comprises the pharmaceutical composition.
  • the composition is disposed in vitro.
  • the composition is disposed in vivo.
  • a brain of a subject comprises the composition.
  • the subject is a human subject.
  • the subject comprises a proteinopathy associated with the pathogenic protein.
  • the proteinopathy comprises an ⁇ -synucleinopathy.
  • the effective amount of the FGL1, or the functional portion or variant thereof substantially treats the proteinopathy in the subject.
  • the contacting step comprises administering the effective amount of the FGL1, or the functional portion or variant thereof, to the subject using at least one administration technique.
  • the administration technique is selected from the group consisting of: a blood exchange, a stereotaxic injection, an intrastriatal injection, an intramuscular injection, an intradermal injection, a transmucosal injection, a subcutaneous injection, and an intravenous injection.
  • a cell comprises the LAG3.
  • the cell comprises a mammalian cell.
  • the cell comprises a neuronal cell.
  • the effective amount of the FGL1, or the functional portion or variant thereof inhibits uptake of the pathogenic protein into the cell and/or inhibits spreading of the pathogenic protein to other cells in the composition.
  • An aggregation comprises the pathogenic protein.
  • the pathogenic protein comprises a prion-like protein.
  • the prion- like protein comprises a monomeric ⁇ -synuclein protein ( ⁇ -syn), an oligomeric ⁇ -syn, a preformed-fibrillar (PFF) ⁇ -syn, a functional portion or variant thereof, or any combination thereof.
  • the prion-like protein comprises a monomeric a microtubule- associated protein tau (tau), an oligomeric tau, a preformed-fibrillar (PFF) tau, a functional portion or variant thereof, or any combination thereof.
  • tau microtubule- associated protein tau
  • PFF preformed-fibrillar
  • the FGL1, or the functional portion or variant thereof comprises a recombinant FGL1, or functional portion or variant thereof, or an exogenous FGL1, or functional portion or Atty Dkt. No.0184.0261-PCT (C17635_P17635-02) variant thereof.
  • the FGL1, or the functional portion or variant thereof comprises a splice variant.
  • a vector comprises the FGL1, or the functional portion or variant thereof. The vector is selected from the group consisting of: an exosome vesicle, a liposome, a lipid nanocrystal, and a lipid nanoparticle.
  • a vector comprises a synthetic polynucleotide encoding the FGL1, or the functional portion or variant thereof, and wherein the method comprises contacting the vector with the composition such that the synthetic polynucleotide is expressed sufficiently to produce the effective amount of the FGL1, or the functional portion or variant thereof.
  • An expression cassette comprises the synthetic polynucleotide.
  • the vector is selected from the group consisting of: a virus, a cell, an exosome vesicle, a plasmid, a phagemid, a cosmid, a liposome, an expression cassette, a lipid nanocrystal, a lipid nanoparticle, and an artificial chromosome.
  • FIGS.1A-1E The expression of FGL1 decreases with aging.
  • A The liver FGL1 level by western blot in the young (3-month-old) and aged mice (20- month-old).
  • B The quantification of liver FGL1.
  • C The expression of FGL1 in the young and aged serum by ELISA.
  • D The plasma FGL1 level by immunoblot in the aged mice (18-month-old), young mice (2, 3-month-old) and aged mice (18-month- old) by HBE with young mice (2, 3-month-old).
  • FIGS.2A-2E Effect of FGL1 on ⁇ -syn PFF-biotin uptake.
  • A The cell binding assay in SH-SY5Y cells treated with Lag3, FGL1 and ⁇ -syn PFF biotin.
  • B The quantitative analysis of ⁇ -syn PFF-biotin binding signal
  • C Schematic showing the experimental design.
  • the primary culture of cortical neurons were treated with FGL1-human Ig and mouse ⁇ -syn PFF-biotin (1 ⁇ M) at DIV14.
  • D The cells were used for neuron, biotin and Hoechst nuclear immunostaining in each group.
  • E The Atty Dkt. No.0184.0261-PCT (C17635_P17635-02) corresponding quantitative analysis of ⁇ -syn PFF-biotin signal. Scale bar, 20 ⁇ m.
  • FIGS.3A and 3B Ratio of FGL1 detected in CSF vs plasma.
  • A Schematic showing the experimental design.
  • a near infrared (IR) dye IRDye680RD was used to label FGL1.
  • IR near infrared
  • the CSF and plasma were collected at 3, 7 and 10 days for analysis.
  • the labeled FGL1 was increased at the 7 day by plate reader.
  • FIGS.4A-4E AAV-FGL1 reduces the pathological ⁇ -synuclein in the mice cerebral cortex.
  • A Schematic showing the experimental design.
  • the cortex sections were used for FGL1 (red) and Hoechst nuclear staining (in blue) immunostaining.
  • C The corresponding quantitative analysis of FGL1 signal.
  • the cortex were immunostained for pathologic ⁇ -synuclein and Hoechst nuclear staining.
  • FIGS.5A-5G AAV-FGL1 reduces the pathological ⁇ -synuclein in the primary culture of cortical neurons.
  • A Schematic showing the experimental design. The primary culture of cortical neurons were treated with AAV-Control, AAV- FGL1 (secreted) and AAV-FGL1 (none secreted) at DIV5. The cells were treated ⁇ - syn PFF at DIV7.
  • the pathological of ⁇ -syn were tested at DIV14.
  • B, C, D, E, F The cells were used for pathological of ⁇ -syn (red), neuron (green) and Hoechst nuclear staining (blue) immunostaining in each group. The corresponding quantitative analysis of FGL1 signal.
  • the cortex was immunostained for pathologic ⁇ -synuclein (red) and Hoechst nuclear staining (blue).
  • G The corresponding quantitative analysis of pathological ⁇ -synuclein signal. Scale bars, 50 ⁇ m. One-way ANOVA with Atty Dkt.
  • FIGS.6A-6E The pathological of ⁇ -synuclein in the parabiosis groups.
  • A Schematic showing the experimental design. The 16-month-old mice were paired with 16-month-old mice as isochronic parabiosis group, and the 16- month-old mice were paired with 2-month-old mice as heterochronic parabiosis group. After one month, the paired aged mice were injected ⁇ -syn PFF (5 ⁇ g/ml) in the striatum.
  • FIGS.7A and 7B Characterization of human iPSC-EVs.
  • FIGS.8A and 8B iPS cells with enforced FGL1 expression show significantly higher level of FGL1 proteins in EVs.
  • A Human iPS cells were transfected with synthetic FGL1 mRNA. After 24h, the level of FGL1 proteins was significantly higher than control cells transfected by GFP mRNA.
  • FIGS.9A and 9B iPS cells neon transfected with FGL1 showed secreted FGL1 proteins in EVs.
  • A Human iPS cells were neon transfected (1100v) with plasmid FGL1. After 72h, the level of FGL1 protein was tested in the supernate and exosome by ELISA.
  • B The ratio of the amount of exosome-FGL1 with secreted-FGL1. Atty Dkt.
  • a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
  • the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Further, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In describing and claiming the methods, systems, and computer readable media, the following terminology, and grammatical variants thereof, will be used in accordance with the definitions set forth below.
  • ⁇ -synuclein As used herein, the term “ ⁇ -synuclein,” “ ⁇ -syn,” “alpha- synuclein,” or “SNCA” refers to a protein member of the synuclein family, which also includes beta- and gamma-synuclein. Synucleins are abundantly expressed in the brain and alpha- and beta-synuclein inhibit phospholipase D2 selectively. SNCA may serve to integrate presynaptic signaling and membrane trafficking. Defects in SNCA have been implicated in the pathogenesis of, for example, Parkinson disease.
  • SNCA peptides are also a major component of amyloid plaques in the brains of patients with Alzheimer's disease. Alternatively spliced transcripts encoding different isoforms have been identified for this gene.
  • a human ⁇ -synuclein NCBI Gene ID No. is 6622. ⁇ -synuclein can be present in various forms, such as monomeric ⁇ -syn, oligomeric ⁇ - syn, and preformed-fibrillar (PFF) ⁇ -syn. Atty Dkt.
  • Binding typically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; “indirect” binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can be assessed in any of a variety of contexts—including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and/or in a biological system or cell).
  • determining FGL1 expression refers to measuring FGL1 expression levels by any means or methods known to the person skilled in the art, in which FGL1 expression may be determined on a protein or mRNA level. Typically, the means or methods to measure FGL1 expression are able to quantitatively detect or determine FGL1 expression, e.g., human FGL1 expression. Quantitatively detecting or determining FGL1 expression includes detecting or determining relative or absolute amounts.
  • Methods suitable for quantitatively detecting and determining FGL1 expression include contacting the sample with an anti-FGL1 antibody and detecting the binding between FGL1 and the antibody, such as by ELISA or by IHC, or detecting the amount of mRNA encoding FGL1 protein in the sample, such as by PCR.
  • Elevate refers to an increase by at least about 10% as compared to a control, for example an increase by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 75%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 200%, or at least about 300%, or any integer decrease between 10-300% as compared to a control.
  • a “control” is a reference value or a reference sample for base line expression from at least one subject not having cancer or a non-cancerous tissue sample, wherein the non- cancerous tissue sample preferably originates from the same organ as the cancer and more preferably from the cancer patient.
  • the methods and sample types used for establishing a cut-off value of a marker like FGL1 and for measuring the FGL1 level in a sample obtained from an individual or patient to be analyzed match each Atty Dkt. No.0184.0261-PCT (C17635_P17635-02) other or are the same.
  • Cut-off values i.e., values above which elevated expression or overexpression (e.g., elevated expression of FGL1 compared to a control) is acknowledged can be obtained in a control group.
  • the control group on which the cut-off value is based is chosen to match the group of individuals/patients under investigation.
  • Encode refers broadly to any process whereby the information in a polymeric macromolecule is used to direct the production of a second molecule that is different from the first.
  • a DNA molecule can encode an RNA molecule (e.g., by the process of transcription that uses a DNA- dependent RNA polymerase enzyme).
  • an RNA molecule can encode a polypeptide, as in the process of translation.
  • an RNA molecule can encode a DNA molecule, e.g., by the process of reverse transcription incorporating an RNA-dependent DNA polymerase.
  • a DNA molecule can encode a polypeptide, where it is understood that “encode” as used in that case incorporates both the processes of transcription and translation.
  • Exosome As used herein, “exosome” or “exosome vesicle” or “EV’ refers to a nanometer-size extracellular vesicle that transports proteins, RNAs, and/or lipids from a cellular origin.
  • Expression when used in reference to a nucleic acid herein, refers to one or more of the following events: (1) production of an RNA transcript of a DNA template (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or 3′ end formation); (3) translation of an RNA into a polypeptide; and/or (4) post- translational modification of a polypeptide.
  • Expression Cassette As used herein, the term "expression cassette” or "expression vector” as used herein refers to a nucleotide sequence which is capable of affecting expression of a protein coding sequence in a host compatible with such sequences.
  • Expression cassettes typically include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, Atty Dkt. No.0184.0261-PCT (C17635_P17635-02) e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be included, e.g., enhancers. "Operably linked', refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence.
  • expression cassettes include plasmids, recombinant viruses, any form of a recombinant vector, and the like.
  • Fibrinogen-like Protein 1 As used herein, the term “fibrinogen-like protein 1” or “FGL1” (synonyms LFIRE-1, HFREPI, Hepassocin, HP-041) refers to a protein of the fibrinogen family of proteins, which also includes fibrinogen-like protein 2 and clotting factors V, VIII, and XIII. However, the term “FGL1 expression” relates to both, protein and mRNA expression in humans unless otherwise stated. FGL1 is encoded by the FGL1 gene. FGL-1 contains a fibrinogen related domain in its C- terminal portion, which contains the four conserved cysteines that are common to all members of the fibrinogen family.
  • FGL-1 lacks the platelet-binding site, cross-linking region, and thrombin-sensitive site which allow the other members of the fibrinogen family to aid in fibrin clot formation.
  • FGL1 is upregulated in regenerating liver and is abundantly associated with the fibrin matrix after clot formation. While the majority of FGL1 is found in plasma, approximately 20% of FGL1 remains in the serum after blood coagulation.
  • Human FGL1 precursor has the amino acid sequence as provided by the NCBI Reference Sequence database under ID number NP_004458.3, wherein amino acids 1 to 22 correspond to the signal peptide and amino acids 23 to 312 correspond to the mature protein.
  • the C-terminal globular domain of fibrinogen corresponds to amino acids 78 to 304.
  • the FGL1 is mammalian. In some embodiments, the FGL1 is murine, porcine, ovine, bovine, human, or combinations thereof.
  • Functional Portion Or Variant Thereof refers to a modified or unmodified portion or fragment, or a sequence variant (e.g., amino acid sequence variant or encoding polynucleotide sequence variant) of FGL1 retains its ability to inhibit a binding interaction between a pathogenic protein associated with a proteinopathy (e.g., a prion-like protein, such as ⁇ -synuclein or tau) and a lymphocyte-activation protein 3 (LAG3) present in the brain of a subject.
  • a pathogenic protein associated with a proteinopathy e.g., a prion-like protein, such as ⁇ -synuclein or tau
  • LAG3 lymphocyte-activation protein 3
  • a “functional portion or variant thereof” of FGL1 has at least 80%, 85%, 90%, 95%, 99% identity with a wild-type FGL1 amino acid sequence.
  • a “functional portion or variant thereof” of a polynucleotide encoding FGL1 has at least 80%, 85%, 90%, 95%, 99% identity with a wild-type FGL1 gene.
  • a “functional portion” of FGL1 or polynucleotide encoding FGL1 can comprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of a corresponding full-length FGL1 or polynucleotide encoding FGL1 molecule.
  • Gene refers to a DNA locus of heritable genomic sequence which affects an organism's trait by being expressed as a functional product or by regulation of gene expression.
  • Genes and polynucleotides may include introns and exons as in genomic sequence, or just the coding sequences as in cDNAs, such as an open reading frame (ORF), comprising a start codon (methionine codon) and a translation stop codon. Genes and polynucleotides can also include regions that regulate their expression, such as transcription initiation, translation and transcription termination. Thus, also included are regulatory elements such as a promoter. [0039] Host cell: As used herein, the phrase “host cell” refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced.
  • host cells may be used to produce the fibrinogen-like protein 1 (FGL1), or a functional portion or variant thereof, (e.g., wild-type, recombinant, or modified protein molecules) referenced herein by standard production techniques.
  • FGL1 fibrinogen-like protein 1
  • a functional portion or variant thereof e.g., wild-type, recombinant, or modified protein molecules referenced herein by standard production techniques.
  • progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
  • host cells include any prokaryotic and eukaryotic cells suitable for expressing an exogenous DNA (e.g., a recombinant nucleic acid sequence).
  • exemplary cells include those of prokaryotes and eukaryotes (single-cell or multiple- cell), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), Atty Dkt. No.0184.0261-PCT (C17635_P17635-02) mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P.
  • the cell is a human, monkey, ape, hamster, rat, or mouse cell.
  • the cell is eukaryotic and is selected from the following cells: Chinese Hamster Ovary or CHO cells (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS cells (e.g., COS-7), retinal cells, Vero cells, CV1 cells, kidney cells (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa cells, HepG2 cells, W138 cells, MRC 5 cells, Colo205 cells, HB 8065 cells, HL-60 cells, BHK21 cells, Jurkat cells, Daudi cells, A431 (epidermal) cells, CV-1 cells, U937 cells, 3T3 cells, L cells, C127 cells, SP2/0 cells, NS-0 cells, MMT 060562 cells, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and a cell line derived from an aforementioned cell.
  • the cell comprises one or more viral genes, e.g., a retinal cell that expresses a viral gene (e.g., a PER.C6TM cell).
  • Isolated refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) designed, produced, prepared, and/or manufactured by the hand of man.
  • Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated.
  • isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
  • a substance is “pure” if it is substantially free of other components.
  • a substance may still be considered “isolated” or even “pure”, after having been combined with certain other components such as, for Atty Dkt. No.0184.0261-PCT (C17635_P17635-02) example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients.
  • carriers or excipients e.g., buffer, solvent, water, etc.
  • a biological polymer such as a polypeptide or polynucleotide that occurs in nature is considered to be “isolated” when, a) by virtue of its origin or source of derivation is not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; c) is expressed by or is otherwise in association with components from a cell or other expression system that is not of the species that produces it in nature.
  • a polypeptide that is chemically synthesized or is synthesized in a cellular system different from that which produces it in nature is considered to be an “isolated” polypeptide.
  • a polypeptide that has been subjected to one or more purification techniques may be considered to be an “isolated” polypeptide to the extent that it has been separated from other components a) with which it is associated in nature; and/or b) with which it was associated when initially produced.
  • Lymphocyte-Activation Protein 3 refers to an inhibitory molecule involved in the regulation of T cell activation, proliferation and homeostasis. It is also referred to as CD223.
  • LAG3 is a CD4-related activation-induced cell surface molecule that acts as an immune checkpoint receptor. LAG3 is expressed on activated T cells, natural killer cells (NK cells), B cells and plasmacytoid dendritic cells (pDCs). The protein negatively regulates proliferation, activation and homeostasis in a similar fashion to CTLA-4 and PD-1. It has been reported to play a role in Treg suppressive function and helps maintaining CD8+ T cell in a tolerogenic state.
  • LAG3 is structurally related to CD4 and binds with high affinity to MHC class II molecules.
  • a single lysine residue (K468) within a conserved “KIEELE” motif is essential for interaction with downstream signaling molecules. It has four extracellular Ig-like domains, with conserved structural motifs between the D1 and D3 domains as well as the D2 and D4 domains.
  • the first domain is an Ig-like V-type domain and the following three Atty Dkt. No.0184.0261-PCT (C17635_P17635-02) domains are Ig-like C2-type domains.
  • the human LAG3 has approximately 70% homology with murine LAG3.
  • nucleic acid refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base-pairing.
  • Nucleic acids can also include nucleotide analogs (e.g., bromodeoxyuridine (BrdU)), and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages).
  • nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, cfDNA, ctDNA, or any combination thereof.
  • Prion-like Protein refers to a neurodegenerative disease-related protein that shares similarities with prion replication and propagation processes, but which has noninfectious characteristics, unlike a prion.
  • Protein is used interchangeably with “polypeptide” and refers to polymers of amino acids of any length. These terms also include proteins that are post-translationally modified through reactions that include, but are not limited to, glycosylation, acetylation, phosphorylation, glycation or protein processing. Modifications and changes, for example fusions to other proteins, amino acid sequence substitutions, deletions or insertions, can be made in the structure of a polypeptide while the molecule maintains its biological functional activity.
  • Proteinopathy As used herein, “proteinopathy” or “protein conformational disorder,” or “protein misfolding disease,” is a class of diseases in which certain proteins become structurally abnormal, and thereby disrupt the function of cells, tissues and organs of the body.
  • proteinopathies include diseases such as Creutzfeldt–Jakob disease and other prion diseases, Alzheimer's disease, Parkinson's disease, Lewy body dementia (LBD), amyloidosis, multiple system atrophy, and a wide range of other neurodegenerative disorders.
  • the proteinopathy is “ ⁇ -synucleinopathy” which are a class of diseases involving misfolded prion-like neuronal protein ⁇ -synuclein.
  • an agent or entity is “pure” if it is substantially free of other components. For example, a preparation that contains more than about 90% of a particular agent or entity is typically considered to be a pure preparation. In some embodiments, an agent or entity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.
  • Recombinant As used herein, the term “recombinant” or “modified” is intended to refer to polypeptides (e.g., a fibrinogen-like protein 1 (FGL1), or a functional portion or variant thereof) that are designed, engineered, prepared, expressed, created or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell, polypeptides isolated from a recombinant, combinatorial polypeptide library or polypeptides prepared, expressed, created or isolated by any other means that involves splicing selected sequence elements to one another. In some embodiments, one or more of such selected sequence elements is found in nature.
  • polypeptides e.g., a fibrinogen-like protein 1 (FGL1), or a functional portion or variant thereof
  • FGL1 fibrinogen-like protein 1
  • recombinant means such as polypeptides expressed using a recombinant expression vector transfected into a host
  • one or more of such selected sequence elements is designed in silico.
  • one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a Atty Dkt. No.0184.0261-PCT (C17635_P17635-02) natural or synthetic source.
  • one or more such selected sequence elements results from the combination of multiple (e.g., two or more) known sequence elements that are not naturally present in the same polypeptide.
  • the term "recombinant" refers to (i) nucleic acid molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) nucleic acid molecules that result from the replication of those described in (i) above.
  • the replication can be in vitro replication or in vivo replication.
  • sample refers to a tissue or organ from a subject; a cell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; or a solution containing one or more molecules derived from a cell or cellular material (e.g., a polypeptide), which is assayed as described herein.
  • a sample may also be any body fluid or excretion (for example, but not limited to, blood, urine, stool, saliva, tears, bile) that contains cells, cell components, or non- cellular fractions.
  • Subject means any member of the animal kingdom. In some embodiments, “subject” refers to humans. In some embodiments, “subject” refers to non-human animals. In some embodiments, subjects include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, the non-human subject is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a ferret, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig).
  • a mammal e.g., a rodent, a mouse, a rat, a rabbit, a ferret, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig.
  • a subject may be a transgenic animal, genetically engineered animal, and/or a clone. In some embodiments, the subject is an adult, an adolescent or an infant. In some embodiments, terms “individual” or “patient” are used and are intended to be interchangeable with “subject.”
  • Tau As used herein, the term “tau,” “microtubule associated protein tau,” or “MAPT” refers to a protein encoded by a gene whose transcript undergoes complex, regulated alternative splicing, giving rise to several mRNA species. MAPT transcripts are differentially expressed in the nervous system, depending on stage of Atty Dkt.
  • a human tau NCBI Gene ID No. is 4137. Tau can be present in various forms, such as monomeric tau, oligomeric tau, and preformed-fibrillar (PFF) tau.
  • PFF preformed-fibrillar
  • Vector in the context of FGL1 therapeutic proteins and polynucleotides encoding FGL1 therapeutic proteins refers to a biological carrier or delivery entity used to introduce the proteins or polynucleotides into target organs or cells of a subject, such as the brain of the subject.
  • a “vector” is a nucleic acid that can be used to introduce a heterologous or synthetic polynucleotide into a cell.
  • plasmid refers to a linear or circular double stranded DNA molecule into which additional nucleic acid segments can be ligated.
  • vectors are a viral vector (e.g., replication defective retroviruses, adenoviruses and adeno- associated viruses), in which additional DNA or RNA segments can be introduced into the viral genome.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno- associated viruses
  • additional DNA or RNA segments can be introduced into the viral genome.
  • viral vectors include cells, exosome vesicles, cosmids, liposomes, expression cassettes, lipid nanocrystals, lipid nanoparticles, and artificial chromosomes, among others.
  • Exemplary vectors in the context of FGL1 therapeutic proteins include exosome vesicles, liposomes, lipid nanocrystals, and lipid nanoparticles, among others.
  • the present disclosure provides therapeutic proteins, namely, fibrinogen- like protein 1 (FGL1), or functional portions or variants thereof, (collectively, sometimes referred to herein as the “FGL1 therapeutic proteins”) for inhibiting prion- like proteinopathies, including Alzheimer’s disease (AD), Lewy body dementia (LBD) and Parkinson’s disease (PD), among other aging-related neurodegenerative disorders.
  • FGL1 therapeutic proteins inhibit the prion-like Atty Dkt. No.0184.0261-PCT (C17635_P17635-02) proteinopathies in the brains of patients, thereby interfering with the pathogenesis of these and other misfolded protein-related neurological disorders.
  • FGL1 can also be used as a blood biomarker to predict the risk of proteinopathy in AD, LBD, PD, and other proteinopathy patients, as well as in the normal aging population.
  • AAV adeno-associated virus
  • FGL1 can also be used as a blood biomarker to predict the risk of proteinopathy in AD, LBD, PD, and other proteinopathy patients, as well as in the normal aging population.
  • AD Alzheimer's disease
  • PGP progressive supranuclear palsy
  • CBD corticobasal degeneration
  • APD Argyrophilic gain disease
  • CTE chronic traumatic encephalopathy
  • LBD is another frequently observed dementia, including dementia with Lewy body (DLB) and PD with dementia (PDD).
  • ⁇ -amyloid plaques ⁇ -amyloid plaques
  • tau tangles ⁇ -synuclein ( ⁇ -syn) inclusions.
  • ⁇ -syn ⁇ -synuclein
  • PD is yet another commonly observed neurodegenerative disease with motor and non-motor dysfunction. It is correlated to ⁇ -syn pathology distribution.
  • ⁇ - syn and tau are prion-like proteins that spread from tissue-to-tissue, region-to-region, and cell-to-cell, thereby significantly driving disease progression.
  • PFF preformed fibrillar
  • the inoculation of, for example, a preformed fibrillar (PFF) of ⁇ -syn and tau can successfully recapitulate neurodegenerative disease progression, including pathology propagation, behavioral deficit phenotypes, neuroinflammation, and neurodegeneration.
  • PFF preformed fibrillar
  • Lymphocyte-activation gene 3 is the receptor of, for example, ⁇ -syn PFF, and depletion and inhibition of LAG3 significantly reduces the pathogenic ⁇ -syn transmission in vitro and in vivo.
  • FGL1 is Atty Dkt. No.0184.0261-PCT (C17635_P17635-02) a major inhibitory ligand of LAG3. Accordingly, in some embodiments, the FGL1 therapeutic proteins of the present disclosure act as aging-related rejuvenation factors that block pathogenic proteins uptake and propagation via LAG3 and other pathways.
  • the present disclosure provides methods of treating a proteinopathy (e.g., AD, LBD, PD, or the like) in a subject (e.g., a human subject).
  • the methods include administering an effective amount of a fibrinogen-like protein 1 (FGL1), or a functional portion or variant thereof, to the subject having the proteinopathy.
  • the effective amount elevates a level of the FGL1, or the functional portion or variant thereof, in a brain of the subject sufficient to inhibit a binding interaction between a pathogenic protein associated with the proteinopathy (e.g., ⁇ - syn, tau, etc.) and a lymphocyte-activation protein 3 (LAG3) present in the brain of the subject.
  • a pathogenic protein associated with the proteinopathy e.g., ⁇ - syn, tau, etc.
  • LAG3 lymphocyte-activation protein 3
  • a neuronal cell comprises the LAG3 on its surface.
  • the present disclosure provides methods of inhibiting a binding interaction.
  • the methods include contacting an effective amount of a fibrinogen-like protein 1 (FGL1), or a functional portion or variant thereof, with a composition that comprises a pathogenic protein (e.g., a prion-like protein, such as ⁇ -syn, tau, etc.) and a lymphocyte-activation protein 3 (LAG3).
  • the effective amount of the FGL1, or the functional portion or variant thereof reduces binding of the pathogenic protein to the LAG3 in the composition relative to the composition in the absence of the FGL1, or the functional portion or variant thereof.
  • the pathogenic protein is present as an aggregation of multiple pathogenic proteins.
  • the contacting step comprises administering the effective amount of the FGL1, or the functional portion or variant thereof, to the subject using an administration technique, such as a blood exchange, a stereotaxic injection, an intrastriatal injection, an intramuscular injection, an intradermal injection, a transmucosal injection, a subcutaneous injection, or an intravenous injection, among other techniques.
  • the effective amount of the FGL1, or the functional portion or variant thereof typically inhibits uptake of the pathogenic protein into the cell and/or inhibits spreading of the pathogenic protein to other cells in the composition. Atty Dkt.
  • the present disclosure provides methods of inhibiting a binding interaction.
  • the methods include contacting an effective amount of an age-related rejuvenation factor, or a functional portion or variant thereof, (e.g., FGL1 or another age-related rejuvenation factor) with a composition that comprises a pathogenic protein (e.g., ⁇ -syn, tau, etc.) and a lymphocyte-activation protein 3 (LAG3).
  • an age-related rejuvenation factor or a functional portion or variant thereof, (e.g., FGL1 or another age-related rejuvenation factor)
  • a composition that comprises a pathogenic protein (e.g., ⁇ -syn, tau, etc.) and a lymphocyte-activation protein 3 (LAG3).
  • a pathogenic protein e.g., ⁇ -syn, tau, etc.
  • LAG3 lymphocyte-activation protein 3
  • the present disclosure provides a use of a fibrinogen-like protein 1 (FGL1), or a functional portion or variant thereof, or a synthetic polynucleotide encoding the FGL1, or the functional portion or variant thereof, for the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a proteinopathy (e.g., AD, LBD, PD, or the like).
  • a proteinopathy e.g., AD, LBD, PD, or the like.
  • the present disclosure provides methods of forming exosome vesicles (EVs).
  • the methods include transfecting induced pluripotent stem (iPS) cells (e.g., human iPS cells) with a synthetic polynucleotide encoding a fibrinogen-like protein 1 (FGL1), or a functional portion or variant thereof, such that the iPS cells express the FGL1, or the functional portion or variant thereof, to produce transfected iPS cells; and purifying EVs that comprise the FGL1, or the functional portion or variant thereof, from the transfected iPS cells to produce purified EVs that comprise the FGL1, or the functional portion or variant thereof.
  • iPS induced pluripotent stem
  • FGL1 fibrinogen-like protein 1
  • the present disclosure provides methods of generating a recombinant adeno-associated virus (AAV) comprising an AAV capsid.
  • the methods include culturing a host cell containing: (a) a nucleic acid sequence encoding an AAV capsid protein; (b) a functional rep gene; (c) a minigene comprising AAV inverted terminal repeats (ITRs) and a transgene encoding a fibrinogen-like protein 1 (FGL1), or a functional portion or variant thereof; and (d) sufficient helper functions to permit packaging of the minigene into the AAV capsid; and purifying the AAV capsid from the host cell.
  • ITRs AAV inverted terminal repeats
  • FGL1 fibrinogen-like protein 1
  • the present disclosure provides methods of predicting a risk of a proteinopathy (e.g., an aging-associated neurodegeneration, such as AD, LBD, PD, or the like) in a subject.
  • the methods include determining a fibrinogen-like protein 1 (FGL1) expression level in a sample obtained from a subject. The sample is indicative of the FGL1 expression level in a brain of the subject.
  • FGL1 fibrinogen-like protein 1
  • the methods also include identifying the subject as likely to develop, or having, the proteinopathy when the FGL1 expression level in the brain of the subject is below a control expression level, or identifying the subject as unlikely to develop, or not having, the proteinopathy when the FGL1 expression level in the brain of the subject is above the control expression level.
  • the methods include administering an effective amount of an exogenous FGL1, or a functional portion or variant thereof, to the subject when the FGL1 expression level in the brain of the subject is below the control expression level, which effective amount elevates a level of the exogenous FGL1, or the functional portion or variant thereof, in the brain of the subject sufficient to inhibit a binding interaction between a pathogenic protein associated with the proteinopathy (e.g., ⁇ -syn, tau, etc.) and a lymphocyte- activation protein 3 (LAG3) present in the brain of the subject.
  • a pharmaceutical composition is presented.
  • the pharmaceutical composition comprises a fibrinogen-like protein 1 (FGL1), or a functional portion or variant thereof, of use in the treatment of a proteinopathy in a subject.
  • the pharmaceutical composition comprises exosome vesicles (EVs) that comprise the FGL1, or the functional portion or variant thereof.
  • the pharmaceutical composition comprises adeno-associated virus (AAV) capsids that comprise a transgene or other synthetic polynucleotide encoding the FGL1, or the functional portion or variant thereof.
  • the pharmaceutical composition comprises one or more blood components suitable for administering the pharmaceutical composition to the subject via blood exchange.
  • the proteinopathy comprises Alzheimer's disease (AD), Parkinson's disease (PD), and/or Lewy body dementia (LBD).
  • a kit comprises the pharmaceutical composition. Atty Dkt. No.0184.0261-PCT (C17635_P17635-02) [0063]
  • the compositions of the present disclosure are disposed in vitro (e.g., in a container or the like), whereas in other embodiments, the compositions are disposed in vivo (e.g., in the brain of a subject after being administered an FGL1 therapeutic protein as described herein).
  • the FGL1, or the functional portion or variant thereof comprises a recombinant FGL1, or the functional portion or variant thereof, or an exogenous FGL1, or the functional portion or variant thereof.
  • the FGL1, or the functional portion or variant thereof comprises a splice variant.
  • a vector comprises the FGL1, or the functional portion or variant thereof.
  • the vector comprises an exosome vesicle, a liposome, a lipid nanocrystal, or a lipid nanoparticle.
  • a vector comprises a synthetic polynucleotide encoding the FGL1, or the functional portion or variant thereof.
  • the methods disclosed herein typically comprise administering the vector to a subject such that the synthetic polynucleotide is expressed sufficiently to produce the effective amount of the FGL1, or the functional portion or variant thereof, for example, in the brain of the subject.
  • an expression cassette comprises the synthetic polynucleotide.
  • the vector comprises a virus, a cell, an exosome vesicle, a plasmid, a phagemid, a cosmid, a liposome, an expression cassette, a lipid nanocrystal, a lipid nanoparticle, or an artificial chromosome.
  • vectors are polynucleotides that can be single- stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. It is generally preferred that the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions.
  • the nucleic acid vectors of the present disclosure are recombinant in which (i) nucleic acid molecules are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) nucleic acid molecules that result from the replication of those described in (i) above.
  • the replication can be in vitro replication or in vivo replication.
  • the nucleic acids can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art.
  • a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine-substituted nucleotides).
  • modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2- thiouracil, beta-D-mannosylqueosine
  • nucleic acids of the invention can be purchased from companies, such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).
  • nucleic acid vectors may be optimized (e.g., codon optimized). Without being bound to a particular theory, it is believed that optimization of the nucleic acid sequence increases the translation efficiency of the mRNA transcripts. Optimization of the nucleic acid sequence may involve substituting a Atty Dkt. No.0184.0261-PCT (C17635_P17635-02) native codon for another codon that encodes the same amino acid, but can be translated by tRNA that is more readily available within a cell, thus increasing translation efficiency.
  • the polynucleotides encoding an FGL1 therapeutic protein e.g., an expression cassette
  • an FGL1 therapeutic protein e.g., an expression cassette
  • a recombinant expression vector typically includes a genetically- modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell.
  • the vectors of the present disclosure are not naturally occurring as a whole. However, parts of the vectors can be naturally occurring.
  • the recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides.
  • the recombinant expression vectors can comprise naturally occurring, non-naturally occurring internucleotide linkages, or both types of linkages. Typically, the non- naturally occurring or altered nucleotides or internucleotide linkages do not hinder the transcription or replication of the vector.
  • the recombinant expression vector of the invention can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host.
  • Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses.
  • the vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA).
  • Bacteriophage vectors such as ⁇ GT10, ⁇ GT11, ⁇ ZapII (Stratagene), ⁇ EMBL4, and ⁇ NM1149, also can be used. Examples of plant Atty Dkt.
  • No.0184.0261-PCT (C17635_P17635-02) expression vectors include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech).
  • animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech).
  • the plasmid can be one of the pcDNA3 family of plasmids known in the art.
  • the plasmid can be the pNGVL4a expression vector.
  • the recombinant expression vectors of the present disclosure can be prepared using standard recombinant DNA techniques as known to those of ordinary skill in the art.
  • Constructs of expression vectors which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell.
  • Replication systems can be derived, e.g., from ColEl, 2 ⁇ plasmid, ⁇ , SV40, bovine papilloma virus, and the like.
  • the recombinant expression vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA-based.
  • a recombinant expression vector of the present disclosure can include one or more marker genes, which allow for selection of transformed or transfected hosts.
  • Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like.
  • Suitable marker genes for the inventive expression vectors include, for instance, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.
  • a recombinant expression vector can comprise a native or nonnative promoter operably linked to the nucleotide sequence encoding the FGL1, or the functional portion or variant thereof, or to the nucleotide sequence which is complementary to or which hybridizes to the nucleotide sequence encoding the FGL1, or the functional portion or variant thereof.
  • the selection of promoters e.g., strong, weak, inducible, tissue-specific and developmental-specific, is within the ordinary skill of the artisan.
  • the combining of a nucleotide sequence with a promoter is also within the skill of the artisan.
  • the promoter can be a non-viral Atty Dkt.
  • the present disclosure also provides a synthetic polypeptide molecule comprising at least one of the FGL1 polypeptides described herein along with at least one other polypeptide.
  • the other polypeptide can exist as a separate polypeptide of the fusion protein, or can exist as a polypeptide, which is expressed in frame (in tandem) with one of the FGL1 polypeptides described herein.
  • the other polypeptide can encode any peptidic or proteinaceous molecule, or a portion thereof. Suitable methods of making fusion proteins are known in the art, and include, for example, recombinant methods. [0100]
  • the FGL1 therapeutic proteins of the present disclosure (including functional portions and functional variants thereof) can be obtained by methods known in the art. Suitable methods of de novo synthesizing polypeptides and proteins are described in references, such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2005; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed.
  • polypeptides and proteins can be recombinantly produced using the nucleic acids described herein using standard recombinant methods. See, for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994.
  • FGL1 therapeutic proteins of the present disclosure can be isolated and/or purified from a source, such as a plant, a bacterium, an insect, a mammal, e.g., a rat, a human, etc. Methods of isolation and purification are well-known in the art.
  • the FGL1 therapeutic proteins can be commercially synthesized by companies, such as Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD), and Multiple Peptide Systems (San Diego, CA).
  • the FGL1 therapeutic proteins of the present disclosure can be synthetic, recombinant, isolated, and/or purified. Atty Dkt.
  • the present disclosure provides pharmaceutical compositions that comprise or are capable of expressing (e.g., upon administration to a subject) an FGL1 therapeutic protein described herein.
  • pharmaceutical compositions are administered or introduced into a subject, for example, a subject receiving treatment for a proteinopathy, and the FGL1 therapeutic proteins are allowed to come into contact with the one or more disease related cells or population of cells in vivo (e.g., cells comprising LAG3 on their surfaces).
  • the pharmaceutical compositions of the present disclosure include a pharmaceutically acceptable carrier.
  • the carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration.
  • the pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well known to those skilled in the art and are readily available to the public.
  • the pharmaceutically acceptable carrier is one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use.
  • the choice of carrier will be determined in part by the chemical properties of the particular pharmaceutical composition as well as by the particular method used to administer the pharmaceutical composition.
  • compositions are formulated for blood exchange, stereotaxic, intrastriatal, parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, or intraperitoneal administration. More than one route can be used to administer the pharmaceutical compositions of the present disclosure, and in certain instances, a particular route can provide an immediate and more effective response than another route.
  • injectable formulations are in accordance with the present disclosure. Effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Atty Dkt.
  • compositions of the present disclosure use synthetic or naturally occurring compounds (e.g., cationic lipids, cationic polymers, lipid-polymer hybrid systems) as carriers to deliver the FGL1 therapeutic proteins or polynucleotides encoding those proteins into target cells or organs.
  • synthetic or naturally occurring compounds e.g., cationic lipids, cationic polymers, lipid-polymer hybrid systems
  • the amount or dose of the FGL1 therapeutic proteins or polynucleotides encoding FGL1 therapeutic proteins administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the subject over a reasonable time frame.
  • the dose will be determined by the efficacy of the given FGL1 therapeutic protein or the given polynucleotide encoding FGL1 therapeutic protein and the condition of the subject, as well as the body weight of the subject to be treated.
  • the attending physician will decide the dosage of the given FGL1 therapeutic protein or the given polynucleotide encoding FGL1 therapeutic protein with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, to be administered, route of administration, and the severity of the condition being treated.
  • the present disclosure provides a pharmaceutical composition comprising the FGL1 therapeutic protein or a polynucleotide encoding an FGL1 therapeutic protein disclosed herein in combination with at least one additional biologically active agent.
  • an “active agent” and a “biologically active agent” are used interchangeably herein to refer to a chemical or biological compound that induces a desired pharmacological and/or physiological effect, wherein the effect may be prophylactic or therapeutic.
  • the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of those active agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, analogs and the like.
  • the active agent can be a biological entity, such as a virus or cell, whether naturally occurring or manipulated, such as transformed.
  • the biologically active agent may vary widely with the intended purpose for the composition.
  • the term active is art-recognized and refers to any moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject.
  • biologically active agents examples include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances which affect the structure or function of the body; or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment.
  • drugs include following: anti- inflammatory agents such as steroids, non-steroidal anti-inflammatory agents, anti- pyretic and analgesic agents, antigenic materials, and anti-viral drugs.
  • the present disclosure provides a method for treating a proteinopathy in a subject in need thereof comprising administering to the subject an effective amount of an FGL1 therapeutic protein or a polynucleotide encoding an FGL1 therapeutic protein disclosed herein in combination with one or more additional biologically active agents (e.g., either simultaneously or serially with at least one other).
  • kits that contain the compositions or pharmaceutical compositions used with the methods, as described herein, to practice those methods.
  • the kits can contain various combinations of pharmaceutical compositions and the like.
  • the kit Atty Dkt. No.0184.0261-PCT (C17635_P17635-02) can contain instructional material teaching methodologies, e.g., means to administer the compositions used to practice the methods disclosed herein, means to inject or infect cells, patients or animals with the pharmaceutical compositions of the present disclosure, means to monitor the resultant therapeutic response and assess the reaction of the individual to which the compositions have been administered, and the like.
  • the term “about,” when referring to a value can be meant to encompass variations of, in some embodiments, ⁇ 100% in some embodiments ⁇ 50%, in some embodiments ⁇ 20%, in some embodiments ⁇ 10%, in some embodiments ⁇ 5%, in some embodiments ⁇ 1%, in some embodiments ⁇ 0.5%, and in some embodiments ⁇ 0.1% from the Atty Dkt. No.0184.0261-PCT (C17635_P17635-02) specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
  • EXAMPLE 1 The protective role of aging-related FGL1 for prion-like proteinopathies [0119] INTRODUCTION [0120]
  • PFF preformed fibrillar
  • ⁇ -syn and tau have successfully recapitulated neurodegenerative disease progression, including pathology propagation, behavioral deficit phenotypes, neuroinflammation, and neurodegeneration.
  • Tremendous efforts have been made to study the cell-to-cell transmission mechanism of pathogenic ⁇ -syn and tau.
  • Lymphocyte-activation gene 3 (Lag3) is the receptor of ⁇ -syn PFF, and depletion and Atty Dkt.
  • No.0184.0261-PCT (C17635_P17635-02) inhibition of Lag3 can significantly reduce the pathogenic ⁇ -syn transmission in vitro and in vivo.
  • fibrinogen-like protein 1 FGL1 was identified a major inhibitory ligand of Lag3.
  • FGL1 is mainly secreted by liver and abundantly detected in human plasma.
  • a significant reduction of FGL1 expression in various human cells has been shown and associated with the aging process. Accordingly, FGL1 is highly related to aging.
  • the propagation of pathogenic ⁇ -syn and tau have been extensively studied, and aging can significantly induce ⁇ -syn and tau spreading. However, the underlying aging-related mechanisms remain poorly understood.
  • FGL1 is an aging-related rejuvenation factor, which can block pathogenic proteins uptake and propagation via Lag3 and other pathways.
  • the inhibitory efficacy of endogenous FGL1 decreases when FGL1 levels decline in aging.
  • HBE heterochronic blood exchange
  • RESULTS The expression of FGL1 decreases with aging
  • Previous studies have shown that FGL1 is aging-related.
  • FGL1 effectively inhibits neuronal uptake of ⁇ -syn PFF and consequential pathology propagation via Lag3
  • FGL1 is the major ligand of Lag3
  • FGL1 is brain permeable and aging-dependent mediator of ⁇ - synucleinopathy
  • FGL1 is brain permeable and aging-dependent mediator of ⁇ - synucleinopathy
  • HBE Heterochronic blood exchange
  • FGL1 can be packaged into exosome vesicles (EVs) [0134] EVs have been found as a potential carrier for the delivery of proteins, RNAs and chemical compounds to various organs.
  • EVs have shown capability to cross the blood brain barrier and deliver therapeutic loads to the brain, thus providing a potential platform for the development of brain disease therapy.
  • EVs can be purified from various human cells (e.g. induced pluripotent stem (iPS) cells, mesenchymal stem cells, etc.) and other resources.
  • iPS induced pluripotent stem
  • Fig.7 we have established a pipeline to manufacture human iPS cell-derived EVs with a panel of quality control assays.
  • Fig.8 We established a strategy to achieve enforced FGL1 expression in human iPS cells using cell transfection by synthetic mRNAs coding the FGL1 gene.
  • mice were removed after transcardial perfusion with 4% PFA in 0.1 M sodium phosphate buffer (pH 7.2), followed by post- fixation in 4% PFA overnight and transfer to 30% sucrose, before being processed for serial sections.
  • Immunohistochemistry [0146] Serial brain sections (30 ⁇ m) were prepared using microtome (Micro HM450, Thermo Fisher Scientific).
  • Plasmid FGL1 neon transfected in the iPS cells [0154] The plasmid of FGL1 (5 ⁇ g) was neon transfected (1100v, 30ms, 1 pulse) in the 1x 10 6 iPS cells. After 72 hours, collected the medium and isolated exosome for ELISA test. Atty Dkt. No.0184.0261-PCT (C17635_P17635-02) [0155] Some further aspects are also defined in the following clauses: [0156] Clause 1: A method of treating a proteinopathy in a subject.
  • the method comprising administering an effective amount of a fibrinogen-like protein 1 (FGL1), or a functional portion or variant thereof, to the subject having the proteinopathy, which effective amount elevates a level of the FGL1, or the functional portion or variant thereof, in a brain of the subject sufficient to inhibit a binding interaction between a pathogenic protein associated with the proteinopathy and a lymphocyte-activation protein 3 (LAG3) present in the brain of the subject, thereby treating the proteinopathy in the subject.
  • FGL1 fibrinogen-like protein 1
  • LAG3 lymphocyte-activation protein 3
  • Clause 4 The method of any of Clauses 1-3, wherein the administering step comprises using an administration technique is selected from the group consisting of: a blood exchange, a stereotaxic injection, an intrastriatal injection, an intramuscular injection, an intradermal injection, a transmucosal injection, a subcutaneous injection, and an intravenous injection.
  • Clause 5 The method of any of Clauses 1-4, wherein a cell comprises the LAG3.
  • Clause 6 The method of any of Clauses 1-5, wherein the cell comprises a mammalian cell.
  • Clause 7 The method of any of Clauses 1-6, wherein the cell comprises a neuronal cell.
  • Clause 8 The method of any of Clauses 1-7, wherein the effective amount of the FGL1, or the functional portion or variant thereof, inhibits uptake of the pathogenic protein into the cell and/or inhibits spreading of the pathogenic protein to other cells in the brain of the subject.
  • Clause 9 The method of any of Clauses 1-8, wherein an aggregation comprises the pathogenic protein. Atty Dkt. No.0184.0261-PCT (C17635_P17635-02)
  • Clause 10 The method of any of Clauses 1-9, wherein the pathogenic protein comprises a prion-like protein.
  • Clause 11 The method of any of Clauses 1-10, wherein the prion-like protein comprises a monomeric ⁇ -synuclein protein ( ⁇ -syn), an oligomeric ⁇ -syn, a preformed-fibrillar (PFF) ⁇ -syn, a functional portion or variant thereof, or any combination thereof.
  • Clause 12 The method of any of Clauses 1-11, wherein the prion-like protein comprises a monomeric a microtubule-associated protein tau (tau), an oligomeric tau, a preformed-fibrillar (PFF) tau, a functional portion or variant thereof, or any combination thereof.
  • Clause 13 The method of any of Clauses 1-12, wherein the FGL1, or the functional portion or variant thereof, is human.
  • Clause 14 The method of any of Clauses 1-13, wherein the FGL1, or the functional portion or variant thereof, comprises a recombinant FGL1, or functional portion or variant thereof, or an exogenous FGL1, or functional portion or variant thereof.
  • Clause 15 The method of any of Clauses 1-14, wherein the FGL1, or the functional portion or variant thereof, comprises a splice variant.
  • Clause 16 The method of any of Clauses 1-15, wherein a vector comprises the FGL1, or the functional portion or variant thereof.
  • Clause 17 The method of any of Clauses 1-16, wherein the vector is selected from the group consisting of: an exosome vesicle, a liposome, a lipid nanocrystal, and a lipid nanoparticle.
  • Clause 18 The method of any of Clauses 1-17, wherein a vector comprises a synthetic polynucleotide encoding the FGL1, or the functional portion or variant thereof, and wherein the method comprises administering the vector to the subject such that the synthetic polynucleotide is expressed sufficiently to produce the effective amount of the FGL1, or the functional portion or variant thereof, in the brain of the subject.
  • Clause 19 The method of any of Clauses 1-18, wherein an expression cassette comprises the synthetic polynucleotide. Atty Dkt. No.0184.0261-PCT (C17635_P17635-02) [0175]
  • Clause 20 The method of any of Clauses 1-19, wherein the vector is selected from the group consisting of: a virus, a cell, an exosome vesicle, a plasmid, a phagemid, a cosmid, a liposome, an expression cassette, a lipid nanocrystal, a lipid nanoparticle, and an artificial chromosome.
  • Clause 21 A method of inhibiting a binding interaction.
  • the method comprising contacting an effective amount of a fibrinogen-like protein 1 (FGL1), or a functional portion or variant thereof, with a composition that comprises a pathogenic protein and a lymphocyte-activation protein 3 (LAG3), wherein the effective amount of the FGL1, or the functional portion or variant thereof, reduces binding of the pathogenic protein to the LAG3 in the composition relative to the composition in the absence of the FGL1, or the functional portion or variant thereof, thereby inhibiting the binding interaction.
  • FGL1 fibrinogen-like protein 1
  • LAG3 lymphocyte-activation protein 3
  • Clause 24 The method of any of Clauses 21-23, wherein a brain of a subject comprises the composition.
  • Clause 25 The method of any of Clauses 21-24, wherein the subject is a human subject.
  • Clause 26 The method of any of Clauses 21-25, wherein the subject comprises a proteinopathy associated with the pathogenic protein.
  • Clause 27 The method of any of Clauses 21-26, wherein the proteinopathy comprises an ⁇ -synucleinopathy.
  • Clause 28 The method of any of Clauses 21-27, wherein the effective amount of the FGL1, or the functional portion or variant thereof, substantially treats the proteinopathy in the subject.
  • Clause 29 The method of any of Clauses 21-28, wherein the contacting step comprises administering the effective amount of the FGL1, or the functional portion or variant thereof, to the subject using at least one administration technique.
  • Atty Dkt. No.0184.0261-PCT C17635_P17635-02
  • Clause 30 The method of any of Clauses 21-29, wherein the administration technique is selected from the group consisting of: a blood exchange, a stereotaxic injection, an intrastriatal injection, an intramuscular injection, an intradermal injection, a transmucosal injection, a subcutaneous injection, and an intravenous injection.
  • Clause 31 The method of any of Clauses 21-30, wherein a cell comprises the LAG3.
  • Clause 32 The method of any of Clauses 21-31, wherein the cell comprises a mammalian cell.
  • Clause 33 The method of any of Clauses 21-32, wherein the cell comprises a neuronal cell.
  • Clause 34 The method of any of Clauses 21-33, wherein the effective amount of the FGL1, or the functional portion or variant thereof, inhibits uptake of the pathogenic protein into the cell and/or inhibits spreading of the pathogenic protein to other cells in the composition.
  • Clause 35 The method of any of Clauses 21-34, wherein an aggregation comprises the pathogenic protein.
  • Clause 36 The method of any of Clauses 21-35, wherein the pathogenic protein comprises a prion-like protein.
  • Clause 37 The method of any of Clauses 21-36, wherein the prion-like protein comprises a monomeric ⁇ -synuclein protein ( ⁇ -syn), an oligomeric ⁇ -syn, a preformed-fibrillar (PFF) ⁇ -syn, a functional portion or variant thereof, or any combination thereof.
  • ⁇ -syn monomeric ⁇ -synuclein protein
  • PFF preformed-fibrillar
  • Clause 38 The method of any of Clauses 21-37, wherein the prion-like protein comprises a monomeric a microtubule-associated protein tau (tau), an oligomeric tau, a preformed-fibrillar (PFF) tau, a functional portion or variant thereof, or any combination thereof.
  • Clause 39 The method of any of Clauses 21-38, wherein the FGL1, or the functional portion or variant thereof, is human.
  • Clause 40 The method of any of Clauses 21-39, wherein the FGL1, or the functional portion or variant thereof, comprises a recombinant FGL1, or functional Atty Dkt.
  • Clause 41 The method of any of Clauses 21-40, wherein the FGL1, or the functional portion or variant thereof, comprises a splice variant.
  • Clause 42 The method of any of Clauses 21-41, wherein a vector comprises the FGL1, or the functional portion or variant thereof.
  • Clause 43 The method of any of Clauses 21-42, wherein the vector is selected from the group consisting of: an exosome vesicle, a liposome, a lipid nanocrystal, and a lipid nanoparticle.
  • Clause 44 The method of any of Clauses 21-43, wherein a vector comprises a synthetic polynucleotide encoding the FGL1, or the functional portion or variant thereof, and wherein the method comprises contacting the vector with the composition such that the synthetic polynucleotide is expressed sufficiently to produce the effective amount of the FGL1, or the functional portion or variant thereof.
  • Clause 45 The method of any of Clauses 21-44, wherein an expression cassette comprises the synthetic polynucleotide.
  • Clause 46 The method of any of Clauses 21-45, wherein the vector is selected from the group consisting of: a virus, a cell, an exosome vesicle, a plasmid, a phagemid, a cosmid, a liposome, an expression cassette, a lipid nanocrystal, a lipid nanoparticle, and an artificial chromosome.
  • Clause 47 A method of inhibiting a binding interaction.
  • the method comprising contacting an effective amount of an age-related rejuvenation factor, or a functional portion or variant thereof, with a composition that comprises a pathogenic protein and a lymphocyte-activation protein 3 (LAG3), wherein the effective amount of the age-related rejuvenation factor, or the functional portion or variant thereof, reduces binding of the pathogenic protein to the LAG3 in the composition relative to the composition in the absence of the age-related rejuvenation factor, or the functional portion or variant thereof, thereby inhibiting the binding interaction.
  • No.0184.0261-PCT (C17635_P17635-02) functional portion or variant thereof, for the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a proteinopathy.
  • Clause 49 A method of forming exosome vesicles (EVs).
  • the method comprising: transfecting one or more induced pluripotent stem (iPS) cells with a synthetic polynucleotide encoding a fibrinogen-like protein 1 (FGL1), or a functional portion or variant thereof, such that the iPS cells express the FGL1, or the functional portion or variant thereof, to produce transfected iPS cells; and purifying one or more EVs that comprise the FGL1, or the functional portion or variant thereof, from the transfected iPS cells to produce purified EVs that comprise the FGL1, or the functional portion or variant thereof, thereby forming the EVs.
  • iPS induced pluripotent stem
  • Clause 51 The transfected iPS cells produced by the method of Clause 49 or Clause 50.
  • Clause 52 The purified EVs that comprise the FGL1, or the functional portion or variant thereof, produced by the method of Clause 49 or Clause 50.
  • Clause 53 A pharmaceutical composition comprising the purified EVs that comprise the FGL1, or the functional portion or variant thereof, produced by the method of Clause 49 or Clause 50.
  • Clause 54 A method of generating a recombinant adeno-associated virus (AAV) comprising an AAV capsid.
  • AAV adeno-associated virus
  • the method comprising: culturing a host cell containing: (a) a nucleic acid sequence encoding an AAV capsid protein; (b) a functional rep gene; (c) a minigene comprising AAV inverted terminal repeats (ITRs) and a transgene encoding a fibrinogen-like protein 1 (FGL1), or a functional portion or variant thereof; and (d) sufficient helper functions to permit packaging of the minigene into the AAV capsid; and purifying the AAV capsid from the host cell, thereby generating the recombinant AAV comprising the AAV capsid.
  • Clause 55 The AAV capsid produced by the method of Clause 54.
  • Clause 56 A pharmaceutical composition comprising the AAV capsid produced by the method of Clause 54. Atty Dkt. No.0184.0261-PCT (C17635_P17635-02) [0212]
  • Clause 57 A method of predicting a risk of a proteinopathy in a subject.
  • the method comprising: determining a fibrinogen-like protein 1 (FGL1) expression level in a sample obtained from a subject, which sample is indicative of the FGL1 expression level in a brain of the subject; and identifying the subject as likely to develop, or having, the proteinopathy when the FGL1 expression level in the brain of the subject is below a control expression level, or identifying the subject as unlikely to develop, or not having, the proteinopathy when the FGL1 expression level in the brain of the subject is above the control expression level, thereby the predicting the risk of the proteinopathy in the subject.
  • FGL1 fibrinogen-like protein 1
  • Clause 58 The method of Clause 57, comprising administering an effective amount of an exogenous FGL1, or a functional portion or variant thereof, to the subject when the FGL1 expression level in the brain of the subject is below the control expression level, which effective amount elevates a level of the exogenous FGL1, or the functional portion or variant thereof, in the brain of the subject sufficient to inhibit a binding interaction between a pathogenic protein associated with the proteinopathy and a lymphocyte-activation protein 3 (LAG3) present in the brain of the subject.
  • a pharmaceutical composition comprising a fibrinogen-like protein 1 (FGL1), or a functional portion or variant thereof, of use in the treatment of a proteinopathy in a subject.
  • Clause 60 The pharmaceutical composition of Clause 59, comprising exosome vesicles (EVs) that comprise the FGL1, or the functional portion or variant thereof.
  • Clause 61 The pharmaceutical composition of Clause 59, comprising adeno-associated virus (AAV) capsids that comprise a transgene encoding the FGL1, or the functional portion or variant thereof.
  • Clause 62 The pharmaceutical composition of Clause 59, comprising one or more blood components suitable for administering the pharmaceutical composition to the subject via blood exchange. Atty Dkt.
  • Clause 63 The pharmaceutical composition of any of Clauses 59-62, wherein the proteinopathy comprises Alzheimer's disease (AD), Parkinson's disease (PD), and/or Lewy body dementia (LBD).
  • Clause 64 A kit comprising the pharmaceutical composition of any of Clauses 59-63.

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Abstract

L'invention propose des méthodes de traitement d'une protéinopathie chez un sujet qui comprennent l'administration d'une quantité efficace d'une protéine 1 de type fibrinogène (FGL1), ou d'une partie fonctionnelle ou d'un variant de celle-ci, au sujet atteint de protéinopathie. La quantité efficace élève un niveau du FGL1, ou de sa partie fonctionnelle ou d'un variant de celui-ci, dans un cerveau du sujet suffisant pour inhiber une interaction de liaison entre une protéine pathogène associée à la protéinopathie et une protéine d'activation des lymphocytes 3 (LAGS) présente dans le cerveau du sujet. L'invention propose également des méthodes, des utilisations, des compositions pharmaceutiques et des kits associés.
PCT/US2024/036483 2023-07-03 2024-07-02 Méthodes et aspects associés de traitement et de diagnostic de protéinopathies Pending WO2025010238A2 (fr)

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