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WO2025027015A1 - Compositions et procédés pour le traitement de la sclérose en plaques - Google Patents

Compositions et procédés pour le traitement de la sclérose en plaques Download PDF

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Publication number
WO2025027015A1
WO2025027015A1 PCT/EP2024/071532 EP2024071532W WO2025027015A1 WO 2025027015 A1 WO2025027015 A1 WO 2025027015A1 EP 2024071532 W EP2024071532 W EP 2024071532W WO 2025027015 A1 WO2025027015 A1 WO 2025027015A1
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Prior art keywords
plant
composition
based nanoparticle
virus
plant virus
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Inventor
Valentina GARONZI
Linda Avesani
Roberta ZAMPIERI
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Diamante Srl
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Diamante Srl
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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
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention generally relates to the field of nanomedicine.
  • the present invention is directed to a composition and methods of an engineered virus-based 10 nanoparticle for the treatment of multiple sclerosis.
  • composition for treating multiple sclerosis (MS) is described.
  • Composition includes a plant virus-based nanoparticle engineered to express at least a synthetic peptide 25 associated with MS and a buffer.
  • a method of manufacturing composition for treating MS is described. Method includes infecting a plant with a virus to produce a plant virus-based nanoparticle, sampling symptomatic leaves from the plant, homogenizing the plant virus-based nanoparticle, incubating the plant virus-based nanoparticle, centrifuging the plant virus-based nanoparticle; 30 and gel-filtrating the plant virus-based nanoparticle.
  • Method includes infecting a plant with a virus to produce a plant virus-based nanoparticle, sampling symptomatic leaves from the plant, homogenizing the plant virus-based nanoparticle, incubating the plant virus-based nanoparticle, and filtrating the plant virus-based 5 nanoparticle.
  • another method of manufacturing composition for treating MS is described. Method includes agroinfiltrating a plant with a virus to produce a plant virus-based nanoparticle, sampling symptomatic leaves of the plant, homogenizing the plant virus-based nanoparticle, centrifuging the plant virus-based nanoparticle, and gel-filtrating the plant virus- 10 based nanoparticle.
  • FIG.1 is a schematic illustration of an exemplary embodiment of a composition for treating 20 multiple sclerosis (MS).
  • FIGS.2A-B are schematic illustrations of exemplary embodiments of a plant virus-based nanoparticle
  • FIG.3A is a schematic illustration of a Tomato bushy stunt virus (TBSV);
  • FIG.3B is a schematic illustration of a Cowpea mosaic virus (CPMV);
  • FIG.4 is a flow diagram illustrating an exemplary embodiment of a method of manufacturing the composition for treating MS;
  • FIG.5 is a flow diagram illustrating an exemplary embodiment of another method of manufacturing the composition for treating MS;
  • FIG.6 is an exemplary embodiment of a method of using plant virus-based nanoparticle for 30 treating MS;
  • FIG.7 is an exemplary embodiment of an extraction process of a plant virus-based nanoparticle.
  • composition may include a plant virus-based nanoparticle engineered to express at least a linear 10 peptide associated with MS and a buffer.
  • method may include identifying an immunodominant peptide associated with MS, modifying a plant-virus based nanoparticle to express the identified peptide, synthesizing a plant virus-based nanoparticle containing the peptide, and/or titrating the plant-virus based nanoparticle to a set pH.
  • autoimmune diseases 15 such as MS using plant virus-based nanoparticles.
  • FIG.1 an exemplary embodiment of a composition 100 for treating MS is illustrated.
  • multiple Sclerosis (MS) is a chronic disease that affects the central nervous system, which includes the brain and spinal cord.
  • MS autoimmune disease
  • the immune system targets the protective covering of nerve fibers, known as myelin, in the central nervous system. This attack on myelin results in communication problems between the brain and the rest of the body and may lead to a range of neurological symptoms.
  • MS is characterized by periods of relapse and remission. Some people who suffer from MS may experience fatigue and difficulty walking, while others may have no symptoms at all for most of 25 their lives. Depending on which nerves are affected, symptoms of MS may vary widely in severity and type. In severe cases, MS may lead to significant disability over time and cause partial or complete loss of mobility.
  • composition 100 lies in harnessing the immunomodulatory effect of a virus-based nanoparticle, which carries a synthetic peptide designed to recalibrate aberrant immune responses observed in MS, as described below.
  • an “immunomodulatory effect” is an effect 5 of modulating the immune system.
  • plant virus-based nanoparticle 104 may modulate an immune system to induce tolerance and therefore prevent or treat a case of autoimmune disease, such as without limitation MS.
  • composition 100 comprises a plant virus-based nanoparticle 104.
  • a “nanoparticle” is a particle with a size10 that ranges between 1 and 500 nanometers.
  • a “plant virus- based nanoparticle” is a virus nanoparticle that is capable of infecting and replicating in plants.
  • plant virus-based nanoparticle 104 may include at least a unit or subunit of a plant viral protein.
  • a “plant viral protein” is a protein that constitutes or is produced by a plant virus. Since plant viruses are unable to replicate 15 in mammals, plant virus proteins may be a safer option for medical applications in human hosts. Nanoparticles created from subunits of plant virus proteins may be genetically engineered to express, on their external surface, an antigen-specific peptide related to an autoimmune disease and then be grown in a plant host, as described in detail below in this disclosure.
  • a “virus nanoparticle” is a proteinaceous and often infectious 20 nanoscale structure that is capable of delivering its nucleic acid efficiently into a host plant cell, enabling production of new viruses therein.
  • Virus and “virus nanoparticle” may be used interchangeably throughout this disclosure. Similarly, “plant virus” and “plant virus-based nanoparticle” may be used interchangeably as well.
  • Virus includes a capsid.
  • a “capsid” is a protein shell exposed at the exterior of a virus that possesses a 25 specific geometric pattern.
  • capsid may possess an icosahedral shape.
  • an “icosahedron” is a geometric shape with 20 sides, each composed of an equilateral triangle.
  • capsid may include a filamentous structure.
  • a “filamentous structure” is an elongated, thread-like formation that makes up a capsid of certain viruses.
  • capsid may include a rod-shaped structure.
  • capsid may include a helical structure.
  • a “helical 4 Attorney Docket No.1266-004PCT1 structure” is a type of structure characterized by a cylindrical, elongated shape with a helical symmetry. This structure may be formed by the regular, repeating arrangement of protein subunits around a viral nucleic acid, providing protection, structural integrity, and aiding in the infectivity of a virus.
  • capsid may include a spherical structure. 5 Additionally and/or alternatively, capsid may adopt any geometry not disclosed herein yet deemed possible by a person of ordinary skill in the art upon reviewing the entirety of this disclosure.
  • plant virus protein may be produced through a process of molecular farming.
  • molecular farming is a process of producing pharmaceutically important and commercially valuable proteins in plants. Once extracted, plant virus proteins may be used to create plant virus-based nanoparticle 104. Plant virus-based nanoparticle 104 and/or plant virus protein may be sourced from a variety of plant hosts.
  • Suitable host plants for such purpose may include Nicotiana benthamiana plant, a Nicotiana tabacum plant, a Solanum lycopersicum or 15 Lycopersicon esculentum plant, a Cycorium intybus plant, a Brassica oleracea var. capitata plant, a Beta vulgaris var cicla plant, a Ocimum basilicum plant, a red beet plant, a spinach plant, or the like.
  • a “Nicotiana benthamiana plant” is a close relative of tobacco and a species of Nicotiana indigenous to Australia. It may be used for farming monoclonal antibodies and other recombinant proteins.
  • a 20 “Nicotiana tabacum plant”, commonly known as cultivated tobacco, is a plant species that belongs to the Nicotiana genus in the Solanaceae family. It is a widely grown herbaceous plant primarily used to produce tobacco products. This species is characterized by large, broad leaves and is cultivated in various climates worldwide.
  • a Nicotiana tabacum plant may serve as a model organism and a host for genetic engineering, enabling the expression and study 25 of recombinant proteins, vaccines, and other biologically significant compounds.
  • a “Solanum lycopersicum plant” or “Lycopersicon esculentum plant”, commonly known as the tomato plant is a plant species that belongs to the Solanaceae family and characterized by its production of edible, fleshy fruits. Solanum lycopersicum is noted for its agricultural significance and its utility in genetic engineering and plant breeding. This species 30 may serve as a model organism for studying plant genetics, disease resistance, and metabolic pathways. It may also be utilized in biotechnological applications for the production of 5 Attorney Docket No.1266-004PCT1 recombinant proteins, novel traits, and improved cultivars through genetic modification techniques.
  • a “Cycorium intybus plant” is a hardy plant widely used in folklore medicine to treat various ailments ranging from wounds to diabetes. It is believed to have antimicrobial, anthelmintic, antimalarial, hepatoprotective, antidiabetic, 5 gastroprotective, anti-inflammatory, analgesic, antioxidant, tumor-inhibitory, and antiallergic activities across several different cultures.
  • an “Ocimum basilicum plant” is a member of the Lamiaceae (mint) family and is more commonly referred to as basil.
  • a “red beet plant” is a biennial plant grown as an annual for its edible root and leafy greens and characterized by its swollen root, which is typically deep red or purple in color. It is a promising candidate for some medicinal uses.
  • the 20 phytochemicals present in red beet may provide protection against diseases including cancer and cardiovascular diseases.
  • a “spinach plant” is a leafy green that belongs to the amaranth family and is closely related to beets and quinoa.
  • plant virus-based nanoparticle 104 may contain one or more viruses, such as without limitation Tomato bush stunt 25 virus (TBSV), Cowpea mosaic virus (CPMV), and/or the like.
  • TBSV Tomato bush stunt virus
  • CPMV Cowpea mosaic virus
  • TBSV Tuttle plant
  • TBSV Cowpea mosaic virus
  • TBSV Tuttle virus
  • TBSV is a plant virus from the Tombusvirus group that primarily infects vegetable crops and causes stunting of growth, leaf mottling, and deformed or absent fruit in an infected plant.
  • TBSV is a prototypic member of the Tombusviridae family.
  • TBSV may provide a scaffold for plant virus-based nanoparticle 104 to enable a stable C- 30 terminal display of peptides.
  • CP may include an RNA binding domain (R), a shell domain (S) forming a capsid backbone, and a C-terminal protruding (P) domain that can accommodate exogenous peptides for display.
  • the structure of TBSV may include a single-stranded RNA with a linear genome of about 4,800 nucleotides.
  • TBSV may also possess three symmetrically distinct coat 5 protein monomers.
  • TBSV may replicate, without limitation, using cytoplasmic replication. This virus may penetrate a host cell by uncoating and releasing viral RNA into cytoplasm.
  • TBSV may also spread indirectly, such as without limitation through water, soil, and/or infected seeds. When engineered to express peptides, the behavior of TBSV may experience one or more modifications, such as without limitation a decrease in virus concentration, infection percentage, 10 and/or disease severity.
  • CPMV Cylea mosaic virus
  • Sunn-hemp mosaic virus is a non-enveloped plant virus of the Comovirus group.
  • CPMV genome may include 2 strands of RNA which are 15 separately encapsulated. CPMV may be approximately 30 nanometers in diameter and contain 60 copies each of a Large (L) and Small (S) coat proteins, as explained below in this disclosure.
  • L Large
  • S Small
  • the structure of CPMV is well-characterized to atomic resolution. CPMV nanoparticles may be thermostable and readily isolated from plants. There are many stable mutants of CPMV already prepared that allow specific modification of their capsid surfaces. In some cases, CPMV may 20 include an icosahedral capsid, as described above.
  • plant virus-based nanoparticle 104 is configured to express at least a MS-related peptide 108.
  • a “MS-related peptide” is a short chain of amino acids that may propose a disease-specific treatment for patients with MS.
  • MS-related peptide 108 may include PLP139-25 151, MOG35-55, and/or MOG1-20, among others.
  • “PLP139- 151” is a specific epitope derived from the Proteolipid Protein (PLP) with a sequence of HSLGKWLGHPDKF (SEQ ID NO:1).
  • a “proteolipid protein (PLP)” is a membrane-bound protein integral to the structure and function of the myelin sheath in the central nervous system. PLP is crucial for the formation, maintenance, and compaction of 30 myelin, which insulates nerve fibers and facilitates rapid neural signal transmission. Primarily expressed by oligodendrocytes (a type of glial cell found in the central nervous system (CNS) 7 Attorney Docket No.1266-004PCT1 such as the brain and spinal cord), PLPs are essential for CNS function and stability.
  • MOG35-55 and MOG1-20 are specific epitopes derived from Myelin Oligodendrocyte Glycoprotein, which has a major role in the myelination of nerves in the central nervous system (CNS).
  • MOG35-55 has a sequence of MEVGWYRSPFSRVVHLYRNGK (SEQ 5 ID NO: 2), whereas MOG1-20 has a sequence of GQFRVIGPRHPIRALVGDEV (SEQ ID NO: 3).
  • MOG35-55 and MOG1-20 are isolated from an extensive chain of complex protein amino acids and may be used for treating MS. As a nonlimiting example.
  • PLP139-151, MOG35-55 and MOG1-20 resulted in significant changes in disease biomarkers, suggesting their potential role in the development and progression of MS.
  • PLP139-151, 10 MOG35-55, and MOG1-20 were studied using a model of chronic relapsing experimental allergic encephalomyelitis (R-EAE) in mice, a well-accepted model for human MS.
  • R-EAE chronic relapsing experimental allergic encephalomyelitis
  • Significant weight loss was observed during the initial acute phase of MS, followed by weight gain correlated with disease remittance.
  • numerous mononuclear cells were noted to populate the perivascular space of the anterior spinal artery, indicating initial stages of 15 demyelinating activity.
  • a “demyelination activity” is a process of causing damage of the myelin sheath, consistent with details described above.
  • PLP139-151, MOG35-55, and/or MOG1-20 may therefore hold potential for reducing the symptoms and progression of MS.
  • PLP139-151, MOG35-55 and/or MOG1-20 may specifically play a key role in reducing the incidence of clinical relapses, CNS histopathology, 20 and T cell responses to both initiating and relapse-associated epitopes.
  • MS-related peptide 108 may include an immunodominant peptide.
  • an “immunodominant peptide” is a peptide that is a representative epitope of a given protein antigen to an immune system in the context of a specific autoimmune disease.
  • immunodominant peptide may include a cytoplasmically located protein.
  • a “cytoplasmical location” is a location inside the cytoplasm of a cell.
  • a coat protein of plant virus-based nanoparticle 104 may be engineered to display immunodominant peptides.
  • a “coat protein” is a protein that is a constituent of the capsid of a virus, as described above.
  • a “buffer” is a solution or mixture that contains at least a pair of weak acid, HA, and its conjugate base, A-, (i.e., the weak acid minus one proton) in a molar ratio between 10:1 and 1:10, wherein the solution maintains a stable pH close to the pK a (i.e., the negative log of the acid dissociation constant, K a ) of the weak acid, against addition of acidic or basic chemical species.
  • A- i.e., the negative log of the acid dissociation constant, K a
  • a buffer containing a pair of conjugate base and acid may be written as A-/HA. Additional examples will be provided below.
  • buffer may include an acetate buffer 112 (i.e., CH3COONa/CH3COOH).
  • buffer may include a borate buffer (i.e., Na 2 B 4 O 7 ⁇ 10H 2 O/H 3 BO 3 ).
  • buffer may include a bicarbonate buffer (i.e., NaHCO3/H2CO3 or Na2CO3/NaHCO3, depending on the desired pH).
  • Good’s buffers include MES (C6H13NO4S), ACES (C4H9NO4S), PIPES (C8H18N2O6S2), MOPS (C7H15NO4S), TES (C6H15NO6S), HEPES (C8H18N2O4S), Tricine (C6H13NO5), TRIS (C4H11NO3), Bicine (C6H13NO4), TAPS (C7H17NO6S), CHES (C8H17NO3S), CAPS (C9H19NO3S), AMPSO (C9H19NO4S), Gly-Gly (C4H8N2O3), ADA (C4H7NO4), BES (C6H15NO5S), MOPSO (C7H15NO5S), EPPS (C9H20N2O4S), HEPPS (C11H24N2O4S), CAPSO (C9H19NO4S), HEPPSO (C9H20N2O
  • buffer may include a phosphate buffer (i.e., NaH2PO4/H3PO4, Na2HPO4/NaH2PO4, or Na3HPO4/ Na2HPO4, depending on the desired pH).
  • buffer may include a phosphate- buffered saline (PBS) solution, a commonly used buffer in biological research and pharmaceutical formulations that typically contains 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , and 1.8 mM KH 2 PO 4 .
  • PBS phosphate- buffered saline
  • Sodium acetate may be a hygroscopic powder that absorbs moisture from air.
  • Sodium acetate is a strong electrolyte that fully dissolves and disassociates in water to form sodium ions (Na + ) and acetate ions (CH 3 COO-).
  • Na + is the principal cation of extracellular fluid and plays a large part in fluid and electrolyte replacement therapies.
  • Sodium acetate may be used as an electrolyte replenisher in isosmotic solution for parenteral replacement of acute losses of extracellular fluid without disturbing normal electrolyte balance.
  • a solution of sodium acetate and acetic acid may act as a buffer to maintain a relatively constant pH.
  • composition 100 may be taken in any suitable form of dosage and/or delivery, including without limitation oral dosage, intravenous dosage, subcutaneous dosage, intraperitoneal dosage, or intradermal dosage.
  • oral dosage is an ingestion of a composition through the mouth.
  • Oral dosage of composition 100 may include use of pills, syrup, tablets, thin film, liquid solution, powder, solid crystals, natural or herbal plants, seeds, or food, pastes, or the like.
  • an “intravenous dosage” is an administration of a composition using injection.
  • Intravenous dosage may include without limitation intravenous injection.
  • Composition 100 may be given intravenously in any suitable manner, including as a bolus and/or as an infusion.
  • other injection methods such as intramuscular injection, intraperitoneal injection, 5 subcutaneous injection, and/or transcutaneous injection may be used.
  • a “subcutaneous dosage” is an administration of a pharmaceutical composition by injecting it into the subcutaneous tissue, which is the layer of tissue directly beneath the skin. This route of administration allows for the absorption of the active ingredient into the bloodstream at a controlled rate.
  • Subcutaneous dosages are commonly used for vaccines, insulin, 10 and other medications requiring slow, sustained release. Subcutaneous dosage may offer advantages such as ease of administration, improved patient compliance, and minimized pain compared to intramuscular or intravenous injections.
  • an “intraperitoneal dosage” is an administration of a pharmaceutical composition by injecting it directly into the peritoneal cavity, which is the space within the abdomen that houses various 15 organs such as the intestines, liver, and stomach. This route may allow for rapid absorption of an active ingredient into the bloodstream and is often used for delivering drugs that require swift systemic effects or for treatments targeting abdominal organs. Intraperitoneal administration may be particularly useful in research settings and certain clinical applications, providing a method for efficient and effective drug delivery.
  • an “intradermal 20 dosage” is an administration of a pharmaceutical composition by injecting it directly into the dermis, which is the layer of skin located between the epidermis and subcutaneous tissue. This method may be employed to deliver small volumes of medication, typically for vaccines, allergy tests, or local anesthesia.
  • the intradermal route may allow for a controlled release and localized effect of an active ingredient, providing advantages such as enhanced immune response, reduced 25 systemic side effects, and precise targeting of an injection site.
  • composition 100 may include one or more anti-sclerosis ingredients.
  • an “anti- sclerosis ingredient” is a medically active chemical that may be used to treat and/or prevent MS.
  • Anti- sclerosis ingredient may include a non-steroidal anti-inflammatory drug (NSAID), a 30 corticosteroid, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, a tumor necrosis 11 Attorney Docket No.1266-004PCT1 factor inhibitor, a T-cell costimulatory blocking agent, a B-cell depleting agent, an Interleukin-1 receptor antagonist therapy, and/or any other immunomodulatory and/or cytotoxic agent.
  • NSAID non-steroidal anti-inflammatory drug
  • Embodiment 200a may be a structural representation of CPMV.
  • plant virus-based nanoparticle 104 may include a large coat protein subunit 204.
  • plant virus-based nanoparticle 104 may include 60 copies of large coat protein subunit 204.
  • plant virus-based nanoparticle 104 may include a small coat protein subunit 208.
  • plant virus-based nanoparticle 104 may include 60 copies of small coat protein subunit 208.
  • plant virus-based nanoparticle 104 may include at least a MS-related peptide 108.
  • At least a MS-related peptide 108 may include immunodominant peptide 212, such as without limitation PLP139-151, MOG35-55, and/or MOG1-20.
  • immunodominant peptide 212 such as without limitation PLP139-151, MOG35-55 and/or MOG1-20, may be embedded within coat protein.
  • immunodominant peptide 212 such as without limitation PLP139- 151, MOG35-55, and/or MOG1-20, may be located outside of coat protein.
  • immunodominant peptide 212 may be exposed on the surface of large coat protein subunit 204.
  • immunodominant peptide 212 may be located inside of a nanoparticle.
  • immunodominant peptide 212 may be placed inside of coat protein.
  • plant virus-based nanoparticle 104 may include an icosahedral structure.
  • plant virus-based nanoparticle 104 may include a filamentous structure.
  • plant virus-based nanoparticle 104 may include a rod-shaped structure.
  • plant virus-based nanoparticle 104 25 may include a helical structure. In one or more embodiments, plant virus-based nanoparticle 104 may include a spherical structure. In one or more embodiments, plant virus-based nanoparticle 104 may be homogeneous or uniform in size. In some embodiments, plant virus-based nanoparticle 104 may be homogeneous or uniform in shape. Additionally, and/or alternatively, plant virus-based nanoparticle 104 may adopt any geometry not disclosed herein yet deemed 30 possible by a person of ordinary skill in the art upon reviewing the entirety of this disclosure.
  • Plant virus-based nanoparticle 104 disclosed herein may be consistent with details described with respect to FIG.2A.
  • Embodiment 200b may be a structural representation of TBSV.
  • plant virus-based nanoparticle 104 may 5 include TBSV, as described above.
  • plant virus-based nanoparticle 104 may include a single protein subunit 216.
  • plant virus- based nanoparticle 104 may include 180 copies of a single protein subunit 216.
  • plant virus-based nanoparticle 104 may include at least a MS-related peptide 108. In one or more embodiments, at least a MS-related peptide 108 may include immunodominant 10 peptide 212. In one or more embodiments, immunodominant peptide 212 may be exposed on the surface of a single protein subunit 216.
  • FIGS.3A-B exemplary structures of TBSV (300a) and CPMV (300b) are illustrated in FIG.3A and 3B, respectively.
  • FIG.4 a flow diagram illustrating an exemplary embodiment of 15 method 300 for preparing plant virus-based nanoparticle 104 is illustrated.
  • Plant virus-based nanoparticle 104 may include any type of plant virus-based nanoparticle described in this disclosure without limitation.
  • method 400 includes infecting a plant with a virus to produce plant virus-based nanoparticle 104.
  • infection is a process of delivering viral genes into a host, wherein the viral genes are capable of replication to20 produce new copies of the corresponding virus. Infection allows a plant to produce plant virus- based nanoparticle 104. In one or more embodiments, infection may be done by spontaneous infiltration.
  • spontaneous infiltration is a type of infiltration that is associated with a negative Gibbs free energy change and occurs naturally without energy input or intervention.
  • infiltration is a process through 25 which one or more substances penetrate or permeate from the surface of a plant into its tissues.
  • spontaneous infiltration may include spraying infectious viral genes onto a plant, spraying viral solution obtained from previously infected leaves, immersion of plants into a viral solution obtained from previously infected leaves and the like.
  • infection may be done by infiltration of viral genes into a plant.
  • infection may be done by infiltration of viral solution obtained from previously infected leaves.
  • infection may be done by forced 13 Attorney Docket No.1266-004PCT1 infiltration.
  • forced infiltration is a type of infiltration that requires a force, a pressure, an energy input, or a similar form of intervention to be applied.
  • forced infiltration may include syringe infiltration, vacuum infiltration, and the like.
  • vacuum infiltration may include vacuum infiltration 5 using a vacuum pump, vacuum infiltration using a syringe, and the like.
  • samples are taken from leaves that are symptomatic.
  • symptomatic is a descriptor that describes one or more 15 characteristics of an object that appear different due to viral infection. As a nonlimiting example, it may take three to six days for a TBSV- and/or a CPMV-based nanoparticle to form and accumulate in a plant.
  • method 400 includes homogenizing the plant virus-based nanoparticle 104.
  • homogenizing is a process of blending elements into a uniform mixture with a consistent or substantially consistent 25 composition across its entirety.
  • a homogenization process may include combining a tissue of plant containing plant virus-based nanoparticles 104 with an extraction buffer solution.
  • an “extraction buffer solution” is an aqueous solution capable of breaking open cells and releasing elements therein.
  • extraction buffer solution may include salts to regulate its acidity.
  • a sodium acetate solution may be used as extraction buffer solution.
  • method 400 may include centrifuging plant virus-based nanoparticle 104.
  • centrifuging is a process of separating multiple components in a mixture based on their difference in density by applying a centrifugal force to the mixture using a centrifuge device.
  • a “centrifuge device” is a device comprising a rotating element attached to a stationary axis and configured to a spin sample under a high rotational speed in order to achieve separation between various elements therein.
  • the amount of time to apply for this centrifuging step may vary from one type of plant virus-based nanoparticle 104 to another. In some cases, this step may be repeated multiple times to achieve an improved yield. As a 15 nonlimiting example, for TBSV, centrifuging step may be repeated two or three times.
  • method 400 may include filtrating plant virus-based nanoparticle 104.
  • filtrating plant virus-based nanoparticle 104 may include filtrating the plant virus-based nanoparticle 104 using tangential flow filtration (TFF), nanofiltration (NF), and/or gel-filtration chromatography, among 20 other similar separation/purification techniques.
  • tangential flow filtration also known as cross-flow filtration, is a separation technique utilized to filter and concentrate biomolecules in solution.
  • a feed solution may flow tangentially across the surface of a filter membrane, while an applied pressure forces some of the fluid through the membrane as filtrate (permeate). A tangential motion may help reduce membrane 25 fouling and allow continuous filtration.
  • NF nanofiltration
  • NF may 30 utilize a semi-permeable membrane to selectively allow certain molecules, such as monovalent ions and small organic molecules, to pass through while rejecting larger molecules, multivalent 15 Attorney Docket No.1266-004PCT1 ions, and contaminants.
  • gel-filtration chromatography is a type of 5 separation and purification technique based on a differing ability of chemical species to retain in pores of a gel-filtration medium. Gel-filtration chromatography is also known as size-exclusion chromatography. A column used for gel-filtration chromatography may be packed with fine, porous beads composed of dextran polymers, agarose, polyacrylamide, and/or the like. The pore sizes of these beads are used to estimate the dimensions of macromolecules and separate them 10 accordingly.
  • gel-filtration chromatography is the fractionation of proteins and other water-soluble polymers.
  • Gel-filtration chromatography may be contrasted 15 with gel permeation chromatography, which is a similar separation and purification technique often used to analyze the molecular weight distribution of organic-soluble polymers.
  • FIG.5 a flow diagram illustrating another exemplary embodiment of method 500 for preparing plant virus-based nanoparticle 104 is illustrated.
  • Plant virus-based nanoparticle 104 may include any type of plant virus-based nanoparticle described in this 20 disclosure without limitation.
  • method 500 includes agroinfiltrating a plant with a virus to produce plant virus-based nanoparticle 104.
  • agroinfiltration is a method used in plant biology to induce transient expression of genes in a plant in order to produce a desired protein.
  • agroinfiltration may be used for CPMV and/or for TBSV.
  • 25 Agrobacterium tumefaciens may be directly injected into a plant leaf or brought into association with plant cells immobilized on a porous support.
  • bacteria may transfer a desired gene into plant cells via transfer of T-DNA.
  • Agroinfiltration may be beneficial when compared to more traditional plant transformations due to its speed and convenience, although yields of the recombinant protein may generally also be higher and more consistent.
  • other production processes may also be used to produce plant virus-based 16 Attorney Docket No.1266-004PCT1 nanoparticle 104. Once a plant has undergone such production processes, it may produce plant virus-based nanoparticles 104 to be used in composition 100.
  • method 500 includes sampling leaves of the plant. This step may be performed utilizing any process of sampling consistent with details 5 described above.
  • method 500 includes homogenizing the plant virus-based nanoparticle. This step may be performed utilizing any process of homogenization as explained above. As a nonlimiting example, for CPMV, a phosphate buffer or a PBS buffer may be used, consistent with details described above. 10 With continued reference to FIG.5, at step 520, method 500 includes centrifuging the plant virus-based nanoparticle 104. This step may be performed utilizing any process of centrifugation as described above. It is worth noting that production of a CPMV-based nanoparticle may not necessarily involve an incubation step described above for method 400. With continued reference to FIG.5, at step 525, method 500 includes filtrating plant 15 virus-based nanoparticle 104.
  • This step may be performed utilizing any process of filtration as described above.
  • FIG.6 an exemplary embodiment of a method 600 of using plant virus- based nanoparticle 104 for treating MS is illustrated.
  • Genetic sequences that encode MS-related peptides 108 such as PLP139-151, MOG35-55, and/or MOG1-20, among others, may be cloned 20 into an expression vector 604 harboring a viral genome or CP gene.
  • an “expression vector” is typically a plasmid or virus designed for gene expression in cells. Expression vector is also known as expression construct.
  • PLP139-151, MOG35-55, and/or MOG1-20 genes may be isolated from TBSV, an expression vector that is infective, and incorporated into CPMV, an expression vector that is non-infective. 25 Resulting vectors may then undergo either in vitro retro-transcription or an Agrobacterium tumefaciens transformation. For in vitro retro-transcription, plant may undergo infection using an infective RNA. For Agrobacterium tumefaciens transformation, plant may undergo agroinfiltration, as previously described. Referring now to FIG.7, an exemplary embodiment of a production process 700 of a 30 plant virus-based nanoparticle 104 is presented.
  • plant virus-based nanoparticle 104 infects plant, the plant may start to produce genetically modified plant viruses.
  • 17 Attorney Docket No.1266-004PCT1 plant may function as a bioreactor.
  • samples containing such plant virus-based nanoparticles 104 may be homogenized with extraction buffer solution, as described above.
  • samples may be centrifuged and filtrated following procedures described above to yield plant virus-based 5 nanoparticles 104, which may be used for downstream therapeutic applications, such as treatment of MS.
  • phrases such as “at least one of” or “one or more of” may occur followed by a conjunctive list of elements or features.
  • the term “and/or” 20 may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features.
  • the phrases “at least one of A and B;” “one or more of A and B;” and “A and/or B” are each intended to mean “A 25 alone, B alone, or A and B together.”
  • a similar interpretation is also intended for lists including three or more items.
  • the phrases “at least one of A, B, and C;” “one or more of A, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.”
  • use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such 30 that an unrecited feature or element is also permissible.

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Abstract

L'invention concerne une composition pour le traitement de la sclérose en plaques (SEP), comprenant des nanoparticules à base de phytovirus modifiées pour exprimer au moins un peptide lié à la SEP et un tampon, dont un procédé de fabrication comprend l'infection d'une plante avec un virus pour produire les nanoparticules à base de phytovirus, l'échantillonnage de feuilles symptomatiques à partir de la plante, l'homogénéisation des nanoparticules à base de phytovirus, l'incubation des nanoparticules à base de phytovirus, la centrifugation des nanoparticules à base de phytovirus et la filtration des nanoparticules à base de phytovirus.
PCT/EP2024/071532 2023-08-01 2024-07-30 Compositions et procédés pour le traitement de la sclérose en plaques Pending WO2025027015A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150125536A1 (en) * 2013-11-04 2015-05-07 Uti Limited Partnership Methods and compositions for sustained immunotherapy
CA3009799A1 (fr) * 2016-01-04 2017-07-13 Cour Pharmaceuticals Development Company Inc. Particules encapsulant des proteines hybrides contenant des epitopes lies
US20220153859A1 (en) * 2018-05-07 2022-05-19 Anokion Sa Glycophorin a antigen-binding proteins

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150125536A1 (en) * 2013-11-04 2015-05-07 Uti Limited Partnership Methods and compositions for sustained immunotherapy
CA3009799A1 (fr) * 2016-01-04 2017-07-13 Cour Pharmaceuticals Development Company Inc. Particules encapsulant des proteines hybrides contenant des epitopes lies
US20220153859A1 (en) * 2018-05-07 2022-05-19 Anokion Sa Glycophorin a antigen-binding proteins

Non-Patent Citations (1)

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Title
MOORMAN CODY D. ET AL: "Emerging Therapeutics for Immune Tolerance: Tolerogenic Vaccines, T cell Therapy, and IL-2 Therapy", FRONTIERS IN IMMUNOLOGY, vol. 12, 1 January 2021 (2021-01-01), Lausanne, CH, pages 657768 - 657768, XP055811870, ISSN: 1664-3224, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8039385/pdf/fimmu-12-657768.pdf> DOI: 10.3389/fimmu.2021.657768 *

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