WO2025051994A1 - Nanoparticules lipidiques ionisables - Google Patents

Nanoparticules lipidiques ionisables Download PDF

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WO2025051994A1
WO2025051994A1 PCT/EP2024/075075 EP2024075075W WO2025051994A1 WO 2025051994 A1 WO2025051994 A1 WO 2025051994A1 EP 2024075075 W EP2024075075 W EP 2024075075W WO 2025051994 A1 WO2025051994 A1 WO 2025051994A1
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group
moiety
formula
lipid
ionizable
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Lavaniya KUNALINGAM
Delphine Compere
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Coave Therapeutics SA
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Coave Therapeutics SA
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • A61K49/0043—Fluorescein, used in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0089—Particulate, powder, adsorbate, bead, sphere
    • A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
    • A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle

Definitions

  • the present invention relates to ionizable lipid nanoparticles (LNPs) and more in particular to ionizable lipid nanoparticles comprising an ionizable lipid and a squaramide modified lipid, preferably a squaramide modified phospholipid.
  • LNPs ionizable lipid nanoparticles
  • the provided ionizable LNPs are useful as a delivery system, particularly for use in gene therapy, cell targeted therapy or gene editing.
  • BACKGROUND OF INVENTION [0002]
  • the effective targeted delivery of biologically active substances such as small molecule drugs, proteins, and nucleic acids represents a continuing medical challenge.
  • nucleic acids are thus based on the genetic modification of cells to produce a therapeutic effect by the delivery of nucleic acids ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ .
  • Gene therapy is thus based on the genetic modification of cells to produce a therapeutic effect by the delivery of nucleic acids ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ .
  • Gene therapy is thus based on the genetic modification of cells to produce a therapeutic effect by the delivery of nucleic acids ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ .
  • the whole or part of a gene is defective or missing from birth, or a gene can change or mutate during life. Any of these variations can disrupt how proteins are synthetized, which can contribute to health
  • Gene editing is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living cell or organism. Unlike early genetic engineering techniques that randomly inserts genetic material into a host genome, genome editing targets the insertions to site-specific locations.
  • the basic mechanism involved in genetic manipulations through programmable endonucleases is the recognition of target genomic loci and binding of effector DNA-binding domain (DBD), double-strand breaks (DSBs) in target DNA by the restriction endonucleases (FokI and Cas), and the repair of DSBs through homology-directed recombination (HDR) or non-homologous end joining (NHEJ).
  • DBD effector DNA-binding domain
  • Cas double-strand breaks
  • HDR homology-directed recombination
  • NHEJ non-homologous end joining
  • endonucleases meganucleases, zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated nucleases (Cas).
  • Lipid nanoparticles also referred as ionizable LNPs are often biodegradable and biocompatible and present low immunogenicity, in general.
  • ionizable LNPs consists of a few components, plus the payload, which is typically a nucleic acid sequence.
  • the main functional component is the ionizable cationic lipid, which usually represents around 50% of the entire structure.
  • Another attractive feature is the possibility of modifying the surface of the lipid nanoparticles with targeting moieties to achieve selective, cell- ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ (Xu et al., ACS Nano 2022, 16, 7168-7196).
  • the arylene or the heteroarylene group Ar is a phenylene or pyridylene group, optionally comprising one or more substitutions.
  • said arylene or heteroarylene group Ar comprises one or more substitutions selected from the group consisting of halogen, C1-6 alkyl, C1-6 haloalkyl, C1-6 acyl and C1-6 alkoxy.
  • Z is a saccharide or a peptide
  • L comprises a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers.
  • the agent is selected from the group consisting of a nucleic acid, a chemotherapeutic agent, a small molecule drug, a protein and a peptide, or a combination thereof.
  • the method for delivering an agent comprises contacting the target cell with an ionizable LNP, wherein the ionizable LNP comprises the agent (e.g.
  • N is a nitrogen atom from the functional moiety RL-NH-
  • N* is a nitrogen atom of an amino group of a phospholipid Pl, comprising the group RL a cell- type targeting ligand or a receptor targeting ligand of the target cell wherein Pl is a phospholipid moiety
  • E-*NH- is an extender moiety comprising an extender group E and a group -*NH-
  • RL-NH- is a functional moiety including a nitrogen containing group -NH- and a group RL, and wherein the functional moiety RL-NH- of the compound of formula (I) comprises a cell-type targeting ligand or a receptor targeting ligand of the target cell.
  • Another aspect of the invention refers to a method for manufacturing an ionizable LNP as defined and described in classes and subclasses disclosed in the present invention, wherein said method comprises: reacting a surface exposed squarate moiety of an ionizable LNP comprising a squarate modified lipid, preferably a squarate modified phospholipid, with a compound of formula (IIb), or a pharmaceutically acceptable salt thereof, at a pH between 7 and 9.5, preferably between 8 and 9: RL-NH2 (IIb) so that the group RL is conjugated to the ionizable LNP via a squaramide moiety of formula (SQ) as disclosed herein; or reacting a surface exposed primary amine moiety of an ionizable LNP comprising a primary amine modified lipid, preferably a primary amine modified phospholipid, with a compound of formula (IIIb), or a pharmaceutically acceptable salt thereof, at a pH between 7 and 9.5, preferably
  • the squarate modified lipid is a compound of formula (IIa), or a pharmaceutically acceptable salt thereof
  • Another aspect of the invention refers to a method for manufacturing an ionizable LNP as defined and described in classes and subclasses disclosed in the present invention, wherein said method comprises: putting into contact an ionizable lipid, a non-cationic lipid, a sterol, a PEGylated lipid and a compound of formula (IIa), or a pharmaceutically acceptable salt thereof, with an agent (such as a nucleic acid), as defined and described in classes and subclasses disclosed in the present invention, to form an ionizable lipid nanoparticle; conjugating the compound of formula (IIa), or a pharmaceutically acceptable salt thereof, of the formed ionizable lipid nanoparticle with a compound of formula (IIb), or a pharmaceutically acceptable salt thereof, at a pH between 7 and 9.5, preferably between 8 and 9: RL-NH
  • the compounds of formula (IIa) and (IIb), or their pharmaceutically acceptable salts thereof are useful to obtain the compounds of formula (I), as defined and described in classes and subclasses in the present invention.
  • the extender group E of the extender moiety E-*NH- comprises a PEG covalently linked to the phospholipid moiety Pl by a -(CH2)q-C(O)-X- group, or a bioisostere moiety thereof and the compound of formula (IIa) is represented by a formula (IIa 1 ): wherein Pl, X; p, q and R2 are as defined and described in classes and subclasses disclosed in the present invention.
  • the extender group E of the extender moiety E-*NH- comprises an aryl group Ar and a PEG, wherein the aromatic group Ar and the PEG are linked by an amide group or a bioisostere moiety thereof, and wherein the PEG is covalently linked to the phospholipid moiety Pl by a -(CH2)q-C(O)-X-group, or a bioisostere moiety thereof and the compound of formula (IIa) is represented by a formula (IIa2) or formula (IIa3): wherein Pl, X, p, m1, m2, q, Ar and R2 are as defined and described in classes and subclasses disclosed in the present invention.
  • the functional moiety RL-NH- comprises a group Z, one or more spacers L, and the compound of formula (IIb), or a pharmaceutically acceptable salt thereof, is represented by formula (IIb1): (IIb 1 )
  • the compound of formula (IIb 1 ), or a pharmaceutically acceptable salt thereof comprises one or more than one spacer L is selected from the group consisting of L1, L2 and L3 and, is selected from the group consisting of formula (IIb2), (IIb3), (IIb4), (IIb5) and (IIb6): (IIb 2 ) wherein Z, L1, L2 and L3 are as defined and described in classes and subclasses in the present invention.
  • the compound of formula (IIb) is a compound selected from the group consisting of formula (IIc) (IId) and (IIe): (IIc), , or a pharmaceutically acceptable salt thereof, wherein n, m , m , 1 1 2 Z, Ar and R are as defined and described in classes and subclasses disclosed in the present invention.
  • the compounds of formula (IIIa) and (IIIb), or their pharmaceutically acceptable salts thereof are useful to obtain the compounds of formula (I), as defined and described in classes and subclasses in the present invention.
  • the extender group E of the extender moiety E-*NH- comprises a PEG covalently linked to the phospholipid moiety Pl by a -(CH 2 )q-C(O)-X- group, or a bioisostere moiety thereof and the compound of formula (IIIa) is represented by a formula (IIIa1): wherein Pl, X, p and q are as defined and described in classes and subclasses disclosed in the present invention.
  • the extender group E of the extender moiety E-*NH- comprises an aryl group Ar and a PEG, wherein the aromatic group Ar and the PEG are linked by an amide group or a bioisostere moiety thereof, and wherein the PEG is covalently linked to the phospholipid moiety Pl by a -(CH2)q-C(O)-X-group, or a bioisostere moiety thereof and compound of formula (IIIa) is represented by a formula (IIIa2) or (IIIa3): wherein Pl, X, Ar, p, m1, m2 and q are as defined and described in classes and subclasses disclosed in the present invention.
  • the functional moiety RL-NH- comprises a group Z, one or more spacers L, and the compound of formula (IIIb), or a pharmaceutically acceptable salt thereof, is represented by formula (IIIb1): (IIIb 1 ). wherein R2, Z and L are as defined and described in classes and subclasses in the present invention.
  • the compound of formula (IIIb1), or a pharmaceutically acceptable salt thereof comprises one or more than one spacer L is selected from the group consisting of L1, L2 and L3 and, is selected from the group consisting of formula (IIIb2), (IIIb3), (IIIb4), (IIIb5) and (IIIb6): , and wherein R2, Z, L 1 , L 2 and L 3 are as defined and described in classes and subclasses in the present invention.
  • the compound of formula (IIIb) is a compound selected from the group consisting of formula (IIIc) (IIId) and (IIIe): or a pharmaceutically acceptable salt thereof, wherein n, m 1 , m 2 , Z, Ar, R1 and R2 are as defined and described in classes and subclasses disclosed in the present invention.
  • Yet another aspect of the present invention refers to a method for manufacturing an ionizable LNP as defined and described in classes and subclasses disclosed in the present invention, wherein said method comprises putting in contact a compound of formula (I), an ionizable cationic lipid, a non-cationic lipid, a sterol and a PEGylated lipid, with an agent (such as a nucleic acid), as defined and described in classes and subclasses disclosed in the present invention.
  • an agent such as a nucleic acid
  • the present disclosure relates to ionizable lipid nanoparticles comprising a compound of formula (I), as previously defined, which couples different types of ligands using a squaramide moiety of formula (SQ) as disclosed herein.
  • SQ squaramide moiety of formula
  • the present disclosure therefore recognizes a particular remaining need to provide suitable coupling chemistries that (i) maintain the integrity and functionality of the ligand as well as of the nanoparticle once the once said ligand is conjugated to said nanoparticle; (ii) expose the ligand at the surface of the particle; (iii) stabilize the ligand in the blood and/or biological medium; ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ target.
  • the present disclosure recognizes the need that the grafting of a ligand to the ionizable ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ to control the localization of the conjugation on the ligand to ensure that the ligand is oriented outward; (ii) simplicity and reproducibility; (iii) easy scale-up and characterization; and (iv) a high yield to limit costs.
  • the squaramide moiety is a conformationally rigid cyclobutene ring derived from squaric acid (diketoclyclobutenediol) which benefits from unique physical and chemical properties which make it surprisingly useful for developing ionizable lipid nanoparticles where a wide range of ligands may be grafted. Moreover, by selecting appropriate pH conditions the first and second substitution of the squarate can be controlled, allowing thus to provide a more selective substitution, resulting in a more flexible scaffold for developing lipid nanoparticles with a wide range of ligands when compared to other solutions (linkers) known in the prior art.
  • Aryl groups include for example phenyl, naphthyl, indenyl, or benzocyclobutenyl groups, optionally substituted by one or more groups optionally comprising one or more substitutions selected from the group consisting of halogen, C1-6 alkyl, C1-6 haloalkyl, C1-6 acyl and C1-6 alkoxy.
  • a preferred aryl group used herein is phenyl.
  • the present disclosure also includes compounds that differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13C- or 14C-enriched carbon are within the scope of this disclosure.
  • Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present disclosure.
  • compounds of this disclosure comprise one or more deuterium atoms.
  • agent refers to any compound or molecule that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.
  • agents include, but are not limited to, nucleic acids, chemotherapeutic agents, small molecule drugs, proteins and peptides, antibodies, antibody fragments, among others.
  • Nucleic acids [0101] In some embodiments the agent is a therapeutic nucleic acid (TNA) is encapsulated in the LNP.
  • TAA therapeutic nucleic acid
  • Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigene, viral DNA (e.g., Lentiviral or AAV genome) or non-viral synthetic DNA vectors, closed-ended linear duplex DNA (ceDNA/CELiD), plasmids, bacmids, DOGGYBONETM DNA vectors, minimalistic immunological-defined gene expression (MIDGE)-vector, nonviral ministring DNA vector (linear-covalently closed DNA vector), or dumbbell-shaped DNA minimal vector (“dumbbell DNA”).
  • viral DNA e.g., Lentiviral or AAV genome
  • non-viral synthetic DNA vectors closed-ended linear duplex DNA (ceDNA/CELiD)
  • plasmids e.g., plasmids
  • bacmids e.g., DOGGYBONETM DNA vectors
  • DOGGYBONETM DNA vectors e.g., minimalistic immunological-defined gene expression (MIDGE)-vector
  • Illustrative therapeutic nucleic acids of the present disclosure can include, but are not limited to, minigenes, plasmids, minicircles, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), ribozymes, closed ended double stranded DNA (e.g., ceDNA, CELiD, linear covalently closed DNA ("ministring"), doggyboneTM, protelomere closed ended DNA, or dumbbell linear DNA), dicer-substrate dsRNA, small hairpin RNA (shRNA), LNAs, asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, CRISPR/Cas9 technology and sgRNA, and DNA viral vectors, viral RNA vector, and any combination thereof.
  • siRNA small interfering RNA
  • miRNA microRNA
  • ASO antisense oligonucleotides
  • siRNA or miRNA that can downregulate the intracellular levels of specific proteins through a process called RNA interference (RNAi) are also contemplated by the present disclosure to be nucleic acid therapeutics.
  • RNAi RNA interference
  • siRNA or miRNA After siRNA or miRNA is introduced into the cytoplasm of a host cell, these double-stranded RNA constructs can bind to a protein called RISC.
  • the sense strand of the siRNA or miRNA is removed by the RISC complex.
  • the RISC complex when combined with the complementary mRNA, can induce either a translation blockade or mRNA cleavage and release the cut strands.
  • RNAi is by inducing specific destruction of mRNA that results in downregulation of a corresponding protein.
  • Antisense oligonucleotides (ASO) and ribozymes that inhibit mRNA translation into protein can be nucleic acid therapeutics.
  • these single stranded deoxy nucleic acids have a complementary sequence to the sequence of the target protein mRNA, and Watson - capable of binding to the mRNA by Crick base pairing. This binding prevents translation of a target mRNA, modulates splicing and/or triggers RNaseH degradation of the mRNA transcript.
  • the antisense oligonucleotide has increased specificity of action (i.e., down-regulation of a specific disease-related protein).
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas9 makes cuts in the DNA and allows new genetic sequences to be inserted.
  • Single- guide RNAs are used to direct Cas9 to the specific spot in DNA where cuts are desired.
  • the TNA is encapsulated in the LNP.
  • the TNA is selected from the group consisting of minigenes, plasmids, minicircles, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), ribozymes, closed-ended (ceDNA), ministring, doggyboneTM protelomere closed ended DNA (ceDNA), or dumbbell linear DNA, dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, DNA viral vectors, viral RNA vector, non- viral vector and any combination thereof.
  • the TNA is ceDNA.
  • non- fusogenic cationic lipid is meant a cationic lipid that can condense and/or encapsulate the nucleic acid cargo, but does not have, or has very little, fusogenic activity.
  • the cationic lipid and ionizable lipid are typically employed to condense the nucleic acid cargo, at low pH and to drive membrane association and fusogenicity.
  • Cationic lipids are lipids comprising at least one quaternary amino group that is permanently positively charged and ionizable lipids are lipids comprising a secondary or tertiary amine group which becomes protonated under acidic conditions, for example at pH of 6.5 or lower.
  • the ionizable LNP comprises 1% or more of an ionizable lipid relative to the total weight of the LNP (w/w). In some embodiments the ionizable LNP comprises at least 5%, preferably at least 15%, more preferably at least 25%, even more preferably at least 35%, even yet more preferably at least 45% of an ionizable lipid relative to the total weight of the LNP (w/w). In one embodiment, the ionizable lipid, or the addition of ionizable lipid and cationic lipid, represents 1-90% (mol), for example 20- 90% (mol) of the total lipid present in the lipid particles (e.g., lipid nanoparticles).
  • the cationic lipid is selected from the group consisting of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); N-[1- (2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP); 1,2-dioleoyl- sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-dilauroyl-sn-glycero-3- ethylphosphocholine (DLEPC); 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC); 1,2-dimyristoleoyl- sn-glycero-3-ethylphosphocholine (14:1), N1-[2-((1S)-1- [(3-aminopropyl)amino]-4-
  • DOTMA 1,2-d
  • the ionizable lipid is selected from the group consisting of 1,2-dilinoleyloxy-3- dimethylaminopropane (DLinDMA), 2,2-dilinoley1-4-(2dimethylaminoethyl)-[1,3]- dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-y1-4- (dimethylamino)butanoate (DLin-MC3-DMA), 1,2-Dioleoyloxy-3- dimethylaminopropane (DODAP), 1,2-Dioleyloxy-3-dimethylaminopropane (DODMA), Morpholinocholesterol (Mo-CHOL), and (R)-5-(dimethylamino)pentane-1,2-diyl dioleate hydrochloride (DODAPen-Cl).
  • DLinDMA 1,2-dilinoleyloxy-3
  • the ionizable lipid is DLin-MC3-DMA.
  • the ionizable lipid is MC3 (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31-tetraen-19-y1-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3) having the following structure: [0115]
  • the lipid DLin-MC3-DMA is described in Jayaraman et al., Angew. Chem. Int. Ed Engl. (2012), 51(34): 8529-8533.
  • the ionizable lipid is the lipid ATX-002 as described in WO2015/074085: [0117] In some embodiments, the ionizable lipid is (13Z,16Z)-N,N-dimethy1-3- nonyldocosa-13,16-dien-1-amine (Compound 32), as described in WO2012/040184. [0118] In some embodiments, the ionizable lipid is Compound 6 or Compound 22 as described in WO2015/199952: Non-cationic lipids [0119] In one embodiment, the lipid particles (LNPs) may further comprise a non- cationic lipid.
  • the non-cationic lipid can serve to increase fusogenicity and also increase stability of the LNP during formation.
  • Non-cationic lipids include amphipathic lipids, neutral lipids and anionic lipids. Accordingly, the non-cationic lipid can be a neutral uncharged, zwitterionic, or anionic lipid.
  • Non-cationic lipids are typically employed to enhance fusogenicity.
  • Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn- glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE- mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimy
  • acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.
  • non-cationic lipids suitable for use in the LNPs include nonphosphorous lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, sphingomyelin, and the like.
  • nonphosphorous lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isoprop
  • the target cell is of a particular tissue type, and the cell- type targeting ligand binds to a marker protein, surface antigen, receptor protein, that is expressed by cells of the target tissue [0142] ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ (chemical or biological) that is able to bind to a specific receptor and direct (or target) the LNP to this receptor and/or drive subsequent LNP-receptor internalization, increasing efficiency in LNP transport and/or transduction or transfection to/of the targeted cells or tissues.
  • a marker protein surface antigen, receptor protein
  • Z comprises, or consists of, a cell-type targeting ligand or a receptor targeting ligand derived from proteins such as transferrin or a peptide derived thereof (e.g. the THR peptide), Epidermal Growth Factor (EGF), and basic Fibroblast Growth Factor FGF.
  • Z comprises, or consists of, a cell-type targeting ligand or a receptor targeting ligand derived from vitamins such as folic acid.
  • Z comprises, or consists of, a cell-type targeting ligand or a receptor targeting ligand derived from a muscle targeting peptide (MTP).
  • MTP muscle targeting peptide
  • Z is a cancer cell targeting peptide and comprises a peptide such as RGD, including cyclic RGD.
  • Z comprises, or consists of, a cell-type targeting ligand or a receptor targeting ligand derived from small molecules or hormones such as naproxen, ibuprofen, cholesterol, progesterone, or estradiol.
  • Z comprises an antibody or antigen-binding portion thereof.
  • an antibody may be or comprise, for example, a single chain antibody or variable domain, such as a camelid antibody, a heavy-chain antibody, a nanobody, a shark antibody, etc.
  • Z comprises or consists of a saccharide selected from the group consisting of monosaccharides, oligosaccharides and polysaccharides; preferably the saccharide is a monosaccharide, wherein said monosaccharide is preferably selected from the group consisting of mannose, galactose, N-acetylglucosamine, N- acetylgalactosamine, fucose, fructose, glucose, xylose, trehalose, desosamine, glucuronic acid, S6-galactose, S6-N-acetylgalactosamine, P6-mannose, P6-glucose, sialic acidand P1-fructose, more preferably selected from the group consisting of mannose, fructose, glucose, N-acetylglucosamine, N-acetylgalactosamine, trehalose, glucuronic acid, S6
  • Deoxymonosaccharides are common derivatives of monosaccharides encompassed in the present invention, i.e., monosaccharides that have had a hydroxyl group replaced with a hydrogen atom.
  • Examples of deoxymonosaccharides include, but are not limited to, deoxyribose, fucose, fuculose, rhamnose, quinovose, pneumose.
  • 2-amino-2-deoxymonosaccharides are also common derivatives of monosaccharides encompassed in the present invention, i.e., monosaccharides that have had a hydroxyl group replaced with an amino group.
  • Examples of monosaccharides containing a phosphate group include, but are not limited to, glucose-6-phosphate, mannose-6-phosphate and fructose-1-phosphate
  • Examples of monosaccharides containing a sulfate group include, but are not limited to, galactose-6-sulfate (S6-galactose), N-acetylgalactosamine-6-sulfate (S6-N-acetylgalactosamine).
  • Examples of monosaccharides containing a carboxyl group include, but are not limited to, glucuronic acid and sialic acid.
  • the monosaccharides and derivatives thereof mentioned herein also encompass acyclic (open-chain) forms and cyclic forms.
  • the monosaccharides and derivatives thereof mentioned herein also encompass D-stereoisomers and L-stereoisomers, as well as mixtures of D- and L- stereoisomers (e.g., racemic mixtures).
  • an oligosaccharide according to the present invention comprises at least two, three, four, five, six, seven, eight, nine or ten monosaccharides, e.g., selected from the monosaccharides disclosed hereinabove, including their derivatives.
  • such oligosaccharide(s) can be a homooligosaccharide (i.e., composed of units of the same monosaccharide, including their derivatives) or heterooligosaccharides (i.e., composed of units of at least two different monosaccharides, including their derivatives).
  • examples of oligosaccharides include, but are not limited to, disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, heptasaccharides, octasaccharides, nonasaccharides, and decasaccharides.
  • disaccharides include, but are not limited to, cellobiose, chitobiose, gentiobiose, gentiobiulose, isomaltose, kojibiose, lactose, lactulose, laminaribiose, maltose, maltulose, mannobiose, melibiose, melibiulose, nigerose, palatinose, rutinose, rutinulose, sophorose, sucrose, trehalose, turanose, and xylobiose.
  • trisaccharides include, but are not limited to, kestose, maltotriose, maltotriulose, melezitose, nigerotriose, and raffinose.
  • tetrasaccharides include, but are not limited to, lychnose, maltotetraose, nigerotetraose, nystose, sesamose, and stachyose.
  • oligosaccharides include, but are not limited to, acarbose, fructooligosaccharide, galactooligosaccharide, isomaltooligosaccharide, and maltodextrin.
  • oligosaccharides can be multi-antennary structures whereby some or all monosaccharides in the oligosaccharide are not linked to one another through O-glycosidic bonds but with branched linker structures.
  • tri-antennary N-acetylgalactosamine which is a ligand for asialoglycoprotein receptor ASGPR (see e.g., Zhou et al., Development of Triantennary N-Acetylgalactosamine Conjugates as Degraders for Extracellular Proteins; ACS Cent. Sci.2021).
  • a polysaccharide comprises more than ten monosaccharides (such as, e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more), e.g., selected from monosaccharides disclosed hereinabove, including their derivatives.
  • monosaccharides such as, e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more
  • polysaccharides can be homopolysaccharides or heteropolysaccharides.
  • a preferential saccharide or derivative thereof according to the present invention is mannose, galactose, N-acetylglucosamine, or N-acetylgalactosamine.
  • a saccharide or derivative thereof is mannose.
  • a saccharide or derivative thereof is galactose.
  • a saccharide or derivative thereof is N-acetylglucosamine.
  • a saccharide or derivative thereof is N-acetylgalactosamine.
  • a saccharide or derivative thereof according to the present invention is a deoxymonosaccharide.
  • a deoxymonosaccharide is preferably fucose.
  • a saccharide or derivative thereof is a saccharide containing a non-hydroxyl group which is a dialkyl amino group.
  • a saccharide containing a non-hydroxyl group which is a dialkyl amino group is a desosamine.
  • a saccharide or derivative thereof is a saccharide containing a non-hydroxyl group which is a sulfate group.
  • L may be any chemical chain which can comprise heteroatoms as well as cyclic moieties such as aryl and/or heteroaryl groups.
  • L may comprise up to 1000 carbon atoms and even more. The length and the chemical nature of L may be optimized depending on the group Z which is intended to be coupled to the LNP and the biological effect which is sought.
  • L is a chemical chain group comprising from 2 to 1000 carbon atoms, preferably from 2 to 500 carbon atoms, from 2 to 300 carbon atoms, e.g. from 2 to 100 carbon atoms, 2 to 40 carbon atoms, from 4 to 30 carbon atoms, or from 4 to 20 carbon atoms.
  • L comprises a C 2-20 straight or branched alkyl chain.
  • L is a polyether (e.g., polyethylene or polypropylene glycol).
  • alkyl amides including, but not limited to, ⁇ (CH2)y ⁇ C(O)NH ⁇ (CH2)p ⁇ and ⁇ (OCH 2 CH 2 ) y ⁇ C(O)NH ⁇ (OCH 2 CH 2 ) p ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ y ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ p ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ y ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ p ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ or more.
  • L is an alkyl amide of formula ⁇ (CH2)y ⁇ C(O)NH ⁇ (CH2)p ⁇ or of formula ⁇ (OCH 2 CH 2 ) y ⁇ C(O)NH ⁇ (OCH 2 CH 2 ) p ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ y ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • amides having the linking units of alkyl or ether bonds including, but not limited to, ⁇ R3 ⁇ C(O)NH ⁇ R4 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ R3 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ 4 ⁇ ⁇ ⁇ ⁇ each independently selected from alkyls (e.g., C 1-20 , C 1-12 , C 1-6 alkyl), ethers, or polyethers (e.g., PEGs having a molecular weight between about 200 to 2,000 g/mol).
  • alkyls e.g., C 1-20 , C 1-12 , C 1-6 alkyl
  • ethers e.g., C 1-12 , C 1-6 alkyl
  • polyethers e.g., PEGs having a molecular weight between about 200 to 2,000 g/mol
  • L may also comprise an alkylene diamine, e.g., ⁇ NH-(CH 2 ) r -NH ⁇ , ⁇ ⁇ ⁇ ⁇ ⁇ r ⁇ is an integer from 2 to 20, for instance from 2 to 10, or an integer selected from 2, 3, 4, or 5.
  • L is a polymer of alkylene diamines (also known as polyamines), e.g., a compound of formula ⁇ NH-[(CH2)r-NH]t-, ⁇ ⁇ ⁇ ⁇ ⁇ r ⁇ is as defined above and herein, and ⁇ t ⁇ is an integer of at least 2, for example of at least 3, 4, 5, 10 or more.
  • L may also comprise polyamides such as poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA), (e.g., pHPMA having a molecular weight between about 200 and about 5000 g/mol).
  • pHPMA poly(N-(2-hydroxypropyl)methacrylamide)
  • L is a polyethylene glycol (PEG), comprising from 1 to 40 ethylene glycol monomers, e.g. from 2 to 10, such as e.g. ⁇ (OCH2CH2)2 ⁇ (referred to ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ (OCH2CH2)3 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ (OCH 2 CH 2 ) 3 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ , ⁇ (OCH 2 CH 2 ) 4 ⁇ (referred to herein as ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ,
  • L may comprise one or more arylene or a heteroarylene groups Ar.
  • the arylene or a heteroarylene group Ar is a 6- to 10-membered aromatic carbocyclic group or a 5- or 12-membered heterocyclic group comprising one or more heteroatoms selected from the group consisting of N, O, S and Se.
  • the group Ar is substituted by an acyl or an amide moiety, or a bioisostere thereof.
  • the arylene or a heteroarylene group Ar is selected from the group consisting of phenylene and pyridylene.
  • L comprises an optionally substituted phenylene moiety.
  • L comprises an optionally substituted pyridylene moiety.
  • said phenylene or pyridylene groups are substituted by one or more moieties selected from the group consisting of halogen, C1-6 alkyl, C1-6 haloalkyl, C1-6 acyl and C1-6 alkoxy.
  • L may comprise an alkylene, ether, polyether, alkylene amide, arylene group, heteroarylene group, an acyl group or a combination thereof.
  • L comprises a polyether, arylene group, heteroarylene group, acyl group or a combination thereof.
  • L comprises an arylene or a heteroarylene group Ar.
  • said arylene or a heteroarylene group Ar is a 6- to 10-membered aromatic carbocyclic group or a 5- or 12-membered heterocyclic group comprising one or more heteroatoms selected from the group consisting of N, O, S and Se.
  • the arylene or a heteroarylene group Ar is selected from the group consisting of phenylene and pyridylene optionally substituted by one or more moieties selected from the group consisting of halogen, C1-6 alkyl, C1-6 haloalkyl, C1-6 acyl and C 1-6 alkoxy.
  • L comprises a PEG.
  • R1 is selected from the group consisting of H, C1-6 alkyl, C 1-6 haloalkyl and Z-(OCH 2 ⁇ CH 2 )n ⁇ , wherein n is selected from 1 to 40. In a preferred embodiment, R1 is selected from the group consisting of H, C 1-6 alkyl and C 1-6 haloalkyl. [0204] In some embodiments, R2 is selected from the group consisting of linear C1-12 alkyl, branched C3-12 alkyl, linear C1-12 haloalkyl, branched C3-12 haloalkyl and benzyl. In some preferred embodiments R2 is methyl, ethyl or benzyl, more preferably ethyl.
  • the phospholipid moiety Pl is a phosphatidylcholine, comprising two fatty acids as described above herein linked to the extender group E by the terminal nitrogen atom of the choline moiety.
  • Extender moiety E-NH- and extender group E [0209]
  • the extender group E of the extender moiety E-*NH- comprises one or more groups selected from the group consisting of a polyethylene glycol (PEG) or a polypropylene glycol (PPG) and an aromatic moiety.
  • the aromatic group Ar and the PEG are linked by an amide group or a bioisostere moiety thereof.
  • the extender group E of the extender moiety E-*NH- comprises a PEG covalently linked to the phospholipid moiety Pl by a -(CH 2 )q-C(O)-X- group, or a bioisostere moiety thereof, wherein q and X are as defined in classes and subclasses disclosed in the present invention.
  • the extender group E of the extender moiety E-*NH- comprises a PEG with 1 to 200, preferably 20 to 80, ethylene glycol units (-OCH 2 CH 2 ), i.e. the group E comprises a group (OCH2CH2)p, wherein p is 1 to 200, preferably 20 to 80, representing p the average number of PEG units in a range of molecular weights and oligomeric form.
  • the extender group E of the extender moiety E-*NH- comprises an aryl group Ar and a PEG, wherein the aromatic group Ar and the PEG are linked by an amide group or a bioisostere moiety thereof, and wherein the PEG is covalently linked to the phospholipid moiety Pl by a -(CH2)q-C(O)-X-group, or a bioisostere moiety thereof, wherein q and X are as defined in classes and subclasses disclosed in the present invention.
  • the extender group E comprises a PEG and the phospholipid is a phosphatidylcholine, wherein the PEG is linked to the terminal nitrogen of the choline moiety of the phosphatidylcholine moiety by a -(CH 2 )q-C(O)-X-group, wherein q and X are as defined in classes and subclasses disclosed in the present invention.
  • the extender group E comprises a PEG and an aromatic group Ar
  • the phospholipid moiety Pl is a phosphatidylcholine moiety, wherein the aromatic group Ar and the PEG are linked by an amide group or a bioisostere moiety thereof, and wherein the PEG is covalently linked to the terminal nitrogen of the choline moiety of the phosphatidylcholine moiety by a -(CH 2 )q-C(O)-X-group, wherein q and X are as defined in classes and subclasses disclosed in the present invention.
  • Some embodiments refer to a compound of formula (I): wherein Pl is a phospholipid moiety; E-*NH- is an extender moiety comprising an extender group E and a group -*NH-; and RL-NH- is a functional moiety including a nitrogen containing group -NH- and a group RL, and wherein said functional moiety RL-NH- comprises a steric shielding agent, a labelling agent, a cell-type targeting ligand or a receptor targeting ligand, a drug moiety or a combination thereof.
  • the extender group E of the extender moiety E-*NH- comprises a polyethylene glycol (PEG) or a polypropylene glycol (PPG).
  • the extender group E of the extender moiety E-*NH- comprises a PEG covalently linked to the phospholipid moiety Pl by a -(CH 2 )q-C(O)-X-group, or a bioisostere moiety thereof and the compound of formula (I) is represented by formula (Ia1): p is 1 to 200; preferably 20 to 80; q is 0 or 1; X is O or NH when q is 1 and X is NH when q is 0 or 1; preferably X is NH; and Pl and RL-NH are as defined and described in classes and subclasses disclosed in the present invention.
  • the extender group E of the extender moiety E-*NH- comprises an aryl group Ar and a PEG, wherein the aromatic group Ar and the PEG are linked by an amide group or a bioisostere moiety thereof, and wherein the PEG is covalently linked to the phospholipid moiety Pl by a -(CH 2 )q-C(O)-X-group, and the compound of formula (I) is represented by formula (Ia 2 ) or (Ia3): wherein p is 1 to 200; preferably 20 to 80; q is 0 or 1; m1 is 0, 1 or 2, m2 is 0, 1 or 2; X is O or NH when q is 1 and X is NH when q is 0 or 1; preferably X is NH; and Pl, Ar and RL-NH are as defined and described in classes and subclasses disclosed in the present invention.
  • L is an optionally substituted group selected from the group consisting of saturated or unsaturated, linear or branched C 2 -C 40 hydrocarbon chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyl diamines and combinations thereof; preferably L is polyethylene glycol.
  • L is a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers.
  • the polyethylene glycol (PEG) is PEG3, PEG4, or PEG5.
  • the compound of formula (I), as defined in the present disclosure comprises more than one spacer L selected from the group consisting of L1, L2 and L3, and said compound of formula (I) is selected from the group consisting of formula (Ib1), (Ib2), (Ib3), (Ib4) and (Ib5): wherein Pl, E, Z, L 1 , L 2 and L 3 are as defined and described in classes and subclasses disclosed in the present invention.
  • L1 is an optionally substituted group selected from the group consisting of saturated or unsaturated, linear or branched C 2 -C 40 hydrocarbon chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyl diamines and combinations thereof; preferably L1 is polyethylene glycol.
  • L1 is a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers.
  • the polyethylene glycol (PEG) is PEG3, PEG4, or PEG5.
  • L2 comprises one or more arylene or a heteroarylene groups Ar, as defined herein.
  • L 2 comprises a phenylene group or a pyridylene group.
  • L1 is a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers;
  • L 2 comprises one or more arylene or a heteroarylene groups;
  • L 3 is a C 1-6 alkylene group, L 3 is covalently linked to L 2 by one carbon atom of the arylene group or by one carbon atom or one heteroatom of the heteroarylene group;
  • L1 and L2 or L1 and L3 are covalently linked by an amide moiety, or a bioisostere moiety thereof, preferably an amide moiety -N(R1)C(O)-, or a bioisostere moiety thereof, wherein R1 is selected from the group consisting of H, C1-6 alkyl, C1-6 haloalkyl, Z-(OCH2 ⁇ CH2)n ⁇ , Z- NH-(CH2)r ⁇ (OCH2-
  • L1 is a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers;
  • L 2 comprises one or more arylene or a heteroarylene groups;
  • L 3 is a C 1-6 alkylene group;
  • L 3 is covalently linked to L 2 by one carbon atom of the arylene group or by one carbon atom or one heteroatom of the heteroarylene group;
  • L1 and L3 are covalently linked by an amide moiety -N(R1)C(O)-, or a bioisostere moiety thereof, wherein R1 is selected from the group consisting of H, C 1-6 alkyl, C 1-6 haloalkyl, Z- (OCH2 ⁇ CH2)n ⁇ , Z-NH-(CH2)r ⁇ (OCH2-CH2)n ⁇ , and Z-C(O) ⁇ (CH2)r ⁇ (OCH2-CH2)n ⁇ , wherein r is selected from 1 to 3, n is selected from 0 to 40, and Z is
  • L 1 and L 2 are covalently linked by an amide moiety -N(R1)C(O)-, or a bioisostere moiety thereof, wherein R1 is selected from the group consisting of H, C1-6 alkyl, C1-6 haloalkyl, Z-(OCH2 ⁇ CH2)n ⁇ , Z-NH-(CH2)r ⁇ (OCH2- CH 2 )n ⁇ , and Z-C(O) ⁇ (CH 2 )r ⁇ (OCH 2 -CH 2 )n ⁇ , wherein r is selected from 1 to 3, n is selected from 0 to 40, and Z is as defined and described in classes and subclasses disclosed in the present invention; preferably R1 is selected from the group consisting of H, C 1-6 alkyl, C 1-6 haloalkyl and Z-(OCH 2 ⁇ CH 2 )n ⁇ ,wherein n is selected from 1 to 40 and more preferably R1 is H or Z-(OCH
  • n 3, 4 or 5, r is 2 and R1 is H.
  • L 1 and L 3 are covalently linked by an amide moiety -N(R1)C(O)-, or a bioisostere moiety thereof, wherein R1 is selected from the group consisting of H, C1-6 alkyl, C1-6 haloalkyl, Z-(OCH2 ⁇ CH2)n ⁇ , Z-NH-(CH2)r ⁇ (OCH2- CH2)n ⁇ , and Z-C(O) ⁇ (CH2)r ⁇ (OCH2-CH2)n ⁇ , wherein r is selected from 1 to 3, n is selected from 0 to 40, and Z is as defined and described in classes and subclasses disclosed in the present invention; preferably R1 is selected from the group consisting of H, C1-6 alkyl, C1-6 haloalkyl and Z-(OCH2 ⁇ CH2)n ⁇ , wherein n is selected from 1 to 40 and more
  • n 3, 4 or 5, r is 2 and R1 is H.
  • Examples of bioisostere moieties of the amide -N(R1)C(O)- may be selected from -C(O)N(R1)-, -N(R3)C(O)N(R1)-, -N(R1)C(O)N(R3)-, -N(R1)C(S)-, -C(S)N(R1)-, -N(R1)C(S)N(R3)-, -N(R3)C(S)N(R1)-, -S(O) 2 -N(R1)-, -N(R1)-S(O) 2 - and a triazolyl group, among others, wherein R3 and R1 are each independently selected from the group consisting of H, C1-6 alkyl, C1-6 haloalkyl, aryl, alkylaryl, Z-(OCH2 ⁇ CH2)n ⁇ , Z
  • L 2 comprises an arylene or a heteroarylene group Ar
  • L1 and the squaramide moiety of formula (SQ), or L1 and L3 when present, are covalently bonded to said arylene or a heteroarylene group Ar in positions orto, meta or para.
  • L3 and the squaramide moiety of formula (SQ) are covalently bonded to said arylene or a heteroarylene group Ar in positions orto, meta or para.
  • L2 comprises an arylene or a heteroarylene group Ar and L3 is a group C1-6 alkylene
  • one or more groups L3 are covalently bonded to said arylene or a heteroarylene group Ar in positions orto, meta or para.
  • L 1 or L 3 may be selected from alkyl (e.g., C 1-20 , C 1-12 , C 1- 6 alkyl), ether, polyether, polyester, alkyl amide, or a combination thereof.
  • ⁇ combination ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ L 1 or L 3 may comprise several hydrocarbon chains, oligomer chains or polymeric chains (e.g.2, 3, 4, 5 or 6) linked by any appropriate group, such as -O-, -S-, -NHC(O)-, -OC(O)-, -C(O)-O-C(O)-, -NH-, -NH-CO-NH-, -O-CO-, -NH-(CS)-NH-, -NH-CS- phosphodiester or phosphorothioate groups.
  • L3 comprises a C2-20 straight or branched alkyl chain.
  • L 1 or L 3 may also comprise polyesters such as polycaprolactone (e.g., polycaprolactone having a molecular weight between about 200 and about 5000 g/mol) or poly(D,L-lactic-co-glycolic acid) (PLGA) (e.g., PLGA having a molecular weight between about 200 and about 5000 g/mol).
  • L1 or L3 may be selected from an optionally substituted group comprising, or consisting of, saturated or unsaturated, linear or branched C2-C40 hydrocarbon chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkylene diamines, and combinations thereof.
  • alkyl amides including, but not limited to, ⁇ (CH2)m3 ⁇ C(O)NH ⁇ (CH2)m4 ⁇ and ⁇ (OCH 2 CH 2 ) m3 ⁇ C(O)NH ⁇ (OCH 2 CH 2 ) m4 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ 3 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ 4 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ m3 ⁇ ⁇ ⁇ ⁇ ⁇ m4 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ or more.
  • L1 or L3 is an alkyl amide of formula ⁇ (CH2)m3 ⁇ C(O)NH ⁇ (CH2)m4 ⁇ or ⁇ (OCH2CH2)m3 ⁇ C(O)NH ⁇ (OCH 2 CH 2 ) m4 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ m 3 ⁇ ⁇ ⁇ ⁇ ⁇ m 4 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ an integer from 1-10, an integer from 1-6, and integer from 3-6, and integer from 3-5, or an integer independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • amides having the linking units of alkyl or ether bonds including, but not limited to, ⁇ R 5 ⁇ C(O)NH ⁇ R 6 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ 5 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ 6 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • L1 may also comprise an acyl group -C(O)-(CH2)r, or an alkylene amine, e.g., ⁇ NH-(CH 2 ) r -, or an alkylene diamine, e.g., ⁇ NH-(CH 2 ) r -NH ⁇ , where ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ to 10, or an integer selected from 2, 3, 4, or 5.
  • L1 is a polymer of alkylene diamines (also known as polyamines), e.g., a compound of formula ⁇ NH-[(CH 2 ) r -NH] t ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • the compound of formula (IIb) is selected from the group consisting of (IIc), (IId) and (IIe): , [0252]
  • the present invention provides a compound of formula (IIIa) which is useful in obtaining a compound of formula (I), as disclosed in the present invention: (IIIa) wherein Pl and E are as defined and described in classes and subclasses disclosed in the present invention.
  • an incubation can last from several hours to several days, for instance from 6 to 72 hours, e.g. from 12 to 48 hours or from 16 to 24 hours. In some embodiments, the incubation is ended when a sufficient yield of coupling is achieved.
  • the temperature of incubation is typically from 4°C to 50°C. In some preferential embodiments, the incubation is performed at room temperature, i.e. at a temperature from 18 °C to 30 °C, e.g. at around 20°C. In some embodiments, the incubating solution can be stirred.
  • lipid nanoparticles can be formed by any method known in the art.
  • ionizable LNPs of the present invention can be used as a combination of both a therapeutic and diagnostic tool, e.g., theragnostic use.
  • Modifications of biological functionalities and/or properties of LNPs [0291]
  • chemical modifications of the components of the ionizable LNPs may modify one, or several, of its biological functionalities and/or properties.
  • biological functionalities and/or properties can depend on the nature of functional moiety RL which is introduced to modify the ionizable LNPs in the present invention.
  • ionizable LNPs-directed neutralizing antibodies does not generate ionizable LNPs-directed neutralizing antibodies; and/or - an increased efficiency of the ionizable LNPs; and/or - an increased transfection efficacy of the ionizable LNPs towards a specific cell, tissue, and/or organ; and/or - a reduced cellular toxicity when transfecting cells in culture; and/or - an induced cellular targeted mortality of cancer cells; and/or - enabling the visualization/monitoring of the ionizable LNPs upon in vivo administration or upon modification of cells in vitro; and/or - enabling theragnostic applications; e.g. combining a therapeutic agent and a diagnostic agent.
  • the ionizable LNPs of the present invention show a preferential tropism for an organ or cell selected from liver, heart, brain, joints, retina, and/or skeletal muscle.
  • the ionizable LNPs of the invention show a preferential tropism for cultured cells selected from, but not limited to, hepatocytes, cardiomyocytes, myocytes, neurons, motor neurons, retinal pigmented cells, photoreceptors, chondrocytes, hematopoietic stem cells (HSC), and/or induced pluripotent stem cells (iPS).
  • the present invention relates to an ionizable LNPs according to the present invention, for use in transfecting a cell of a subject.
  • an agent as defined herein, such as a nucleic acid, small molecule, or a protein (antibody or fragment thereof) into a cell.
  • the transduced/transfected nucleic acid or protein of interest may be of any type and is selected depending on the sought effect.
  • the ionizable LNP according to the present invention when used for transduced/transfecting a cell, it comprises a gene or a protein.
  • the LNP according to the invention can comprise an exogenous gene expression cassette.
  • said cassette may comprise a promoter, a gene of interest, and a terminator.
  • Nucleic acids useful in the disclosure typically include a first region of linked nucleosides encoding a polypeptide of ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ -terminus of the ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ - ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ -terminus of the ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • a nucleic acid further includes a poly-A region or a Kozak sequence ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ -UTR).
  • a nucleic acid e.g., an mRNA
  • the gene editing tools can be of any type, and encompass, without being limited to, CRISPR and its associated systems (including without limitation a Cas protein such as a Cas9 protein, or fusion protein thereof, or a Cas mRNA such as a Cas9 mRNA, a crRNA and tracrRNA, the latter two being either separate or linked together in a single gRNA), TALEN, Zinc Finger Nuclease, meganuclease, as well as RNA and DNA encoding said gene editing proteins and their associated systems.
  • CRISPR and its associated systems including without limitation a Cas protein such as a Cas9 protein, or fusion protein thereof, or a Cas mRNA such as a Cas9 mRNA, a crRNA and tracrRNA, the latter two being either separate or linked together in a single gRNA
  • TALEN Zinc Finger Nuclease
  • meganuclease as well as RNA and DNA encoding said gene editing proteins and their associated systems.
  • the present invention also refers to a non-therapeutic method for delivering an agent (e.g. a nucleic acid or a protein) to a target cell comprising: contacting the target cell with an ionizable LNP, as defined and described in classes and subclasses disclosed in the present invention, wherein the ionizable LNP comprises the agent (e.g.
  • the present invention also refers to an ionizable LNP for use in a non-therapeutic method for delivering an agent (e.g.
  • a nucleic acid or a protein to a target cell
  • said method comprises: contacting the target cell with an ionizable LNP, as defined and described in classes and subclasses disclosed in the present invention, wherein the ionizable LNP comprises the agent (e.g. the nucleic acid or the protein) to be delivered and a group RL, conjugated to the ionizable LNP via a squaramide wherein N is a nitrogen atom from the functional moiety RL-NH-, and wherein N* is a nitrogen atom of an amino group of a phospholipid lipid Pl, comprising the group RL a cell-type targeting ligand or a receptor targeting ligand of the target cell.
  • an ionizable LNP comprises the agent (e.g. the nucleic acid or the protein) to be delivered and a group RL, conjugated to the ionizable LNP via a squaramide wherein N is a nitrogen atom from the functional moiety RL
  • the method for delivering an agent comprises contacting the target cell with an ionizable LNP, wherein the ionizable LNP comprises the agent (e.g.
  • N is a nitrogen atom from the functional moiety RL-NH-
  • N* is a nitrogen atom of an amino group of a phospholipid Pl, comprising the group RL a cell- type specific ligand of the target cell wherein Pl is a phospholipid moiety
  • E-*NH- is an extender moiety comprising an extender group E and a group -*NH-
  • RL-NH- is a functional moiety including a nitrogen containing group -NH- and a group RL, and wherein the functional moiety RL-NH- of the compound of formula (I) comprises a cell-type targeting ligand or a receptor targeting ligand of the target cell.
  • the present invention also relates to use of a LNP according to the present invention for transfecting a cell of a subject.
  • the present invention also relates to a method for transfecting a cell of a subject, comprising administering an ionizable LNP according to the present invention to said subject.
  • the present invention also relates to a method of delivering an agent (e.g. a gene or a protein, or small molecule) to a target cell, the method comprising contacting a cell with an ionizable LNP, as defined and described in classes and subclasses disclosed in the present, and an agent (e.g.
  • nucleic acid a therapeutic nucleic acid or a protein
  • the ionizable LNP comprises the agent (e.g. the nucleic acid or the protein) to be delivered and a compound of formula (I): wherein Pl is a phospholipid moiety; E-*NH- is an extender moiety comprising an extender group E and a group -*NH-; and RL-NH- is a functional moiety including a nitrogen containing group -NH- and a group RL, and wherein the functional moiety RL-NH- of the compound of formula (I) comprises a cell-type targeting ligand or receptor targeting ligand of the target cell.
  • the present invention also relates to a method for delivering an agent (e.g. a gene or a protein) into a cell of a subject, comprising administering an ionizable LNP, according to the present invention, comprising said agent (e.g. said gene or said protein), or a composition comprising the same, to said subject.
  • an agent e.g. a gene or a protein
  • the present invention further relates to an in vitro or ex vivo method for transducing/transfecting a cell, comprising contacting said cell with an ionizable LNP according to the invention.
  • the cell may be from a subject (e.g., a patient).
  • the cell after transduction/transfection, the cell may be transplanted to a subject in need thereof (e.g., the patient, and/or another subject).
  • an ionizable LNP can be administered to a cell in vivo, ex vivo, or in vitro.
  • the cell may be derived from a mammal (e.g., humans, non-human primates, cows, mice, sheep, goats, pigs, rats, etc.) In some embodiments, the cell may be derived from a human.
  • the cell may be, but is not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, tumor cellsand induced pluripotent stem cells (iPS).
  • hepatocytes epithelial cells
  • hematopoietic cells epithelial cells
  • epithelial cells endothelial cells
  • lung cells bone cells
  • stem cells mesenchymal cells
  • neural cells cardiac cells
  • adipocytes vascular smooth muscle cells
  • cardiomyocytes vascular smooth muscle cells
  • cardiomyocytes vascular smooth muscle cells
  • LNPs described herein may be particularly useful in gene therapy, e.g., to deliver a therapeutic nucleic acid of interest to a subject.
  • the present invention also relates to a LNP according to the present invention, for use in gene therapy.
  • the present invention also relates to a method of gene therapy in a subject in need thereof, comprising administering a LNP according to the present invention to said subject.
  • a LNP of the invention can be delivered by any appropriate route to the subject.
  • appropriate administration routes encompass, without being limited to, inhalational, topical, intra-tissue (e.g.
  • LNPs according to the present invention may be useful for preventing or treating a disease, disorder, or condition.
  • LNPs may be useful in treating a disease, disorder, or condition characterized by missing or aberrant protein or polypeptide activity.
  • the LNP includes an mRNA encoding a missing or aberrant polypeptide may be administered or delivered to a cell. Subsequent translation of the mRNA may produce the polypeptide, thereby reducing or eliminating an issue caused by the absence of or aberrant activity caused by the polypeptide.
  • Diseases, disorders, and/or conditions characterized by dysfunctional or aberrant protein or polypeptide activity for which a LNP according to the present invention may be administered include, but are not limited to, rare diseases, infectious diseases (as both vaccines and therapeutics), cancer and proliferative diseases, genetic diseases (e.g ., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, and metabolic diseases.
  • a subject is ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ treated ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ an L
  • the mammal is a primate, more preferably a human.
  • the present invention further relates to a composition comprising a LNP according to the invention.
  • LNPs in the composition according to the present invention comprises at least one agent (e.g. a gene or a protein).
  • the composition is a pharmaceutical composition comprising a LNP according to the invention and at least one pharmaceutically acceptable vehicle.
  • pharmaceutically acceptable vehicles, excipients, carriers and preservatives that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, proteins (such as, e.g., serum albumin, gelatin, immunoglobulins and the like), buffer substances (such as, e.g., phosphates, citrates or other organic acids, and the like), amino acids (such as, e.g., glycine, glutamine, asparagine, arginine, lysine and the like), antioxidants (such as, e.g., ascorbic acid and the like), chelating agents (such as, e.g., EDTA), sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as, e.g., protamine sulfate, disodium hydrogen phosphate,
  • proteins such as, e
  • a pharmaceutical composition according to the present invention comprises vehicles which are pharmaceutically acceptable for a formulation intended for injection into a subject.
  • these may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • a pharmaceutical composition according to the present invention comprise one or more agents that promote the entry of LNP described herein into a mammalian cell, such as, e.g., natural and/or synthetic polymers, such as poloxamer, chitosan, cyclodextrins, dendrimers, poly(lactic-co-glycolic acid) polymers, and the like.
  • LNPs comprising at least one agent e.g. a gene or a protein
  • the invention thus relates to a medicament comprising LNPs comprising at least one agent (e.g.
  • LNPs according to the present invention are to be administered to a subject in need thereof in a therapeutically effective amount.
  • the dose of LNPs required to achieve a desired effect or a therapeutic effect will vary based on several factors including, but not limited to, the specific route of administration, the level of gene, RNA or protein expression required to achieve a therapeutic effect, the specific disease being treated, and the stability of the gene, RNA, and/or protein product.
  • the volume of LNPs administered to a subject will also depend, among other things, on the size of the subject, the dose of the LNP required to obtain therapeutic effect, the concentration of the LNP, and the proposed route of administration.
  • the rate of administration of LNPs delivered to a subject will also depend, among other things, on the size of the subject, the dose of the LNP required to obtain therapeutic effect, the concentration of the LNP, the volume of the LNP solution, and the proposed route of administration.
  • the total dose or total volume of LNPs may be administered continuously (i.e., wherein the total dose or total volume of modified LNPs is injected in a single shot or infusion); or discontinuously (i.e., wherein fractions of the total dose or total volume of LNPs are injected with intermittent periods between each shot, preferably with short intermittent periods such as periods of time of 15 seconds, 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, or 5 minutes between each shot or infusion).
  • Kits [0331] The present invention also relates to kits and kits-of-parts, for: - transfecting a cell of a subject; and/or - delivering an agent (e.g.
  • kits or kits-of-parts comprise one or more LNPs and/or compositions according to the present invention.
  • the kits or kits-of-parts further comprise a device for delivery of one or more LNPs and/or compositions according to the present invention.
  • the kits further include instructions for delivery of one or more LNPs and/ or compositions according to the present invention.
  • kits comprise instructions for preventing and/or treating a targeted disease, using the compositions, and/or methods described herein.
  • kits described herein may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and/or package inserts with instructions for performing any methods described herein.
  • Step 5 4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)-N-(2-(2-(2- (((3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy) ethoxy)ethoxy)ethyl)benzamide (5B1) [0365] To a mixture of intermediate 4-((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)benzoic acid described in preparation 1 (256 mg), HATU (372 mg) and DIPEA (0.43 mL) in DMF was added under argon atmosphere compound obtained in previous step (254 mg).
  • Step 3 Procedure for grafting 3,4-diethoxycyclobut-3-ene-1,2-dione for compound (6B1) [0368] To a solution of peptide (0.1 mmol) in distilled water (0,5 mL), DIEA was added until a pH between 7 and 7.5 is reached. A solution of 3,4-diethoxycyclobut-3-ene-1,2- dione (1.2 eq.0.12 mmol) in EtOH (0,5mL) was added. The reaction was stirred for 2h at room temperature.
  • Step 4 Purification and salt exchange
  • the raw peptide was purified directly from the reaction mixture using a reverse phase preparative HPLC system (Waters Delta Prep 4000) using a reverse phase column (Vydac Denali prep C-18, 10 ⁇ m, 120 ⁇ , 50 300 mm) and a suitable gradient of ACN+TFA 0.1%/ H2O+TFA 0.1% as eluent.
  • the fractions containing the purified target peptide were identified by UV measurement (UV/Visible Waters 2489 detector) at 214 nm and the selected fractions were then combined and freeze-dried.
  • the exchange of trifluoroacetate salt into acetate was carried out during purification via a proprietary buffer.
  • Step 1 Spacer Boc-Aryl-PEG2-COOH synthesis
  • the peptidyl resin Boc-Aryl-PEG2-resin was synthesized using standard deprotection and coupling cycles.
  • the cleavage from the resin of the protected spacer was performed under selective mild conditions by the treatment of HFIP/DCM (20:80) for 1h at RT, the resin was removed by filtration and the filtrate was evaporated to dryness under vacuum.
  • the protected spacer was precipitated in ice-cold diethyl ether, isolated by centrifugation.
  • Step 2 Peptide DfKRG synthesis and spacer introduction on solid support
  • the peptidyl resin Fmoc-Asp(Otbu)-DPhe-Lys(Alloc)-Arg(Pbf)-Gly-resin was synthesized, followed by the deprotection of the Alloc group by using 0.35 eq. of Palladium (0) and 24 eq. of phenylsilane.
  • the spacer from step 1 (1.5 eq.) was introduced using BOP (1.5 eq.) as activator and DIEA (3eq.) in DMF at RT for 3h.
  • Step 3 Cleavage from the solid support [0375] The cleavage from the resin was performed under selective mild conditions by the treatment of HFIP/DCM (20:80) for 1h at RT, the resin was removed by filtration and the filtrate was evaporated to dryness under vacuum. Crude peptide was precipitated by addition of cold diethylether. Protected peptide was thus obtained by filtration.
  • Step 4 Head to tail cyclization: [0376] Protected peptide H-Asp(OtBu)-DPhe-Lys(Boc-Spacer)-Arg(Pbf)-Gly-OH was solubilized in DMF (at 10 mM).
  • Step 5 Acido labile protecting group removal
  • Protected cyclic peptide was treated with TFA/H 2 O/TIPS (92.5/5/2.5, v/v/v)) solution at room temperature for 2 hours. Mixture was evaporated to dryness under vacuum, and crude peptide was precipitated by addition of cold diethylether and placed in a freeze-dryer to remove as much residual TFA as possible prior to the coupling step of the diethyl squarate motif and used without further purification.
  • Step 6 Procedure for grafting 3,4-diethoxycyclobut-3-ene-1,2-dione for compound (7B1) [0378] To a solution of crude peptide (0.1 mmol) in distilled water (0,5 mL), DIEA was added until a pH between 7 and 7.5 is reached. A solution of 3,4-diethoxycyclobut-3-ene- 1,2-dione (1.2 eq.0.12 mmol) in EtOH (0.5mL) was added. The reaction was stirred for 2h at room temperature. Step 7: Purification and salt exchange.
  • the raw peptide was purified directly from the reaction mixture using a reverse phase preparative HPLC system (Waters Delta Prep 4000) using a reverse phase column (Vydac Denali prep C-18, 10 ⁇ m, 120 ⁇ , 50 300 mm) and a suitable gradient of ACN+TFA 0.1%/ H2O+TFA 0.1% as eluent.
  • the fractions containing the purified target peptide were identified by UV measurement (UV/Visible Waters 2489 detector) at 214 nm and the selected fractions were then combined and freeze-dried.
  • the exchange of trifluoroacetate salt into acetate was carried out during purification via a proprietary buffer.
  • Step 2 Solid support cleavage [0381] After drying in vacuo, the peptidyl resin was exposed to 20% HFIP in DCM solution several times to lead to the sequence cleaved from the resin. The filtrate was concentrated in vacuo, solubilized in ACN/H 2 O followed by lyophilization.
  • Step 3 Backbone cyclisation The protected peptide was dissolved in DMF and treated with DIPEA in the presence of PyBOP leading to the cyclization of the backbone intermediate.
  • Step 4 Side chain of cyclic peptide and tBoc-PEG2 spacer deprotection
  • the side chain protecting group of the peptide and tBoc-PEG2 spacer were cleaved using a mixture of TFA/H 2 O/TIPS during 2 hours. Isolation of the cyclic backbone peptide was performed by precipitation using Et2O/Pentane and the material was washed several times with this mixture to remove protecting groups and maximum of residual TFA. The material was then solubilized in ACN/H 2 O followed by freeze- drying step.
  • Step 5 Peptide purification
  • the cyclized crude material was dissolved in an appropriate co-mixture of ACN/Milli-Q H2O and injected on a C18 preparative HPLC column using acidic eluents (A: MilliQ-H O+0.1% AcOH ; B : ACN+0.05% + 2 AcOH).
  • ESI-MS m/z: [M+H] calcd for C34H54N10O10: 762.40, found: 763.82.
  • EXAMPLE 2 Production and coupling of LNPs [0386] Conjugated LNPs were generated by coupling the squarate moieties of the invention to at least one primary amine as described below. 2.1 Production of mRNA containing LNPs [0387] Lipid nanoparticles were prepared on a Nanoassemblr ⁇ microfluidic system (Precision NanoSystems) according to the manufacturer's instructions. Depending on the desired formulation, an ethanol solution consisting of an ionizable lipid (e.g.
  • Dlin-MC3- DMA, CAS 1224606-06-7 a zwitterionic lipid (e.g., distearoylphosphatidylcholine (DSPC, CAS 816-94-4), a component to provide membrane integrity (such as a sterol, e.g., cholesterol, CAS 54-88-5), PEG-lipids molecule (e.g., 1-(monomethoxy- polyethyleneglycol)-2,3-dimyristoylglycerol, with an average PEG molecular weight of ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ - ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ -62-4) and: a functionalized PEG-lipid molecule of formula (IIIa), in particular, the compound of formula (IIIa 1 ) 10C2, 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N
  • LNP3 was used as a negative control and did not include a functionalized PEG- lipid molecule of formula (IIa) nor a functionalized PEG-lipid molecule of formula (IIIa).
  • an aqueous solution with an mRNA encoding for eGFP protein was prepared in 50 mM citrate buffer at pH 3.0.
  • LNP were prepared at a total lipid to mRNA weight ratio of approximately 10:1.
  • Lipid and mRNA-containing solutions were mixed 1:3 (ethanol: citrate) at a constant ⁇ ow rate of 12 ml/min to form LNPs.
  • Table 3 shows the LNPs prepared and tested.
  • the exposure of mannose moieties at the surface of the LNPs was evaluated using ConA binding assay.
  • the conjugated particles were incubated with the lectin ConA, and the changes in particle size (Z-average diameter) were monitored over time utilizing a Malvern Zetasizer ZS.
  • the results demonstrated that the mannose conjugated LNPs were effectively recognized and bound by the ConA, which resulted in an increase of the particle size. This increase confirms the successful interaction of ConA with the mannose moieties exposed on the surface of the LNPs ( Figure 2B) c.
  • the exposure of mannose moieties at the surface of the LNPs was evaluated using ⁇ v ⁇ 3 integrin binding assay.
  • the conjugated particles were incubated with th ⁇ ⁇ v ⁇ 3 integrin, and the changes in particle size (Z-average diameter) were monitored over time utilizing a Malvern Zetasizer ZS.
  • the results demonstrated that the conjugated LNPs were effectively recognized and bound by the integrin, which resulted in an increase of the particle size.
  • the mild coupling conditions preserved the structural integrity of the ligands, ensuring their functionality and enabling effective interactions with their respective target.
  • the preservation of ligand functionality is crucial to maintain their biological activity and binding specificity after conjugation. Consequently, the LNPs can effectively engage in their intended biological interactions, confirming the robustness and efficacy of the coupling process.

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Abstract

La présente invention concerne des nanoparticules lipidiques ionisables (LNP) comprenant au moins une molécule lipidique comportant une fraction squaramide liée à une fraction fonctionnelle. Les nanoparticules lipidiques (LNP) comprenant au moins une molécule lipidique comportant une fraction squaramide liée à une fraction fonctionnelle, divulguées ici, sont utiles dans l'administration d'un agent à une cellule ou dans la transfection d'une cellule, en particulier pour la thérapie génique ou pour l'édition génique.
PCT/EP2024/075075 2023-09-07 2024-09-09 Nanoparticules lipidiques ionisables Pending WO2025051994A1 (fr)

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