EP4626400A1 - Nouveaux lipides conjugués à un polyglycérol et compositions de nanoparticules lipidiques les comprenant - Google Patents

Nouveaux lipides conjugués à un polyglycérol et compositions de nanoparticules lipidiques les comprenant

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Publication number
EP4626400A1
EP4626400A1 EP23836699.1A EP23836699A EP4626400A1 EP 4626400 A1 EP4626400 A1 EP 4626400A1 EP 23836699 A EP23836699 A EP 23836699A EP 4626400 A1 EP4626400 A1 EP 4626400A1
Authority
EP
European Patent Office
Prior art keywords
lipid
lnp
polymer
alkyl
formula
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23836699.1A
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German (de)
English (en)
Inventor
Nolan GALLAGHER
Andrew MILLSTEAD
Douglas A. Rose
Sandy ZHANG
Daniele VINCIGUERRA
Matthew G. Stanton
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Generation Bio Co
Original Assignee
Generation Bio Co
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Filing date
Publication date
Application filed by Generation Bio Co filed Critical Generation Bio Co
Publication of EP4626400A1 publication Critical patent/EP4626400A1/fr
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A—HUMAN NECESSITIES
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    • 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
    • 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
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers

Definitions

  • RNA and DNA molecules which typically range from 300 kDa to 5,000 kDa, or ⁇ 1-15 kb
  • siRNA small interfering RNAs
  • ASOs antisense oligonucleotides
  • RNA sensing by myeloid dendritic cells MDCs
  • PRR pattern recognition receptor
  • rAAV adeno-associated virus
  • the present disclosure provides a polymer-conjugated lipid, comprising:
  • R 1 is absent, hydrogen, Ci-Ce alkyl, or a hydrophobic tail comprising 10-30 carbon atoms;
  • R 3 is a hydrophobic tail comprising 10-30 carbon atoms
  • the PG derivative is a carboxylated PG.
  • the carboxylated PG is a glutarylated PG.
  • the glutarylated PG is 3 -methyl glutarylated PG.
  • the carboxylated PG is 2-carboxycyclohexane-l -carboxylated PG.
  • the PG or the PG derivative is linear or branched.
  • R 1 is absent, and wherein R 2 and R 3 are each independently a hydrophobic tail comprising 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon atoms.
  • R 2 and R 3 are each independently a hydrophobic tail comprising 16, 17, 18, 19, 20, 21, or 22 carbon atoms.
  • R 2 and R 3 are each independently a hydrophobic tail comprising 18 carbon atoms, and wherein the lipid moiety is dioctadecylamine (DODA).
  • DODA dioctadecylamine
  • the lipid moiety conjugated to a linker is represented by the following structure:
  • the PG or the PG derivative comprises about 5 to 100 monomeric units, or an average of 5 to 100 monomeric units.
  • the PG or the PG derivative comprises about 5, 6, 7, 8, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 monomeric units, or an average of 5, 6, 7, 8, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 monomeric units.
  • the helper lipid represented by Formula (II) is: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.
  • the ionizable lipid is present in the LNP in an amount of about 20 mol% to about 60 mol% of the total lipid present in the LNP. In some embodiments, the ionizable lipid is present in the LNP in an amount of about 35 mol% to about 50 mol% of the total lipid present in the LNP.
  • the present disclosure also provides a lipid nanoparticle (LNP) comprising:
  • TAA therapeutic nucleic acid
  • helper lipid wherein the helper lipid is DSPC;
  • a second lipid-anchored polymer wherein the second lipid-anchored polymer comprises DSPE conjugated to PEG.
  • the linear PG comprises about 34 monomeric units or about 45 monomeric units, or an average of 34 monomeric units, or an average of 45 monomeric units.
  • the TNA is ceDNA.
  • the TNA is a single-stranded nucleic acid or a double-stranded nucleic acid.
  • the single-stranded nucleic is mRNA.
  • the singlestranded nucleic acid is a DNA molecule (ssDNA).
  • the ssDNA comprises at least three stem-loop structures at the 3’ end. In some embodiments, the ssDNA comprises at least four or more stem-loop structures at the 3’ end. In some embodiments, the at least one stem-loop structure at the 3’ end comprises a hairpin DNA structure. In some embodiments, the at least one stem-loop structure at the 3’ end comprises a DNA structure selected from the group consisting of: a cruciform DNA structure, a hammerhead DNA structure, a quadraplex DNA structure, a bulged DNA structure, and a multibranched loop structure.
  • the at least one stem-loop structure at the 3’ end does not comprise the A, A’, D, and D’ regions that would be present in a wild-type AAV ITR. In some embodiments, the at least one stem-loop structure at the 3’ end does not comprise the A, A’, B, B’, C, C’, D, and D’ regions that would be present in a wild-type AAV ITR. In some embodiments, the at least one stem-loop structure at the 3’ end does not comprise a rep binding element (RBE) that would be present in a wild-type ITR. In some embodiments, the at least one stem-loop structure at the 3’ end does not comprise a terminal resolution site (trs) that would be present in a wild-type ITR.
  • RBE rep binding element
  • trs terminal resolution site
  • At least one stem-loop structure at the 3’ end further comprises a functional moiety.
  • the ssDNA molecule further comprises a 5’ end, comprising at least one stem-loop structure.
  • the ssDNA comprises at least two stem-loop structures at the 5’ end.
  • the ssDNA comprises at least three stem-loop structures at the 5’ end.
  • the ssDNA comprises at least four or more stem-loop structures at the 5’ end.
  • the at least one stem-loop structure at the 5’ end comprises a hairpin DNA structure.
  • the at least one stem-loop structure at the 5’ end does not comprise the
  • the at least one stem-loop structure at the 5’ end does not comprise the A, A’, B, B’, C, C’, D, and D’ regions that would be present in a wild-type AAV ITR.
  • the at least one stemloop structure at the 5’ end does not comprise a rep binding element (RBE) that would be present in a wild-type ITR.
  • the at least one stem-loop structure at the 5’ end does not comprise a terminal resolution site (trs) that would be present in a wild-type ITR.
  • the stem structure at the 5’ end comprises four or more nucleotides that are modified to be exonuclease resistant.
  • the nucleotides are phosphorothioate -modified nucleotides.
  • the loop structure at the 5’ end further comprises one or more nucleic acids to stabilize the ends. In some embodiments, the loop structure at the 5’ end further comprises one or more nucleic acids that are chemically modified. In some embodiments, the loop structure at the 5’ end further comprises one or more aptamers. In some embodiments, the loop structure at the 5’ end further comprises one or more synthetic ribozymes.
  • the loop structure at the 5’ end further comprises one or more antisense oligonucleotides (ASOs). In some embodiments, the loop structure at the 5’ end further comprises one or more short-interfering RNAs (siRNAs). In some embodiments, the loop structure at the 5’ end further comprises one or more antiviral nucleoside analogues (AN As).
  • ASOs antisense oligonucleotides
  • shRNAs short-interfering RNAs
  • AN As antiviral nucleoside analogues
  • the loop structure at the 5’ end further comprises one or more triplex forming oligonucleotides. In some embodiments, the loop structure at the 5’ end further comprises one or more gRNAs or gDNAs. In some embodiments, the loop structure at the 5’ end further comprises one or more molecular probes.
  • the ssDNA molecule is devoid of any viral capsid protein coding sequences. In some embodiments, the ssDNA molecule is synthetically produced in vitro. In some embodiments, the ssDNA molecule is synthetically produced in vitro in a cell-free environment.
  • the ssDNA molecule does not activate or minimally activates an immune pathway.
  • the immune pathway is an innate immune pathway.
  • the innate immune pathway is selected from the group consisting of the cGAS/STING pathway, the TLR9 pathway, an inflammasome-mediated pathway, and a combination thereof.
  • the ss DNA molecule is capable of expressing at least one therapeutic protein or a therapeutic fragment thereof.
  • the at least one therapeutic protein is selected from the group consisting of an antibody, an enzyme, a coagulation factor, a transcription factor, a replication factor, a growth factor, a hormone, and a fusion protein.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the LNP of the disclosure and a pharmaceutically acceptable carrier.
  • the present disclosure also provides a method of treating a genetic disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the LNP of the disclosure or the pharmaceutical composition of the disclosure.
  • the subject is a human.
  • the genetic disorder is selected from the group consisting of sickle cell anemia; melanoma; hemophilia A (clotting factor VIII (FVIII) deficiency); hemophilia B (clotting factor IX (FIX) deficiency); cystic fibrosis (CFTR); familial hypercholesterolemia (LDL receptor defect); hepatoblastoma; Wilson’s disease; phenylketonuria (PKU); congenital hepatic porphyria; an inherited disorder of hepatic metabolism; Lesch Nyhan syndrome; a thalassaemia; xeroderma pigmentosum; Fanconi’s anemia; retinitis pigmentosa; ataxia telangiectasia; Bloom’s syndrome; retinoblastoma; a mucopolysaccharide storage disease; a Niemann-Pick Disease; Fabry disease; Schindler disease; GM2-gangliosidosis Type II (Sandhoff).
  • the present disclosure also provides a method of providing anti- tumor immunity to a subject in need thereof, the method comprising administering to the subject an effective amount of the LNP of the disclosure or the pharmaceutical composition of the disclosure.
  • the present disclosure also provides a method of synthesizing a polymer- conjugated lipid of the disclosure, comprising: a) reacting a lipid moiety which is conjugated to a linker with 2,3-epoxy-l-(l- ethoxyethoxyjpropane (EEGE) in the presence of a base under argon atmosphere, or in the presence of an organocatalyst, to produce a lipid moiety conjugated to a linker and polymerized EEGE; and b) subjecting the lipid moiety conjugated to a linker and polymerized EEGE to acidic conditions to produce the polymer-conjugated lipid.
  • EEGE 2,3-epoxy-l-(l- ethoxyethoxyjpropane
  • the base is a phosphazene base. In some embodiments, the phosphazene base is P4-t-Bu.
  • the lipid moiety conjugated to a linker is represented by the following structure:
  • Figure 1A depicts a MALDI-TOF spectrum of DODA-PG34.
  • Figure IB depicts a MALDI-TOF spectrum of DODA-PG45.
  • Figure 1C depicts a MALDI-TOF spectrum of DODA-PG58.
  • Figure IE shows “Scheme 2” showing synthesis of DODA-PG45 and DODA-PG58.
  • Figure 2A shows the total flux measured by the total photon counts per the region of interest, i.e., the liver, measured in mice by In Vivo Imaging System (IVIS) at Day 4 post-dosing for LNP formulations of the disclosure and a negative control (PBS).
  • IVIS In Vivo Imaging System
  • Figure 2B shows the total flux measured by the total photon counts per the region of interest, i.e., the liver, measured in mice by IVIS at Day 7 post-dosing for LNP formulations of the disclosure and a negative control (PBS).
  • Figure 2C shows the total flux measured by the total photon counts per the region of interest, i.e., the liver, measured in mice by IVIS across two collection days (Day 4 and Day 7) post-dosing for LNP formulations of the disclosure and a negative control (PBS).
  • PBS negative control
  • Figure 2D shows percent change in body weight (BW) of mice at Day 1 post-dosing with LNP formulations of the disclosure.
  • Figure 3 shows luciferase activity for LNP formulations of the disclosure containing different lipid-anchored polymers.
  • Figure 4B is a schematic depicting the assay used for evaluating opsonization-driven cell uptake of LNPs.
  • Figure 4C shows DiD fluorescence area normalized to area of live nuclei measured for LNP formulations of the disclosure containing different lipid-anchored polymers.
  • Figure 5 shows DiD fluorescence area normalized to area of live nuclei for LNP formulations of the disclosure containing different amounts of polyglycerol-conjugated lipids and a control.
  • Figure 6 shows the amount of endosomal escape measured as the amount of luciferase expression normalized to DiD uptake in mouse hepatocytes treated with LNP formulations of the disclosure containing different amounts of polyglycerol-conjugated lipids and a control.
  • Figure 7 shows the whole blood clearance of the Control LNP, and the different Lipid Z carrying LNPs of the disclosure.
  • Figure 8A shows the total flux quantified by total photon counts per the region of interest, i.e., the liver, measured in mice by IVIS at Day 7 post-dosing with LNP formulations of the disclosure and a negative control (DPBS).
  • DPBS negative control
  • Figure 10 shows DiD fluorescence area normalized to area of live nuclei for LNP formulations of the disclosure containing different amounts of polyglycerol-conjugated lipids and a control, and formulated with DSPE-PEG5K-N3 using a mole percentage of 0.5%.
  • the present disclosure provides polymer-conjugated lipids, comprising, e.g., a polyglycerol (PG) conjugated to dioctadecylamine (DODA), such as DODA-PG34, DODA-PG45 and DODA- PG58, and methods of their synthesis.
  • PG polyglycerol
  • DODA dioctadecylamine
  • the present disclosure also provides lipid nanoparticles (LNPs) comprising, inter alia, polymer-conjugated lipids of the disclosure, and methods of treatment of various disorders comprising administering to a subject in need thereof LNPs of the disclosure.
  • LNPs lipid nanoparticles
  • the linker may be selected from the group consisting of -(CH 2 ) n -, -C(0)(CH2) n -, - C(O)O(CH 2 ) n -, -0C(0)(CH2) n C(0)0-, and -NH(CH 2 ) n C(O)O-, wherein n is an integer ranging from 1 to 20.
  • the linker is -C(O)(CH 2 ) n -, and wherein n is 2, 3, 4, 5, or 6. In one specific embodiment, n is 4.
  • the terms “lipid particle” or “lipid nanoparticle” or “LNP” refers to a lipid formulation that can be used to deliver a therapeutic agent such as nucleic acid therapeutics to a target site of interest (e.g., cell, tissue, organ, and the like).
  • the lipid particle of the disclosure is a nucleic acid containing lipid particle, which is typically formed from a cationic lipid, a non-cationic lipid, and optionally a conjugated lipid that prevents aggregation of the particle.
  • a therapeutic agent such as a therapeutic nucleic acid may be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation.
  • the lipid particle comprises a nucleic acid (e.g., ceDNA, ssDNA, mRNA, etc.) and a lipid comprising one or more tertiary amino groups, one or more phenyl ester bonds and a disulfide bond.
  • a nucleic acid e.g., ceDNA, ssDNA, mRNA, etc.
  • a lipid comprising one or more tertiary amino groups, one or more phenyl ester bonds and a disulfide bond.
  • lipid particles of the disclosure typically have a mean diameter of from about 20 nm to about 75 nm, about 20 nm to about 70 nm, about 25 nm to about 75 nm, about 25 nm to about 70 nm, from about 30 nm to about 75 nm, from about 30 nm to about 70 nm, from about 35 nm to about 75 nm, from about 35 nm to about 70 nm, from about 40 nm to about 75 nm, from about 40 nm to about 70 nm, from about 45 nm to about 75 nm, from about 50 nm to about 75 nm, from about 50 nm to about 70 nm, from about 60 nm to about 75 nm, from about 60 nm to about 70 nm, from about 65 nm to about 75 nm, from about 65 nm to about 70 nm, or about 20 nm, about 25 nm, about 30 nm to about 75 n
  • the LNPs of the disclosure have a mean diameter selected to provide an intended therapeutic effect.
  • the LNPs of the disclosure have a mean diameter that is compatible with a target organ, such that the LNPs of the disclosure are able to diffuse through the fenestrations of a target organ (e.g., liver) or a target cell subpopulation (e.g., hepatocytes).
  • a target organ e.g., liver
  • a target cell subpopulation e.g., hepatocytes
  • the lipid particles of the disclosure typically have a mean diameter of less than about 75 nm, less than about 70 nm, less than about 65 nm, less than about 60 nm, less than about 55 nm, less than about 50 nm, less than about 45 nm, less than about 40 nm, less than about 35 nm, less than about 30 nm, less than about 25 nm, less than about 20 nm in size.
  • cationic lipid refers to any lipid that is positively charged at physiological pH.
  • the cationic lipid in the lipid particles may comprise, e.g., one or more cationic lipids such as l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA), l,2-di-y-linolenyloxy-N,N-dimethylaminopropane (y- DLenDMA), 2, 2-dilinoleyl-4-(2-dimethylaminoethyl)-[ 1,3] -dioxolane (DLin-K-C2-DMA), 2,2- dilinoleyl-4-dimethylaminomethyl-[ 1,3] -dioxolane (DLin-K-DMA), “SS-cleavable
  • a cationic lipid can also be an ionizable lipid, i.e., an ionizable cationic lipid, i.e.
  • the term “cationic lipids” also encompasses lipids that are positively charged at any pH, .e.g., lipids comprising quaternary amine groups, i.e., quarternary lipids. Any cationic lipid described herein comprising a primary, secondary or tertiary amine group may be converted to a corresponding quaternary lipid, for example, by treatment with chloromethane (CH3CI) in acetonitrile (CH3CN) and chloroform (CHCI3).
  • CH3CI chloromethane
  • CH3CN acetonitrile
  • chloroform CHCI3
  • the term “ionizable lipid” refers to a lipid, e.g., cationic lipid, having at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will be understood by one of ordinary skill in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipids be present in the charged or neutral form.
  • ionizable lipids have a pKa of the protonatable group in the range of about 4 to about 7.
  • ionizable lipid may include “cleavable lipid” or “SS- cleavable lipid”.
  • organic lipid solution refers to a composition comprising in whole, or in part, an organic solvent having a lipid.
  • local delivery refers to delivery of an active agent such as an interfering RNA (e.g., siRNA) directly to a target site within an organism.
  • an agent can be locally delivered by direct injection into a disease site such as a tumor or other target site such as a site of inflammation or a target organ such as the liver, heart, pancreas, kidney, and the like.
  • nucleotides contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups.
  • single-stranded DNA molecule refers to a deoxyribonucleic acid (DNA) molecule comprising at least one single-stranded nucleic acid sequence flanked by at least one stem-loop structure at the 3’ end.
  • the single-stranded DNA molecule further comprises at least one stem-loop structure at the 5’ end.
  • a single-stranded DNA molecule may comprise regions of double-stranded DNA (or partial duplexes), e.g., a stem-loop structure, e.g., an inverted terminal repeat or portion thereof, at the terminal end(s), e.g., the 3’ end and/or the 5’ end.
  • a ssDNA molecule is a synthetic ssDNA molecule.
  • a ssDNA molecule comprises at least one stemloop structure at the 5’ end and at least one stem-loop structure at the 3’ end.
  • single-stranded (ss) synthetic DNA molecules refers to a single-stranded (ss) synthetic DNA molecule (ssDNA), a single-stranded vector and synthetic production methods thereof in an entirely cell-free environment.
  • the production may involve one or more molecules in a manner that does not involve replication or other multiplication of the molecule by or inside of a cell or using a cellular extract.
  • Synthetic production avoids contamination of the produced molecule with cellular contaminants, e.g., cellular proteins or cellular nucleic acid, viral protein or DNA, insect protein or DNA and further minimizes unwanted cellular- specific modification of the molecule during the production process, e.g., methylation or glycosylation or other post-translational modification.
  • cellular contaminants e.g., cellular proteins or cellular nucleic acid, viral protein or DNA, insect protein or DNA
  • the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil, and various types of wetting agents.
  • the term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans, as well as any carrier or diluent that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered compound.
  • Typical gaps, designed and created by the methods described herein and synthetic vectors generated by the methods can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 nt long in length.
  • Exemplified gaps in the present disclosure can be 1 nt to 10 nt long, 1 to 20 nt long, 1 to 30 nt long in length.
  • nick refers to a discontinuity in a double stranded DNA molecule where there is no phosphodiester bond between adjacent nucleotides of one strand typically through damage or enzyme action. It is understood that one or more nicks allow for the release of torsion in the strand during DNA replication and that nicks are also thought to play a role in facilitating binding of transcriptional machinery.
  • the ceDNA is a doggyboneTM DNA. According to some embodiments, the ceDNA comprises one or more phosphorothioate-modified nucleotides. According to some embodiments, the ceDNA comprises no phosphorothioate-modified nucleotides.
  • neDNA or “nicked ceDNA” refers to a closed-ended DNA having a nick or a gap of 1-100 nucleotides in a stem region or spacer region upstream of an open reading frame (e.g., a promoter and transgene to be expressed).
  • inverted terminal repeat refers to a nucleic acid sequence located at the 5’ and/or 3’ terminus of the ssDNA molecules disclosed herein, which comprises at least one stem-loop structure comprising a partial duplex and at least one loop.
  • stem-loop structure refers to a nucleic acid structure comprising at least one double-stranded region (referred to herein as a “stem”) and at least one single-stranded region (referred to herein as a “loop”).
  • a stem-lop structure is a hairpin structure.
  • a stem-loop structure comprises more than one stem and more than one loop.
  • a loop is located at the end of a stem (such that a single loop connects the two strands of a duplex stem, e.g., as in a hairpin structure).
  • the 5’ and/or 3’ terminus of certain ssDNA molecules comprise inverted terminal repeats (ITRs) of about 145 nucleotides at both ends, or fragments thereof.
  • ITRs inverted terminal repeats
  • the terminal 125 nucleotides in each ITR form a palindromic double-stranded T-shaped hairpin structure, in which the A-A' palindrome forms the stem, and the two smaller palindromes, B-B' and the C-C', form the cross-arms of the T.
  • the other 20 nucleotides in ITR remain single-stranded, and are called the D sequence.
  • the D(-) sequence (also referred to herein as “the ssD(-) sequence”) is at the 3' end, and the complementary D(+) sequence (also referred to herein as “the ssD(+) sequence”) is at the 5' end.
  • Second-strand DNA synthesis turns both ssD(-) and ssD(+) sequences into a double- stranded (ds) D( ⁇ ) sequence, each of which comprises a D region and a D’ region.
  • ssD(-) and ssD(+) have been reported to contain one or more transcription factor binding sites and to be required for packaging and replication (Ling et al. J Virol. 2015 Jan 15;89(2):952-61; WO2016081927A2, incorporated by reference in its entirety herein).
  • the ITR may be a viral ITR (e.g., AAV or other dependo virus), a sequence derived or modified from a viral ITR (e.g., truncation, deletion, substitution, insertion and/or addition), or an entirely artificial sequence (e.g., the ITRs contain no sequences derived from a virus).
  • the ITR may further comprise one stem-loop structure (e.g., a “hairpin”), or more than one stem-loop structure.
  • the ITR may comprise two stem-loop structures (e.g., a “hammerhead”, “doggy-bone”, or “dumbbell”), three stem-loop structures (e.g., “cruciform”), or more complex structures (e.g., quadruplex stem-loop structure).
  • the ITR may comprise an aptamer sequence or one or more chemical modifications.
  • the ITR can be made entirely out of an aptamer sequence having at least one stem region and at least one loop region.
  • the term “substantially symmetrical WT-ITRs” or a “substantially symmetrical WT-ITR pair” refers to a pair of WT-ITRs within a single-stranded DNA (ssDNA) molecule that are both wild type ITRs that have an inverse complement sequence across their entire length.
  • an ITR can be considered to be a wild-type sequence, even if it has one or more nucleotides that deviate from the canonical naturally occurring canonical sequence, so long as the changes do not affect the physical and functional properties and overall three-dimensional structure of the sequence (secondary and tertiary structures).
  • the deviating nucleotides represent conservative sequence changes.
  • a sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the canonical sequence (as measured, e.g., using BLAST at default settings), and also has a symmetrical three-dimensional spatial organization to the other WT- ITR such that their 3D structures are the same shape in geometrical space.
  • the substantially symmetrical WT-ITR has the same ssD(-)/ssD(+), A-A’, C-C’ and B-B’ loops in 3D space.
  • a substantially symmetrical WT-ITR can be functionally confirmed as WT by determining that it has an operable Rep binding site (RBE or RBE’) and terminal resolution site (TRS) that pairs with the appropriate Rep protein.
  • RBE or RBE operable Rep binding site
  • TRS terminal resolution site
  • modified ITR or “mod-ITR” or “mutant ITR” are used interchangeably and refer to an ITR with a mutation in at least one or more nucleotides as compared to the WT-ITR from the same serotype.
  • the mutation can result in a change in one or more of ssD(-) or ssD(+), A, A’, C, C’, B, B’ regions in the ITR, and can result in a change in the three-dimensional spatial organization (z.e., its 3D structure in geometric space) as compared to the 3D spatial organization of a WT-ITR of the same serotype.
  • polynucleotide and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes single, double, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer including purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. “Oligonucleotide” generally refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double-stranded DNA.
  • oligonucleotide is also known as “oligomers” or “oligos” and may be isolated from genes, or chemically synthesized by methods known in the art.
  • polynucleotide and nucleic acid should be understood to include, as applicable to the embodiments being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.
  • the nucleic acid is a single-stranded DNA (ssDNA) molecule described by the present disclosure.
  • the subject is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of diseases and disorders.
  • the methods and compositions described herein can be used for domesticated animals and/or pets.
  • a human subject can be of any age, gender, race or ethnic group, e.g., Caucasian (white), Asian, African, black, African American, African European, Hispanic, Mideastern, etc.
  • the subject can be a patient or other subject in a clinical setting. In some embodiments, the subject is already undergoing treatment.
  • the subject is an embryo, a fetus, neonate, infant, child, adolescent, or adult. In some embodiments, the subject is a human fetus, human neonate, human infant, human child, human adolescent, or human adult. In some embodiments, the subject is an animal embryo, or non-human embryo or non-human primate embryo. In some embodiments, the subject is a human embryo.
  • the phrase “subject in need” refers to a subject that (i) will be administered a ceDNA lipid particle (or pharmaceutical composition comprising a ceDNA lipid particle) according to the described disclosure, (ii) is receiving a ceDNA lipid particle (or pharmaceutical composition comprising aceDNA lipid particle) according to the described disclosure; or (iii) has received a ceDNA lipid particle (or pharmaceutical composition comprising a ceDNA lipid particle) according to the described disclosure, unless the context and usage of the phrase indicates otherwise.
  • the terms “effective amount”, which may be used interchangeably with the terms “therapeutic amount”, “therapeutically effective amount”, an “amount effective”, or “pharmaceutically effective amount” of an active agent refers to an amount that is sufficient to provide the intended benefit of treatment or effect, e.g., expression or inhibition of expression of a target sequence in comparison to the expression level detected in the absence of a therapeutic nucleic acid.
  • Suitable assays for measuring expression of a target gene or target sequence include, e.g., examination of protein or RNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art. Dosage levels are based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular active agent employed. Thus, the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods.
  • the terms “effective amount”, “therapeutic amount”, “therapeutically effective amounts” and “pharmaceutically effective amounts” include prophylactic or preventative amounts of the compositions of the described invention.
  • pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of, a disease, disorder or condition in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease, disorder or condition, including biochemical, histologic and/or behavioral symptoms of the disease, disorder or condition, its complications, and intermediate pathological phenotypes presenting during development of the disease, disorder or condition. It is generally preferred that a maximum dose be used, that is, the highest safe dose according to some medical judgment.
  • dose and “dosage” are used interchangeably herein.
  • therapeutic amount refers to non-prophylactic or non-preventative applications.
  • therapeutic effect refers to a consequence of treatment, the results of which are judged to be desirable and beneficial.
  • a therapeutic effect can include, directly or indirectly, the arrest, reduction, or elimination of a disease manifestation.
  • a therapeutic effect can also include, directly or indirectly, the arrest reduction or elimination of the progression of a disease manifestation.
  • therapeutically effective amount may be initially determined from preliminary in vitro studies and/or animal models.
  • a therapeutically effective dose may also be determined from human data.
  • the applied dose may be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other well-known methods is within the capabilities of the ordinarily skilled artisan.
  • General principles for determining therapeutic effectiveness which may be found in Chapter 1 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001), incorporated herein by reference, are summarized below.
  • Pharmacokinetic principles provide a basis for modifying a dosage regimen to obtain a desired degree of therapeutic efficacy with a minimum of unacceptable adverse effects. In situations where the drug's plasma concentration can be measured and related to therapeutic window, additional guidance for dosage modification can be obtained.
  • the terms “treat,” “treating,” and/or “treatment” include abrogating, inhibiting, slowing or reversing the progression of a condition, ameliorating clinical symptoms of a condition, or preventing the appearance of clinical symptoms of a condition, obtaining beneficial or desired clinical results. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s). In one aspect of any of the aspects or embodiments herein, the terms “treat,” “treating,” and/or “treatment” include abrogating, inhibiting, slowing or reversing the progression of a condition, or ameliorating clinical symptoms of a condition.
  • Beneficial or desired clinical results include, but are not limited to, preventing the disease, disorder or condition from occurring in a subject that may be predisposed to the disease, disorder or condition but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), alleviation of symptoms of the disease, disorder or condition, diminishment of extent of the disease, disorder or condition, stabilization (i.e., not worsening) of the disease, disorder or condition, preventing spread of the disease, disorder or condition, delaying or slowing of the disease, disorder or condition progression, amelioration or palliation of the disease, disorder or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.
  • proliferative treatment preventing the disease, disorder or condition from occurring in a subject that may be predisposed to the disease, disorder or condition but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), alleviation of symptoms of the disease, disorder or condition, diminishment of extent of the disease, disorder or condition, stabilization (i.e., not worsening) of
  • the term “combination therapy” refers to treatment regimens for a clinical indication that comprise two or more therapeutic agents.
  • the term refers to a therapeutic regimen in which a first therapy comprising a first composition (e.g., active ingredient) is administered in conjunction with a second therapy comprising a second composition (active ingredient) to a patient, intended to treat the same or overlapping disease or clinical condition.
  • the first and second compositions may both act on the same cellular target, or discrete cellular targets.
  • the phrase “in conjunction with,” in the context of combination therapies means that therapeutic effects of a first therapy overlaps temporarily and/or spatially with therapeutic effects of a second therapy in the subject receiving the combination therapy.
  • the combination therapies may be formulated as a single formulation for concurrent administration, or as separate formulations, for sequential administration of the therapies.
  • alkyl refers to a saturated monovalent hydrocarbon radical of 1 to 20 carbon atoms (z.e., Ci-20 alkyl). “Monovalent” means that alkyl has one point of attachment to the remainder of the molecule. In one embodiment, the alkyl has 1 to 12 carbon atoms (z.e., Cm alkyl) or 1 to 10 carbon atoms (z.e., Ci-10 alkyl).
  • the alkyl has 1 to 8 carbon atoms (z.e., Ci- 8 alkyl), 1 to 7 carbon atoms (z.e., C1-7 alkyl), 1 to 6 carbon atoms (z.e., Ci-6 alkyl), 1 to 4 carbon atoms (z.e., C1-4 alkyl), or 1 to 3 carbon atoms (z.e., Cm alkyl).
  • Examples include, but are not limited to, methyl, ethyl, 1 -propyl, 2-propyl, 1 -butyl, 2-methyl-l -propyl, 2-butyl, 2-methyl -2 -propyl, 1 -pentyl, 2- pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2 -butyl, 3-methyl-l -butyl, 2-methyl-l -butyl, 1-hexyl, 2- hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2- methyl-3-pentyl, 2,3 -dimethyl -2 -butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and the like.
  • a linear or branched alkyl such as a “linear or branched Ci-6 alkyl,” “linear or branched C1-4 alkyl,” or “linear or branched C1-3 alkyl” means that the saturated monovalent hydrocarbon radical is a linear or branched chain.
  • the term “linear” as referring to aliphatic hydrocarbon chains means that the chain is unbranched.
  • alkylene refers to a saturated divalent hydrocarbon radical of 1 to 20 carbon atoms (z.e., Ci-20 alkylene), examples of which include, but are not limited to, those having the same core structures of the alkyl groups as exemplified above. “Divalent” means that the alkylene has two points of attachment to the remainder of the molecule. In one embodiment, the alkylene has 1 to 12 carbon atoms (z.e., C 1 12 alkylene) or 1 to 10 carbon atoms (z.e., Ci-10 alkylene).
  • the alkylene has 1 to 8 carbon atoms (z.e., Ci-8 alkylene), 1 to 7 carbon atoms (z.e., C1-7 alkylene), 1 to 6 carbon atoms (z.e., Ci-6 alkylene), 1 to 4 carbon atoms (z.e., C1-4 alkylene), 1 to 3 carbon atoms (i.e., Cm alkylene), ethylene, or methylene.
  • a linear or branched alkylene, such as a “linear or branched Ci-6 alkylene,” “linear or branched C1-4 alkylene,” or “linear or branched C1-3 alkylene” means that the saturated divalent hydrocarbon radical is a linear or branched chain.
  • alkenyl refers to straight or branched aliphatic hydrocarbon radical with one or more (e.g., one or two) carbon-carbon double bonds, wherein the alkenyl radical includes radicals having “cis” and “trans” orientations, or by an alternative nomenclature, “E” and “Z” orientations.
  • alkenylene refers to aliphatic divalent hydrocarbon radical of 2 to 20 carbon atoms (z.e., C2-20 alkenylene) with one or two carbon-carbon double bonds, wherein the alkenylene radical includes radicals having “cis” and “trans” orientations, or by an alternative nomenclature, “E” and “Z” orientations. “Divalent” means that alkenylene has two points of attachment to the remainder of the molecule. In one embodiment, the alkenylene has 2 to 12 carbon atoms (i.e., C2-12 alkenylene), 2 to 10 carbon atoms (i.e., C2 10 alkenylene). In one embodiment, the alkenylene has 2 to four carbon atoms (C2-4).
  • a linear or branched alkenylene such as a “linear or branched C2-6 alkenylene,” “linear or branched C2-4 alkenylene,” or “linear or branched C2-3 alkenylene” means that the unsaturated divalent hydrocarbon radical is a linear or branched chain.
  • cycloalkylene refers to a divalent saturated carbocyclic ring radical having 3 to 12 carbon atoms as a monocyclic ring, or 7 to 12 carbon atoms as a bicyclic ring. “Divalent” means that the cycloalkylene has two points of attachment to the remainder of the molecule. In one embodiment, the cycloalkylene is a 3- to 7-membered monocyclic or 3- to 6- membered monocyclic.
  • Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, cyclononylene, cyclodecylene, cycloundecylene, cyclododecylene, and the like.
  • the cycloalkylene is cyclopropylene.
  • heterocycle refers to a cyclic group which contains at least one N atom has a heteroatom and optionally 1-3 additional heteroatoms selected from N and S, and are non-aromatic (i.e., partially or fully saturated). It can be monocyclic or bicyclic (bridged or fused).
  • heterocyclic rings include, but are not limited to, aziridinyl, diaziridinyl, thiaziridinyl, azetidinyl, diazetidinyl, triazetidinyl, thiadiazetidinyl, thiazetidinyl, pyrrolidinyl, pyrazolidinyl, imidazolinyl, isothiazolidinyl, thiazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl, azepanyl, azocanyl, and the like.
  • the heterocycle contains 1 to 4 heteroatoms, which may be the same or different, selected from N and S.
  • a group is described as being “optionally substituted,” the group may be either (1) not substituted, or (2) substituted. If a carbon of a group is described as being optionally substituted with one or more of a list of substituents, one or more of the hydrogen atoms on the carbon (to the extent there are any) may separately and/or together be replaced with an independently selected optional substituent.
  • the substituent for the optionally substituted alkyl, alkylene, alkenylene, cycloalkylene, and heterocyclyl described above is selected from the group consisting of halogen, -CN, -NR101R102, -CF3, -ORioo, aryl, heteroaryl, heterocyclyl, -SR101, -SOR101, -SO2R101, and -SO3M.
  • salts refers to pharmaceutically acceptable organic or inorganic salts of an ionizable lipid of the disclosure.
  • Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate “mesylate,” ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (z.e., l,r-methylene
  • the polymer-conjugated lipid of the disclosure comprises PG.
  • the polymer-conjugated lipid of the disclosure comprises PG derivative.
  • the PG derivative comprised in the polymer-conjugated lipid of the disclosure may be a carboxylated PG, e.g., 2-carboxycyclohexane-l-carboxylated polyglycerol.
  • the PG derivative comprised in the polymer-conjugated lipid of the disclosure may also be a glutarylated PG, e.g., 3 -methyl glutarylated PG.
  • the PG derivative comprised in the polymer-conjugated lipid of the disclosure is represented by the following structural formula: wherein: n is an integer ranging from 8 to 100; and
  • R is selected from the group consisting of
  • the lipid moiety comprised in the polymer-conjugated lipid of the disclosure is represented by Formula (I) or a pharmaceutically acceptable salt thereof, wherein:
  • R 1 is absent, hydrogen, Ci-Ce alkyl, or a hydrophobic tail comprising 10-30 carbon atoms;
  • LNPs consisting of: (i) a therapeutic nucleic acid (TNA); (ii) an ionizable lipid; (iii) a sterol; and (iv) a first lipid-anchored polymer, and optionally further consisting of a helper lipid, wherein the first lipid-anchored polymer comprises the polymer-conjugated lipid of the present disclosure.
  • lipid DLin-MC3-DMA The lipid DLin-MC3-DMA is described in Jayaraman et al., Angew. Chem. Int. Ed Engl. (2012), 51(34): 8529-8533, content of which is incorporated herein by reference in its entirety.
  • the ionizable lipid is selected from the group consisting of N-[l-(2,3- dioleyloxy)propyll-N,N,N-trimethylammonium chloride (DOTMA); N-[l-(2,3-dioleoyloxy)propyll- N,N,N-trimethylammonium chloride (DOTAP); 1 ,2-dioleoyl-sn-glycero -3 -ethylphosphocholine (DOEPC); 1 ,2-dilauroyl-sn-glycero-3 -ethylphosphocholine (DLEPC); l,2-dimyristoyl-sn-glycero-3- ethylphosphocholine (DMEPC); 1 ,2-dimyristoleoyl- sn-glycero-3-ethylphosphocholine (14:1), Nl- [2-((lS)-l-[(3-aminopropyl)
  • the condensing agent e.g. a cationic lipid
  • R 2 and R 2’ are each independently C 1-3 alkylene.
  • the linear or branched C 1-3 alkylene represented by R 1 or R 1’ , the linear or branched C 1-6 alkylene represented by R 2 or R 2’ , and the optionally substituted linear or branched C 1-6 alkyl are each optionally substituted with one or more halo and cyano groups.
  • R 1 and R 2 taken together are C1-3 alkylene and R 1’ and R 2’ taken together are C1-3 alkylene, e.g., ethylene.
  • R 3 and R 3’ are each independently optionally substituted C1-3 alkyl, e.g., methyl.
  • R 4 and R 4’ are each –CH.
  • R 2 is optionally substituted branched C1-6 alkylene; and R 2 and R 3 , taken together with their intervening N atom, form a 5- or 6-membered heterocyclyl.
  • R 2’ is optionally substituted branched C1-6 alkylene; and R 2’ and R 3’ , taken together with their intervening N atom, form a 5- or 6-membered heterocyclyl, such as pyrrolidinyl or piperidinyl.
  • R 4 is –C(R a )2CR a , or –[C(R a )2]2CR a and R a is C1-3 alkyl; and R 3 and R 4 , taken together with their intervening N atom, form a 5- or 6-membered heterocyclyl.
  • R 4’ is –C(R a )2CR a , or –[C(R a )2]2CR a and R a is C1-3 alkyl; and R 3’ and R 4’ , taken together with their intervening N atom, form a 5- or 6-membered heterocyclyl, such as pyrrolidinyl or piperidinyl.
  • R 5 and R 5’ are each independently C 1-10 alkylene or C 2-10 alkenylene. In one embodiment, R 5 and R 5’ are each independently C 1-8 alkylene or C 1-6 alkylene. In some embodiments, R 6 and R 6’ , for each occurrence, are independently C1-10 alkylene, C3-10 cycloalkylene, or C 2-10 alkenylene. In one embodiment, C 1-6 alkylene, C 3-6 cycloalkylene, or C 2-6 alkenylene. In one embodiment the C 3-10 cycloalkylene or the C 3-6 cycloalkylene is cyclopropylene. In some embodiments, m and n are each 3.
  • the ionizable lipid in the LNPs of the present disclosure may be selected from any one of the lipids listed in Table 1 below, or a pharmaceutically acceptable salt thereof.
  • Table 1 Formula (B) the ionizable lipid in the LNPs of the present disclosure is represented by Formula (B): or a pharmaceutically acceptable salt thereof, wherein: a is an integer ranging from 1 to 20 (e.g., a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); b is an integer ranging from 2 to 10 (e.g., b is 2, 3, 4, 5, 6, 7, 8, 9, or 10); R 1 is absent or is selected from (C2-C20)alkenyl, -C(O)O(C2-C20)alkyl, and cyclopropyl substituted with (C2-C20)alkyl; and R 2 is (C2-C20)alkyl.
  • the ionizable lipid of Formula (B) is represented by Formula (B-1): or a pharmaceutically acceptable salt thereof, wherein c and d are each independently integers ranging from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and wherein the remaining variables are as described for Formula (B).
  • c and d in Formula (B-1) are each independently integers ranging from 2 to 8, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 8, 4 to 7, 4 to 6, 5 to 8, 5 to 7, or 6 to 8, wherein the remaining variables are as described for Formula (B-1).
  • c in Formula (B-1) is 2, 3, 4, 5, 6, 7, or 8, wherein the remaining variables are as described for Formula (B), or the second or third embodiment of Formula (B).
  • c and d in Formula (B-1) are each independently 1, 3, 5, or 7, wherein the remaining variables are as described for Formula (B), or the second or third embodiment of Formula (B).
  • d in the cationic lipid of Formula (B-l) is 2, 3, 4, 5, 6, 7, or 8, wherein the remaining variables are as described for Formula (B), or the second, third or fourth embodiments of Formula (B).
  • at least one of c and d in Formula (B-l) is 7, wherein the remaining variables are as described for Formula (B), or the second, third or fourth embodiments of Formula (B).
  • Formula (B) the ionizable lipid of Formula (B) or Formula (B-l) is represented by Formula (B-2):
  • b in Formula (B), (B-l), or (B-2) is an integer ranging from 3 to 9, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth or sixth embodiments of Formula (B).
  • b in Formula (B), (B-l), or (B-2) is an integer ranging from 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 5 to 9, 5 to 8,
  • a in Formula (B), (B- 1), or (B-2) is an integer ranging from 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 18, 4 to 17, 4 to 16, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 25 to 8, 5 to 7, 6 to 18, 6 to 17, 6 to 16, 6 to 15, 6 to 14, 6 to 13,
  • a in Formula (B), (B-1), or (B-2) is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18, , wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth or seventh embodiment of Formula (B).
  • R 1 in Formula (B), Formula (B-1), or Formula (B-2) is absent or is selected from (C5-C15)alkenyl, -C(O)O(C4-C18)alkyl, and cyclopropyl substituted with (C4-C16)alkyl, wherein the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh or eighth embodiments of Formula (B).
  • R 5’ in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is a branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C).
  • the ionizable lipid in the LNPs of the present disclosure is Ionizable Lipid 87 : heptadecan-9-yl 9-((4-(dimethylamino)butanoyl)oxy)hexadecanoate or a pharmaceutically acceptable salt or ester thereof, or a deuterated analogue thereof.
  • the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (E-l):
  • R 1 and R 2 are each independently hydrogen or C1-C2 alkyl, or C2-C3 alkenyl; or R’, R 1 , and R 2 are each independently hydrogen, C1-C2 alkyl; and all other remaining variables are as described for Formula (E), Formula (E-1) or the second embodiment of Formula (E).
  • he ionizable lipid e.g., cationic lipid
  • LNPs of the present disclosure he ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by Formula (E-4): (E-4) or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3) or the second, fifth or seventh embodiments of Formula (E).
  • R 6a and R 6b contain an equal number of carbon atoms with each other; or R 6a and R 6b are the same; or R 6a and R 6b are both C 12 alkyl, C 11 alkyl, C 10 alkyl, C 9 alkyl, C 8 alkyl, C 7 alkyl, C 12 alkenyl, C 11 alkenyl, C 10 alkenyl, C 9 alkenyl, C 8 alkenyl, or C 7 alkenyl; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh, ninth, tenth or eleventh embodiments of Formula (E), R 6a and R 6b contain an equal number of carbon atoms with each other; or R 6a and R 6b are the same; or R 6a and R 6b are both C 12 alkyl, C 11 alkyl, C 10 alkyl, C 9 alkyl, C 8 alkyl, C 7 alkyl
  • the ionizable lipid e.g., cationic lipid, in the ENPs of the present disclosure or the cationic lipid of Formula (E), Formula (E-l), Formula (E-2), Formula (E-3), Formula (E-4) is any one lipid selected from the lipids in Table 7 or a pharmaceutically acceptable salt thereof:
  • the LNPs provided by the present disclosure comprise an ionizable lipid that is also a cleavable lipid.
  • cleavable lipid which may be used interchangeably with the term “SS-cleavable lipid” refers to an ionizable lipid comprising a disulfide bond (“SS”).
  • the SS in the cleavable lipid is a cleavable unit.
  • a cleavable lipid comprises an amine, e.g., a tertiary amine, e.g.and a disulfide bond.
  • the SS-cleavable lipid is an SS-cleavable and pH-activated lipid-like material (ssPalm).
  • ssPalm lipids are well known in the art. For example, see Togashi et al., Journal of Controlled Release, 279 (2016) 262-270, the entire contents of which are incorporated herein by reference.
  • the ssPalm is an ssPalmM lipid comprising the structure of Lipid B shown below:
  • the ssPalmE lipid is a ssPalmE-P4-C2 lipid comprising the structure of Lipid C below: Lipid C
  • the ssPalmE lipid is a ssPalmE-Paz4-C2 lipid, comprising the structure of
  • the cleavable lipid is an ss-M lipid.
  • an ss-M lipid comprises the structure shown in Lipid E below:
  • the cleavable lipid is an ss-E lipid.
  • an ss-E lipid comprises the structure shown in Lipid F below:
  • the cleavable lipid is an ss-EC lipid.
  • an ss-EC lipid comprises the structure shown for Lipid G below: Lipid G
  • the cleavable lipid is an ss-LC lipid.
  • an ss-LC lipid comprises the structure shown for Lipid H below:
  • the cleavable lipid is an ss-OC lipid.
  • an ss-OC lipid comprises the structure shown for Lipid J below:
  • the condensing agent e.g. a cationic lipid
  • the LNPs provided by the present disclosure comprise a structural lipid. Without wishing to be bound by a specific theory, it is believed that a structural lipid, when present in an LNP, contributes to membrane integrity and stability of the LNP.
  • the structural lipid is a sterol, e.g., cholesterol, or a derivative thereof.
  • the structural lipid is cholesterol.
  • the structural lipid is a derivative of cholesterol.
  • Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-cholestanol, 5P-coprostanol, cholesteryl-(2’-hydroxy)-ethyl ether, cholesteryl-(4’- hydroxy) -butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5P-cholestanone, and cholesteryl decanoate; and mixtures thereof.
  • the cholesterol derivative is a polar analogue such as cholesteryl-(4’-hydroxy)- butyl ether.
  • cholesterol derivative is cholestryl hemisuccinate (CHEMS).
  • Exemplary cholesterol derivatives are described in International Patent Application Publication No. W02009/127060 and U.S. Patent Application Publication No. US2010/0130588, contents of both of which are incorporated herein by reference in their entirety.
  • the sterol in the LNPs of the present disclosure is selected from the group consisting of cholesterol, beta-sitosterol, stigmasterol, beta-sitostanol, campesterol, brassicasterol, and derivatives thereof, and any combination thereof.
  • the sterol is cholesterol.
  • the sterol is beta-sitosterol.
  • the structural lipid e.g., a sterol
  • the structural lipid constitutes about 20 mol% to about 50 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid, e.g., a sterol, constitutes about 30 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid, e.g., cholesterol, constitutes about 30 mol% of the total lipid present in the LNP.
  • the structural lipid is dexamethasone or dexamethasone -palmitate.
  • the LNPs provided by the present disclosure comprise a helper lipid.
  • the helper lipid is a ceramide.
  • the ceramides in the LNPs of the present disclosure are not conjugated to a polymer, such as polyethylene glycol or PEG.
  • Ceramides are sphingolipids which is a class of cell membrane lipids. Ceramides contain an A-acetylsphingosine (i.e., (E)-A-(l,3-dihydroxyoctadec-4-en-2-yl)acetamide) backbone and a fatty acid linked to the amide group.
  • sequence identity when used in reference to a polypeptide or a protein, refers to the ratio of the number of identical amino acids between the 2 aligned sequences over the aligned length, expressed as a percentage. In some embodiments, the 2 aligned sequences are identical in length, i.e., have the same number of amino acids.
  • the targeting moiety in an LNP of the present disclosure is an ApoE protein conjugate in an ApoB protein conjugate, which is a conjugate of one or more ApoE and/or ApoB protein molecules (native or modified) or a fragment thereof covalently linked to, for example, a lipid-anchored polymer as defined herein.
  • the targeting moiety in an LNP of the present disclosure is an ApoE polypeptide conjugate in an ApoB polypeptide conjugate, which is a conjugate of one or more ApoE and/or ApoB polypeptide molecules or a fragment thereof covalently linked to, for example, a lipid-anchored polymer as defined herein.
  • the targeting moiety is an antibody or an antibody fragment, e.g., an antibody or an antibody fragment that is capable of specifically binding to an antigen present on the surface of a cell.
  • the antibody or an antibody fragment is a monoclonal antibody (mAh), a single chain variable fragment (scFv), a heavy chain antibody (hcAb), a nanobody (Nb), a heavy-chain-only immunoglobulin (HCIg), an immunoglobulin new antigen receptor (IgNAR), variable domain of immunoglobulin new antigen receptor (VNAR), a single -domain antibody, or a variable heavy chain-only antibody (VHH).
  • an LNP of the present disclosure comprises a polymer-conjugated lipid of the present disclosure, and the targeting moiety as defined herein (and including GalNAc, ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide, an antibody or an antibody fragment) is conjugated to the polymer-conjugated lipid of the present disclosure.
  • an LNP of the present disclosure may comprise a polymer-conjugated lipid of the present disclosure as a first lipid-anchored polymer, and a targeting moiety as described herein conjugated to the polymer-conjugated lipid.
  • the polymer in the polymer-conjugated lipid e.g., a PG or a PG derivative, is conjugated to a targeting moiety.
  • the targeting moiety may be conjugated to the polymer-conjugated lipid via a reactive species.
  • the reactive species may be a thiol reagent, a maleimide reagent, or click chemistry reagent, e.g., a reagent selected from the group consisting of an alkyne reagent, such as a dibenzocyclooctyne (DBCO) reagent, a transcyclooctene (TCO) reagent, a tetrazine (TZ) reagent and an azide (AZ) reagent.
  • DBCO dibenzocyclooctyne
  • TCO transcyclooctene
  • TZ tetrazine
  • AZ azide
  • the polymer-conjugated lipid of the present disclosure comprising an azide reagent as the reactive species may be reacted with a targeting moiety functionalized with a DBCO reagent as a complementary reactive species to produce a polymer-conjugated lipid conjugated to a targeting moiety via the reactive species.
  • the polymer-conjugated lipid of the present disclosure comprising a thiol reagent may be reacted with a targeting moiety functionalized with a maleimide reagent to produce a polymer-conjugated lipid conjugated to a targeting moiety via the reactive species.
  • an LNP of the present disclosure may comprise a polymer-conjugated lipid of the present disclosure as a first lipid-anchored polymer, a second lipid-anchored polymer and a targeting moiety as described herein conjugated to the second lipid-anchored polymer.
  • the targeting moiety may be conjugated to the second lipid-anchored polymer via a reactive species.
  • the reactive species may be a thiol reagent, a maleimide reagent, or click chemistry reagent, e.g., a reagent selected from the group consisting of an alkyne reagent, such as a dibenzocyclooctyne (DBCO) reagent, a transcyclooctene (TCO) reagent, a tetrazine (TZ) reagent and an azide (AZ) reagent.
  • DBCO dibenzocyclooctyne
  • TCO transcyclooctene
  • TZ tetrazine
  • AZ azide
  • the second lipid-anchored polymer of the present disclosure comprising an azide reagent as the reactive species may be reacted with a targeting moiety functionalized with a DBCO reagent as a complementary reactive species to produce a second lipid-anchored moiety conjugated to the targeting moiety via a reactive species.
  • the polymer-conjugated lipid of the present disclosure comprising a thiol reagent may be reacted with a targeting moiety functionalized with a maleimide reagent to produce a polymer-conjugated lipid comprising a targeting moiety.
  • the LNP comprises a second lipid-anchored polymer and the targeting moiety as defined herein (and including GalNAc, ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide, an antibody or an antibody fragment) is conjugated to the second lipid-anchored polymer.
  • the second lipid-anchored polymer contains a lipid moiety conjugated to a polymer, optionally via a linker.
  • the second lipid-anchored polymer comprises a moiety selected from the group consisting of 1 ,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), l-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (POPE), 1 -palmitoyl-2-oleoyl-sn- glycero-3-phospho-(l'-rac-glycerol) (POPG), 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1 ,2-dielaidoyl-sn- phosphatidylethanolamine (DEPE), l-stearoyl-2-oleoyl-sn-g-gly
  • the second lipid- anchored polymer comprises a linker-lipid moiety selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, and a derivative thereof.
  • the lipid moiety of the second lipid-anchored polymer comprises DSPE.
  • the ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide, an antibody, or a fragment thereof is covalently linked to a lipid-anchored polymer (e.g., first lipid anchored polymer or second lipid-anchored polymer) or to an LNP of the present disclosure via strain promoted alkyne-azide cycloaddition (SPAAC) chemistry, such as via an azide -modified lipid- anchored polymer (e.g., DSG-PEG2000-azide, DSPE-PEG2000-azide, DSG-PEG3400-azide, DSPE- PEG3400-azide, DSG-PEG5000-azide, DSPE-PEG5000-azide) and a dibenzocyclooctyne (DBCO)- functionalized ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide, an antibody or a fragment thereof.
  • the second lipid-anchored polymer conjugated to a targeting moiety is represented by the following structure:
  • the second lipid-anchored polymer conjugated to a targeting moiety is represented by the following structure:
  • the LNPs of the present disclosure may comprise a first lipid-anchored polymer and a second lipid-anchored polymer.
  • the LNPs of the present disclosure may comprise a first lipid-anchored polymer that does not comprise a targeting moiety, and a second type of lipid-anchored polymer that comprises a targeting moiety, such as GalNAc.
  • the LNPs of the present disclosure may comprise DSG-PEG2000 modified to comprise an additional OCH3 group (DSG-PEG2000-OMe) as a first lipid-anchored polymer and DSPE-PEG2000-GalNAc3 as a second lipid-anchored polymer.
  • the LNPs of the present disclosure may comprise a polymer-conjugated lipid as a first lipid-anchored polymer of the present disclosure and a second-anchored polymer, e.g., a second-anchored polymer conjugated to a targeting moiety.
  • the LNPs of the present disclosure may comprise DODA-PG45 as a first lipid-anchored polymer and DSPE-PEG2000-GH as the second lipid-anchored polymer.
  • the targeting moiety is conjugated to a DSG-anchored polymer.
  • the DSG-anchored polymer is DSG-PEG or a derivative thereof.
  • the LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA); an ionizable lipid; a sterol; a helper lipid, and a a first lipid-anchored polymer, wherein the first lipid-anchored polymer comprises the polymer-conjugated lipid.
  • TAA therapeutic nucleic acid
  • a sterol a sterol
  • a helper lipid a lipid-anchored polymer
  • the first lipid-anchored polymer comprises the polymer-conjugated lipid.
  • the ionizable lipid constitutes about 20 mol% to about 60 mol% of the total lipid present in the LNP. In some embodiments, the ionizable lipid constitutes about 35 mol% to about 50 mol% of the total lipid present in the LNP. In some embodiments, the sterol constitutes about 20 mol% to about 50 mol% of the total lipid present in the LNP. In some embodiments, the sterol constitutes about 30 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the helper lipid constitutes about 1 mol% to about 40 mol% of the total lipid present in the LNP.
  • LNPs of the present disclosure have a mean diameter as determined by light scattering of less than about 90 nm, e.g. , less than about 80 nm or less than about 75 nm. According to some embodiments, LNPs of the present disclosure have a mean diameter as determined by light scattering of between about 50 nm and about 75 nm or between about 50 nm and about 70 nm.
  • LNPs in PBS at a concentration of 0.4 mM total lipid can be prepared using the in-line process as described herein and elsewhere.
  • TNS can be prepared as a 100 mM stock solution in distilled water.
  • Vesicles can be diluted to 24 mM lipid in 2 mL of buffered solutions containing, 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, where the pH ranges from 2.5 to 11.
  • TNS solution An aliquot of the TNS solution can be added to give a final concentration of 1 mM and following vortex mixing fluorescence intensity is measured at room temperature in a SLM Aminco Series 2 Luminescence Spectrophotometer using excitation and emission wavelengths of 321 nm and 445 nm. A sigmoidal best fit analysis can be applied to the fluorescence data and the pKa is measured as the pH giving rise to half-maximal fluorescence intensity.
  • relative activity can be determined by measuring luciferase expression in the liver 4 hours following administration via tail vein injection. The activity is compared at a dose of 0.3 and 1.0 mg ceDNA/kg and expressed as ng luciferase/g liver measured 4 hours after administration.
  • LNP of the present disclosure includes a lipid formulation that can be used to deliver a capsid-free, non-viral DNA vector to a target site of interest (e.g., cell, tissue, organ, and the like).
  • a target site of interest e.g., cell, tissue, organ, and the like.
  • the LNP comprises capsid-free, non-viral DNA vector and a cationic lipid or a salt thereof.
  • the ceDNA vector is preferably duplex, e.g., self-complementary, over at least a portion of the molecule, such as the expression cassette (e.g., ceDNA is not a double stranded circular molecule).
  • the ceDNA vector has covalently closed ends, and thus is resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III), e.g., for over an hour at 37°C.
  • a ceDNA vector comprises, in the 5’ to 3’ direction: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (for example an expression cassette as described herein) and a second AAV ITR.
  • AAV adeno-associated virus
  • ITR inverted terminal repeat
  • nucleotide sequence of interest for example an expression cassette as described herein
  • second AAV ITR for example an expression cassette as described herein
  • the first ITR (5’ ITR) and the second ITR (3’ ITR) are asymmetric with respect to each other - that is, they have a different 3D-spatial configuration from one another.
  • the polynucleotide comprises a first ITR sequence and a second ITR sequence, wherein the nucleotide sequence of interest is flanked by the first and second ITR sequences, and the first and second ITR sequences are asymmetrical relative to each other, or symmetrical relative to each other.
  • the posttranscriptional regulatory element comprises WPRE.
  • the poly adenylation and termination signal comprise BGHpolyA.
  • Any cis regulatory element known in the art, or combination thereof, can be additionally used e.g., SV40 late poly A signal upstream enhancer sequence (USE), or other posttranscriptional processing elements including, but not limited to, the thymidine kinase gene of herpes simplex virus, or hepatitis B virus (HBV).
  • the expression cassette length in the 5’ to 3’ direction is greater than the maximum length known to be encapsidated in an AAV virion. In one embodiment, the length is greater than 4.6 kb, or greater than 5 kb, or greater than 6 kb, or greater than 7 kb.
  • Various expression cassettes are exemplified herein.
  • the expression cassette can comprise more than 4000 nucleotides, 5000 nucleotides, 10,000 nucleotides or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides or 50,000 nucleotides, or any range between about 4000-10,000 nucleotides or 10,000-50,000 nucleotides, or more than 50,000 nucleotides.
  • the expression cassette can comprise a transgene which is a therapeutic nucleic acid sequence in the range of 500 to 50,000 nucleotides in length.
  • the expression cassette can comprise a transgene which is a therapeutic nucleic acid sequence in the range of 500 to 75,000 nucleotides in length.
  • the expression cassette can comprise a transgene which is a therapeutic nucleic acid sequence in the range of 500 to 10,000 nucleotides in length. In one embodiment, the expression cassette can comprise a transgene which is a therapeutic nucleic acid sequence in the range of 1000 to 10,000 nucleotides in length. In one embodiment, the expression cassette can comprise a transgene which is a therapeutic nucleic acid sequence in the range of 500 to 5,000 nucleotides in length.
  • the ceDNA vectors do not have the size limitations of encapsidated AAV vectors, and thus enable delivery of a large-size expression cassette to the host. In one embodiment, the ceDNA vector is devoid of prokaryote-specific methylation.
  • the rigid therapeutic nucleic acid can be a plasmid.
  • ceDNA vectors disclosed herein are used for therapeutic purposes (e.g., for medical, diagnostic, or veterinary uses) or immunogenic polypeptides.
  • the ceDNA expression cassette can include, for example, an expressible exogenous sequence (e.g., open reading frame) that encodes a protein that is either absent, inactive, or insufficient activity in the recipient subject or a gene that encodes a protein having a desired biological or a therapeutic effect.
  • the exogenous sequence such as a donor sequence can encode a gene product that can function to correct the expression of a defective gene or transcript.
  • the expression cassette can also encode corrective DNA strands, encode polypeptides, sense or antisense oligonucleotides, or coding RNAs or non-coding RNAs (e.g., siRNAs, guide RNAs, shRNAs, micro-RNAs, and their antisense counterparts (e.g., antagoMiR)).
  • coding RNAs or non-coding RNAs e.g., siRNAs, guide RNAs, shRNAs, micro-RNAs, and their antisense counterparts (e.g., antagoMiR)
  • expression cassettes can include an exogenous sequence that encodes a reporter protein to be used for experimental or diagnostic purposes, such as P-lactamase, P-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.
  • a reporter protein such as P-lactamase, P-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.
  • the expression cassette can include any gene that encodes a protein, polypeptide or RNA that is either reduced or absent due to a mutation or which conveys a therapeutic benefit when overexpressed is considered to be within the scope of the disclosure.
  • the ceDNA vector may comprise a template or donor nucleotide sequence used as a correcting DNA strand to be inserted after a double-strand break (or nick) provided by a nuclease.
  • the ceDNA vector may include a template nucleotide sequence used as a correcting DNA strand to be inserted after a double-strand break (or nick) provided by a guided RNA nuclease, meganuclease, or zinc finger nuclease.
  • the TNA comprised in an LNP of the present disclosure may be a single-stranded nucleic acid, e.g., a single-stranded DNA or a single-stranded RNA.
  • the TNA may be a single-stranded RNA, e.g., mRNA.
  • the TNA may be a single-stranded DNA (ssDNA) molecule, e.g., a synthetic ssDNA molecule.
  • the TNA is a ssDNA molecule comprising at least one nucleic acid sequence of interest flanked by at least one stem-loop structure at the 3’ end.
  • the ssDNA molecule may further comprise at least one stem-loop structure at the 5’ end.
  • the stem-loop structure at the 3’ end may comprise a partial DNA duplex (e.g., with a free 3’ -OH group) to prime replication or transcription. The partial DNA duplex functions, in part, to hold the stem-loop structure together.
  • the partial DNA duplex comprises between 4-500 nucleotides, for example between 4-10 nucleotides, between 4-25 nucleotides, between 4-50 nucleotides, between 4-100 nucleotides, between 4-200 nucleotides, between 4-300 nucleotides, between 4-400 nucleotides, between 20-25 nucleotides, between 20-50 nucleotides, between 20-100 nucleotides, between 20-200 nucleotides, between 20-300 nucleotides, between 20-400 nucleotides, between 20-500 nucleotides, between 50-100 nucleotides, between 50-200 nucleotides, between SO- SOO nucleotides, between 50-400 nucleotides, between 50-500 nucleotides, 150-200 nucleotides, between 150-300 nucleotides, between 150-400 nucleotides, between 150-500 nucleotides, between 200-300 nucleotides, between 4-400 nucleo
  • the DNA duplex comprises at least 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 150, 200, 250, 300, 350, 400, 450 or 500 nucleotides, and at least one loop on the 3’ end.
  • the loop structure at the 3’ end comprises a minimum of between 3-500 unbound nucleotides, for example between 3-450 nucleotides, between 3-400 nucleotides, between 3-350 nucleotides, between 3-300 nucleotides, between 3-250 nucleotides, between 3-200 nucleotides, between 3-150 nucleotides, between 3-100 nucleotides, between 3-90 nucleotides, between 3-80 nucleotides, between 3-70 nucleotides, between 3-60 nucleotides, between 3-50 nucleotides, between 3-40 nucleotides, between 3-30 nucleotides, between 3-20 nucleotides, between 3-10 nucleotides, between 3-5 nucleotides, between 10-450 nucleotides, between 10-400 nucleotides, between 10-350 nucleotides, between 10-300 nucleotides, between 10-250 nucleotides, between 10-200 nucleotides, between 10-150 nucleot
  • the stem portion of the stem-loop is 4-500 nucleotides in length and the loop portion of the stem-loop is 3-500 nucleotides in length. According to some embodiments, the stem portion of the stem-loop is 4-50 nucleotides in length and the loop portion of the stem-loop is 3-50 nucleotides in length. According to some embodiments, the stem portion of the stem-loop is 4-20 nucleotides in length and the loop portion of the stem-loop is 3-20 nucleotides in length. According to some embodiments, the stem portion of the stem-loop is 4-10 nucleotides in length and the loop portion of the stem-loop is 3-10 nucleotides in length.
  • the loop further comprises one or more nucleic acids or that are used to stabilize the ends. According to other embodiments, the loop further comprises one or more nucleic acids that may be employed in therapeutic methods. According to other embodiments, the loop further comprises one or more nucleic acids that may be employed in diagnostic methods. According to other embodiments, the loop further comprises one or more nucleic acids that that may be employed for research purposes.
  • the minimal nucleic acid structure that is necessary at the 3’ end of the ssDNA is any structure that loops back on itself, i.e., a hairpin structure.
  • the ssDNA described herein may comprise at least one stem-loop structure at the 3’ end.
  • the ssDNA may comprise at least two stem-loop structures at the 3’ end.
  • the ssDNA may comprise at least three stem-loop structures at the 3’ end.
  • the ssDNA may comprise at least four stem-loop structures at the 3’ end.
  • the ssDNA may comprise at least five stem-loop structures at the 3’ end.
  • the nucleotides at the 3’ end form a cruciform DNA structure.
  • a DNA cruciform structure can be formed when both strands form a stem-loop structure at the same location in the molecule, and comprises a four-way junction and two closed hairpin-shaped points.
  • the nucleotides at the 3’ end form a hairpin DNA structure.
  • Hairpin loop structures in nucleic acids consist of a base-paired stem structure and a loop sequence with unpaired or non- Watson-Crick-paired nucleotides.
  • the nucleotides at the 3’ end form a hammerhead DNA structure, made up of three base paired helices, separated by short linkers of conserved sequence.
  • the nucleotides at the 3’ end form a quadraplex DNA structure.
  • G-quadruplexes are four-stranded DNA secondary structures (G4s) that form from certain guanine-rich sequences.
  • the nucleotides at the 3’ end form a bulged DNA structure. According to some embodiments, the nucleotides at the 3’ end form a multibranched loop. According to some embodiments, the nucleotides at the 3’ end do not form a 2 stem-loop structure.
  • the stem structure at the 3’ end comprises about 4 to about 10 phosphorothioate- modified nucleotides, e.g., about 4 to about 5, about 4 to about 6, about 4 to about 7, about 4 to about 8, about 4 to about 9, about 4 to about 10, about 5 to about 6, about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 8 to about 9, about 8 to about 10 or about 9 to about 10.
  • the stem structure comprises more than 10 phosphorothioate-modified nucleotides.
  • the phosphorothioate-modified nucleotides are located adjacent to each other.
  • the one or more phosphorothioate-modified nucleotides of the 3’ end are resistant to exonuclease degradation.
  • Boranophosphate modified DNA is also resistant to nuclease degradation, and may be considered as an alternative to phosphorothioate modification.
  • the loop further comprises one or more aptamers.
  • the aptamer is identified from the Apta-index database of aptamers available to the public (aptagen.com/apta-index).
  • the loop further comprises one or more synthetic ribozymes.
  • the loop further comprises one or more antisense oligonucleotides (ASOs).
  • ASOs antisense oligonucleotides
  • the loop further comprises one or more antiviral nucleoside analogues (AN As).
  • AN antiviral nucleoside analogues
  • the loop further comprises one or more triplex forming oligonucleotides .
  • click azide-alkyne cycloaddition (Kolb et al., Angew. Chem. Int. Ed. Engl. 2001, 40, 2004-2021) is used to modify the nucleotides in the loop.
  • Click chemistry was developed to join together organic molecules under mild conditions in the presence of a diverse range of functional groups.
  • Most click-mediated modifications are performed on the nitrogenous bases by introducing novel base analogues, attaching fluorophores or isotopic elements for molecular imaging, forming inter-strand linkages between oligonucleotides, and for the bioconjugation of molecules.
  • click chemistry is the Cu 1 catalyzed version of Huisgen’s [3 + 2] azide-alkyne cycloaddition reaction (Angew. Chem., Int. Ed. 1963, 2, 633-645), discovered independently by Sharpless and Meldal (the CuAAC reaction) (Angew. Chem., Int. Ed. 2002, 41, 2596-2599).
  • the introduction of active amino or thiol groups into synthesized oligonucleotides provides acceptors for, e.g., subsequent chemical fluorescent labeling.
  • the stem-loop structure may comprise alternative or modified nucleotides, including, but not limited to, ribonucleic acids (RNA), peptide -nucleic acids (PNA), locked nucleic acids (LNA).
  • RNA ribonucleic acids
  • PNA peptide -nucleic acids
  • LNA locked nucleic acids
  • the loop portion of the stemloop structure may comprise a chemical structure that does not comprise nucleic acids.
  • Lipid nanoparticles can form spontaneously upon mixing of a therapeutic nucleic acid (e.g., ceDNA, ssDNA, synthetic AAV, etc., as described herein) and a pharmaceutically acceptable excipient that comprises a lipid.
  • a therapeutic nucleic acid e.g., ceDNA, ssDNA, synthetic AAV, etc., as described herein
  • a pharmaceutically acceptable excipient that comprises a lipid.
  • LNPs can be formed by any method known in the art.
  • the LNPs can be prepared by the methods described, for example, in US2013/0037977, US2010/0015218, US2013/0156845, US2013/0164400, US2012/0225129, and US2010/0130588, content of each of which is incorporated herein by reference in its entirety.
  • LNPs can be prepared using a continuous mixing method, a direct dilution process, or an in-line dilution process.
  • the processes and apparatuses for preparing lipid nanoparticles using direct dilution and in-line dilution processes are described in US2007/0042031, the content of which is incorporated herein by reference in its entirety.
  • the processes and apparatuses for preparing lipid nanoparticles using step- wise dilution processes are described in US2004/0142025, the content of which is incorporated herein by reference in its entirety.
  • the disclosure provides for an LNP comprising a DNA vector, including a ceDNA vector, ssDNA vector, or synthetic AAV, as described herein and an ionizable lipid.
  • a lipid nanoparticle formulation that is made and loaded with therapeutic nucleic acid like ceDNA obtained by the process as disclosed in International Patent Application No. PCT/US2018/050042, filed on September 7, 2018, which is incorporated by reference in its entirety herein.
  • This can be accomplished by high energy mixing of ethanolic lipids with aqueous synthetic AAV at low pH which protonates the ionizable lipid and provides favorable energetics for synthetic AAV/lipid association and nucleation of particles.
  • the particles can be further stabilized through aqueous dilution and removal of the organic solvent.
  • the particles can be concentrated to the desired level.
  • the LNPs can be prepared by an impinging jet process.
  • the particles are formed by mixing lipids dissolved in alcohol (e.g., ethanol) with ceDNA dissolved in a buffer, e.g., a citrate buffer, a sodium acetate buffer, a sodium acetate and magnesium chloride buffer, a malic acid buffer, a malic acid and sodium chloride buffer, or a sodium citrate and sodium chloride buffer.
  • a buffer e.g., a citrate buffer, a sodium acetate buffer, a sodium acetate and magnesium chloride buffer, a malic acid buffer, a malic acid and sodium chloride buffer, or a sodium citrate and sodium chloride buffer.
  • the mixing ratio of lipids to ceDNA can be about 45-55% lipid and about 65-45% ceDNA.
  • the lipid solution can contain an ionizable lipid, a ceramide, a lipid-anchored polymer and a sterol (e.g., cholesterol) at a total lipid concentration of 5-30 mg/mL, more likely 5-15 mg/mL, most likely 9-12 mg/mL in an alcohol, e.g., in ethanol.
  • mol ratio of the lipids can range from about 25-98% for the cationic lipid, preferably about 35-65%; about 0-15% for the non- ionic lipid, preferably about 0-12%; about 0-15% for the PEG or PEG conjugated lipid molecule, preferably about 1-6%; and about 0-75% for the sterol, preferably about 30-50%.
  • this buffered solution can be at a temperature in the range of 15-40°C or 30-40°C.
  • the mixed LNPs can then undergo an anion exchange filtration step. Prior to the anion exchange, the mixed LNPs can be incubated for a period of time, for example 30mins to 2 hours. The temperature during incubating can be in the range of 15-40°C or 30-40°C. After incubating the solution is filtered through a filter, such as a 0.8pm filter, containing an anion exchange separation step. This process can use tubing IDs ranging from 1 mm ID to 5 mm ID and a flow rate from 10 to 2000 mL/min.
  • the ultrafiltration process can use a tangential flow filtration format (TFF) using a membrane nominal molecular weight cutoff range from 30-500 kD.
  • the membrane format is hollow fiber or flat sheet cassette.
  • the TFF processes with the proper molecular weight cutoff can retain the LNP in the retentate and the filtrate or permeate contains the alcohol; citrate buffer and final buffer wastes.
  • the TFF process is a multiple step process with an initial concentration to a ceDNA concentration of 1-3 mg/mL. Following concentration, the LNPs solution is diafiltered against the final buffer for 10-20 volumes to remove the alcohol and perform buffer exchange. The material can then be concentrated an additional 1-3 -fold. The concentrated LNP solution can be sterile filtered.
  • the present disclosure also provides a pharmaceutical composition comprising the LNPs of the present disclosure and at least one pharmaceutically acceptable excipient.
  • encapsulation of TNA (e.g., ceDNA) in the LNPs of the present disclosure can be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that has enhanced fluorescence when associated with nucleic acid, for example, an Oligreen® assay or PicoGreen® assay.
  • encapsulation is determined by adding the dye to the lipid particle formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of nonionic detergent.
  • Detergent- mediated disruption of the lipid bilayer releases the encapsulated TNA (e.g., ceDNA), allowing it to interact with the membrane-impermeable dye.
  • the proportions of the components can vary and the delivery efficiency of a particular formulation can be measured using, for example, an endosomal release parameter (ERP) assay.
  • ERP endosomal release parameter
  • the pharmaceutical compositon comprising LNPs of the disclosure is an aqueous solution. In one embodiment, the pharmaceutical compositon comprising LNPs of the disclosure is a lyophilized powder.
  • the at least one pharmaceutically acceptable excipient in the pharmaceutical compositons of the present disclosure is a sucrose, tris, trehalose and/or glycine.
  • the pharmaceutical compositons comprising LNPs of the disclosure are suitable for administration to a subject for in vivo delivery to cells, tissues, or organs of the subject.
  • the pharmaceutical compositon is suitable for a desired route of therapeutic administration (e.g., parenteral administration).
  • the pharmaceutical compositions for therapeutic purposes can be formulated as a solution, microemulsion, dispersion, liposomes, or other ordered structure suitable for high TNA (e.g., ceDNA) vector concentration.
  • Sterile injectable solutions can be prepared by incorporating the TNA (e.g., ceDNA) vector compound in the required amount in an appropriate buffer with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • LNPs are solid core particles that possess at least one lipid bilayer.
  • the LNPs have a non-bilayer structure, i.e., a non-lamellar (i.e., non-bilayer) morphology.
  • the non-bilayer morphology can include, for example, three dimensional tubes, rods, cubic symmetries, etc.
  • the non-lamellar morphology (i.e., non-bilayer structure) of the LNPs can be determined using analytical techniques known to and used by those of skill in the art.
  • Such techniques include, but are not limited to, Cryo-Transmission Electron Microscopy (“Cryo-TEM”), Differential Scanning calorimetry (“DSC”), X-Ray Diffraction, and the like.
  • Cryo-TEM Cryo-Transmission Electron Microscopy
  • DSC Differential Scanning calorimetry
  • X-Ray Diffraction X-Ray Diffraction
  • the morphology of the lipid particles can readily be assessed and characterized using, e.g., Cryo-TEM analysis as described in US2010/0130588, the content of which is incorporated herein by reference in its entirety.
  • the LNPs having a non-lamellar morphology are electron dense.
  • the LNPs provided by the present disclosure is either unilamellar or multilamellar in structure.
  • the pharmaceutical composition of the disclosure comprises multi-vesicular particles and/or foam-based particles.
  • the rate at which the lipid conjugate exchanges out of the lipid particle and, in turn, the rate at which the LNP becomes fusogenic.
  • other variables including, for example, pH, temperature, or ionic strength, can be used to vary and/or control the rate at which the LNP becomes fusogenic.
  • Other methods which can be used to control the rate at which the LNP becomes fusogenic will be apparent to those of ordinary skill in the art based on this disclosure. It will also be apparent that by controlling the composition and concentration of the lipid conjugate, one can control the lipid particle size.
  • interfering RNA-ligand conjugates and nanoparticle-ligand conjugates may be combined with ophthalmologically acceptable preservatives, co-solvents, surfactants, viscosity enhancers, penetration enhancers, buffers, sodium chloride, or water to form an aqueous, sterile ophthalmic suspension or solution.
  • the unit dosage form is adapted for intrathecal or intracerebroventricular administration.
  • the pharmaceutical composition is formulated for topical administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.
  • the present disclosure provides methods of treating a disorder in a subject that comprise administering to the subject an effective amount of an LNP of the disclosure of the pharmaceutical compositon comprising the LNP of the disclosure.
  • the disorder is a genetic disorder.
  • LNPs of the disclosure There are a number of inherited diseases in which defective genes are known, and typically fall into two classes: deficiency states, usually of enzymes, which are generally inherited in a recessive manner, and unbalanced states, which may involve regulatory or structural proteins, and which are typically but not always inherited in a dominant manner.
  • deficiency state diseases the LNPs and LNP compositions of the disclosure can be used to deliver transgenes to bring a normal gene into affected tissues for replacement therapy, as well, in some embodiments of any of the aspects and embodiments herein, to create animal models for the disease using antisense mutations.
  • the LNPs and LNP compositions of the disclosure can be used to create a disease state in a model system, which could then be used in efforts to counteract the disease state.
  • the LNPs or LNP compositions of the disclosure and methods disclosed herein permit the treatment of genetic diseases.
  • a disease state is treated by partially or wholly remedying the deficiency or imbalance that causes the disease or makes it more severe.
  • the LNPs of the disclosure or the pharmaceutical compositons comrpsing the LNPs of the disclosure can be used to treat, ameliorate, and/or prevent a disease or disorder caused by mutation in a gene or gene product.
  • Exemplary diseases or disorders that can be treated with the LNPs or the LNP compositions of the disclosure include, but are not limited to, metabolic diseases or disorders (e.g., Fabry disease, Gaucher disease, phenylketonuria (PKU), glycogen storage disease); urea cycle diseases or disorders (e.g., ornithine transcarbamylase (OTC) deficiency); lysosomal storage diseases or disorders (e.g., metachromatic leukodystrophy (MLD), mucopolysaccharidosis Type II (MPSII; Hunter syndrome)); liver diseases or disorders (e.g., progressive familial intrahepatic cholestasis (PFIC); blood diseases or disorders (e.g., hemophilia A and B, thalassemia, and anemia); cancers and tumors, and genetic diseases or disorders (e.g., cystic fibrosis).
  • metabolic diseases or disorders e.g., Fabry disease, Gaucher disease, phenylket
  • the LNPs or LNP compositions of the disclosure may be employed to deliver a heterologous nucleotide sequence in situations in which it is desirable to regulate the level of transgene expression (e.g., transgenes encoding hormones or growth factors).
  • a heterologous nucleotide sequence in situations in which it is desirable to regulate the level of transgene expression (e.g., transgenes encoding hormones or growth factors).
  • treatment of OTC deficiency can be achieved by producing functional OTC enzyme; treatment of hemophilia A and B can be achieved by modifying levels of Factor VIII, Factor IX, and Factor X; treatment of PKU can be achieved by modifying levels of phenylalanine hydroxylase enzyme; treatment of Fabry or Gaucher disease can be achieved by producing functional alpha galactosidase or beta glucocerebrosidase, respectively; treatment of MFD or MPSII can be achieved by producing functional arylsulfatase A or iduronate-2-sulfatase, respectively; treatment of cystic fibrosis can be achieved by producing functional cystic fibrosis transmembrane conductance regulator; treatment of glycogen storage disease can be achieved by restoring functional G6Pase enzyme function; and treatment of PFIC can be achieved by producing functional ATP8B1, ABCB11, ABCB4, or TJP2 genes.
  • the transgene encodes a monoclonal antibody specific for one or more desired targets. In some exemplary embodiments, more than one transgene is encoded by the ceDNA vector. In some exemplary embodiments, the transgene encodes a fusion protein comprising two different polypeptides of interest. In some embodiments of any of the aspects and embodiments herein, the transgene encodes an antibody, including a full-length antibody or antibody fragment, as defined herein. In some embodiments of any of the aspects and embodiments herein, the antibody is an antigen-binding domain or an immunoglobulin variable domain sequence, as that is defined herein.
  • this disclosure provides a method of providing anti-tumor immunity in a subject, the method comprising administering to the subject an effective amount of any embodiment of an LNP contemplated herein or any embodiment of a pharmaceutical composition comprising an LNP contemplated herein. Furthermore, this disclosure provides a method of treating a subject having a disease, disorder or condition associated with an elevated expression of a tumor antigen, the method comprising administering to the subject an effective amount of any embodiment of an LNP contemplated herein or any embodiment of a pharmaceutical composition comprising an LNP contemplated herein.
  • the TNA is retained in the spleen for at least about 6 hours, or at least about 9 hours, or at least about 12 hours, or at least about 15 hours, or at least about 18 hours, or at least about 21 hours, or at least about 24 hours, or at least about 27 hours, or at least about 30 hours, or at least about 33 hours, or at least about 36 hours after dosing of an LNP of this disclosure, for example, via intravenous or intratumoral administration.
  • the amount (z.e., number of copies) of the TNA at the start of a 12, 18, or 24-hour time window post-dosing and the amount of the TNA at the end of the time window are within the same order of magnitude (e.g., 10 7 copies, 10 6 copies, 10 5 copies, 10 4 copies, 10 3 copies, 10 2 copies, 10 1 copies, 10° copies, 10 1 copies, 10 2 copies, 10 3 copies, etc. or any other suitable therapeutic levels).
  • the TNA is a messenger RNA (mRNA).
  • solid tumors treatable with an LNP disclosed herein or a pharmaceutical composition comprising the same include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting liver, lung, breast, lymphoid, gastrointestinal (e.g., colon), genitourinary tract (e.g., renal, urothelial cells), prostate and pharynx.
  • Adenocarcinomas include malignancies such as most colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • the tumor or cancer is a melanoma, e.g., an advanced stage melanoma.
  • Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the disclosure.
  • examples of other solid tumors or cancers that can be treated include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood,
  • Non-limiting examples of the blood disease, disorder or condition include acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), Hodgkin lymphoma (HL), multiple myeloma, a myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), adrenoleukodystrophy (ALD), Hurler syndrome, Krabbe disease (Globoid-cell leukodystrophy or GLD), metachromatic leukodystrophy (MLD), severe aplastic anemia (SAA), severe combined immunodeficiency (SCID), sickle cell disease (SCD), thalassemia, Wiskott-Aldrich syndrome, Diamond-Blackfan anemia, essential thrombocytosis, Fanconi anemia, hemophagocytic lymphohistiscytosis (HLH), juvenile myelomonoc
  • an LNP or an LNP composition of the disclosure can be administered to an organism for transduction of cells in vivo. In some embodiments, an LNP or an LNP composition of the disclosure can be administered to an organism for transduction of cells ex vivo.
  • administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • Exemplary modes of administration of an LNP or an LNP composition of the disclosure include oral, rectal, transmucosal, intranasal, inhalation (e.g., via an aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intraendothelial, in utero (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intracranial, intramuscular [including administration to skeletal, diaphragm and/or cardiac muscle], intrapleural, intracerebral, and intraarticular), topical (e.g., to both skin and mucosal surfaces, including airway surfaces, and transdermal administration), intralymphatic, and the like, as well as direct tissue or organ injection (e.g., to liver, eye, skeletal muscle, cardiac muscle, diaphragm muscle or brain).
  • parenteral e.g., intravenous, subcutaneous, intradermal, intracranial,
  • Administration of the LNP or LNP compositions of the disclosure can be to any site in a subject, including, without limitation, a site selected from the group consisting of the brain, a skeletal muscle, a smooth muscle, the heart, the diaphragm, the airway epithelium, the liver, the kidney, the spleen, the pancreas, the skin, and the eye.
  • a site selected from the group consisting of the brain, a skeletal muscle, a smooth muscle, the heart, the diaphragm, the airway epithelium, the liver, the kidney, the spleen, the pancreas, the skin, and the eye.
  • ceDNA permits one to administer more than one transgene in a single vector, or multiple ceDNA vectors (e.g., a ceDNA cocktail).
  • the LNPs or LNP compositions of the disclosure can be administered to the desired region(s) of the CNS by any route known in the art, including but not limited to, intrathecal, intra-ocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intra-aural, intra-ocular (e.g., intra-vitreous, sub-retinal, anterior chamber) and peri-ocular (e.g., sub-Tenon’s region) delivery as well as intramuscular delivery with retrograde delivery to motor neurons.
  • intrathecal intra-ocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intra-aural, intra-ocular (e.g., intra-vitreous, sub-retinal, anterior chamber) and peri-ocular (e.g., sub-Tenon’s region
  • the LNPs of the disclosure or the pharmaceutical compositions comprising the LNPs of the disclosure when administered to a subject, is characterized by a lower immunogenicity than a reference LNP or a pharmaceutical composition comprising a reference LNP.
  • the immunogenicity of the LNP of the disclosure or the pharmaceutical compostion comprising the LNP of the disclosure may be measured by measuring levels of one or more proinflammatory cytokines. Accordingly, in some embodiments, the LNPs of the disclosure or the pharmaceutical compositions comprising the LNPs of the disclosure, when administered to a subject, elicits a lower pro-inflammatory cytokine respose than a reference LNP or a pharmaceutical composition comprising a reference LNP.
  • pro-inflammatory cytokines include, but are not limited to, granulocyte colony stimulating factor (G-CSF), interleukin 1 alpha (IL-la), interleukin 1 beta (IL-ip), interleukin 6 (IL-6), interleukin 8 (IL-8 or CXCL8), interleukin 11 (IL-11), interleukin 17 (IL-17), interleukin 18 (IL-18), interferon a (IFN-a), interferon P (IFN-P), interferon y (IFN-y), C-X-C motif chemokine ligand 10 (CXCL10 or IP-10), monocyte chemoattractant protein 1 (MCP-1), CD40L, CCL2, CCL3, CCL4, CCL5, CCL11, tumor necrosis factor a (TNF-a), and combinations thereof.
  • G-CSF granulocyte colony stimulating factor
  • IL-la interleukin 1 alpha
  • IL-ip interleukin 6
  • EEGE was azeotrope with toluene and desiccated with P 2 O 5 before the rection.
  • DODA_1 was also desiccated with P 2 O 5 .
  • Figure 1A is a MALDI-TOF spectrum of DODA-PG34. Synthesis of DODA-PG41 To a solution of DODA_2 in MeOH was added HCl (0.1 mL, 1M in ethyl acetate) dropwise and stirred for 4 hours at room temperature. Subsequently, the reaction mixture was concentrated via rotavapor.
  • Compound DODA-1 was also co-evaporated with toluene to azeotrope off any water present and kept over P2O5 overnight on high vacuum line. The reaction was carried under inert atmosphere and very dry conditions. Compound DODA-1 (0.2 g, 0.32 mmol, 1 eq.) was dissolved in 2 mL of dry toluene and a catalytic amount of P4-tBu (0.4 mL, 0.8 M in hexane) was added.
  • DODA with 23 units (0.72 grams, 1.6 mmol) was dissolved in 2 mL of dry toluene and treated with 0.3 mL of P4-tBu (0.8M/hexanes) stirring for 20 minutes before 2,3-epoxy-1-(1- ethoxyethoxypropane) was added (2.3 grams, 144 mmol) in 0.5 mL of toluene.
  • Figure 1B is a MALDI-TOF spectrum of DODA-PG45.
  • Synthesis of DODA-PG58 DODA 2A (1.5 g, 0.19 mmol) was dissolved in MeOH (40 mL) and treated with 1N HCl/EtOAc (0.4 mL, 0.4 mmol) and stirred for 4h at ambient temperature.
  • the reaction mixture was concentrated, dissolved in 3 mL of MeOH and treated with 30 mL of ice-cold Et2O.
  • the cloudy-oily mixture was centrifuged at 4.4x10 3 x g for 10 minutes, the solvents were decanted, and the sonication procedure was repeated the same way as described for the analog above providing 770 mg (93%) of DODA-PG58.
  • Example 4 In vivo expression of nucleic acids in LNP formulations containing different anchored polymers
  • CD-I mice males
  • ceDNA nucleic acid carrying a firefly luciferase reporter construct that was formulated in LNPs comprising DSPE- PEG2K-0H or DODA-PG45 (composition in Table 8) at a dose of 0.5 mg/kg (0 day).
  • FIG. 1 shows the total flux measured by the total photon counts per the region of interest, i.e., the liver, measured by IVIS at Day 4 post-dosing for tested LNPs and for a negative control (PBS) injected with saline instead of formulated ceDNA.
  • Figure 2B shows the total flux measured for tested LNPs and negative control at Day 7 post-dosing.
  • Figure 2C shows the total flux measured for tested LNPs and negative control across two collection days (Day 4 and Day 7).
  • FIG. 2C The results shown in Figure 2C indicate that administration of formulated LNPs with different anchored polymers in combination with a targeting ligand, i.e., GalNAc3 (Formulations 180, 182, and 184) results in higher expression of luciferase as compared to untagged LNPs (Formulations 179, 181, and 183) at both Day 4 and Day 7.
  • Figure 2D shows the percentage change in body weight (BW) of mice at Day 1 post-dosing. The results indicate that the tested LNPs with targeting ligand GalNAc3 (Formulations 180, 182, and 184), caused a smaller change in body weight in mice as compared to untagged LNPs.
  • a targeting ligand i.e., GalNAc3
  • Example 4 demonstrate that a GalNAc3 targeted LNP of the disclosure comprising anchored polymers (DSPE-PEG2K-OH or DODA-PG45) when delivered in vivo supports the expression of nucleic acids without triggering any major tolerability issues and other adverse events in
  • Example 4 demonstrate that only half the amount (1.5 mol%) of DODA-PG45 in an untargeted LNP formulated with PG-containing anchored polymer
  • Figure 3 is a bar graph showing luciferase activity for the tested LNP formulations containing different lipid-anchored polymers. The results are shown in Figure 3, and indicate that an LNP formulated with helper lipid DSPC, and anchored polymer DODA-PG34 and DSPE-PEG2K- GalNAc3 (Formulation 227) showed higher luciferase activity than uninhibited control.
  • Example 7 Evaluation of the effect of anchored polymer composition on opsonization-driven LNP uptake in primary mouse hepatocytes
  • Figure 6 is a bar graph showing the amount of endosomal escape measured as the amount of luciferase expression normalized to DiD uptake in mouse hepatocytes treated with LNP formulations containing different amounts of polyglycerol-conjugated lipids and a control. It is demonstrated in Figure 6 that by increasing the amount of DODA-PG, the ability for the LNP to escape the endosome is reduced. This is important when the data from Figure 5 is considered where less PG can result in a higher level of stelthiness. Thus, an LNP containing less DODA-PG can be used to achieve a similar level of stealth while also enhancing the endosomal escape potential of the LNP.
  • Figure 6 shows the inverse relationship between the amount of an polymer-conjugated lipid in an LNP formulation and the level of endosomal escape. Specifically, Figure 6 shows that LNPs formulated with a relavtively low amount (1.45%) of PG maintained a relavtively high level of endosomal escape compared to LNPs formulated with significantly higher amount (2.95%) of PG or PEG.
  • LNPs formulated with PG- containing anchored polymer to achieve this advantageous stealth/endosomal tradeoff as compared to LNPs formulated with PEG-cotanining anchored polymer is further supported by Figure 4 wherein the stealthness of LNPs formulated with PEG suffers as the amount of PEG decreases, in contrast to LNPs formulated with PG for which the stealthness does not suffer as the amount of PG decreases.
  • Example 9 Analysis of whole blood clearance of LNPs formulated with ionizable lipid: Lipid Z, and different polymer-conjugated lipids
  • PK pharmacokinetic
  • novel LNPs formulations containing the ionizable lipid Lipid Z, along with DSPC, cholesterol and different polymer-conjugated lipids as described in Table 11 with a control LNP formulated with ionizable Lipid 87, cholesterol, and DSG-PEG2K-0Me (Formulation 829).
  • Formulations of control LNP and Lipid Z carrying LNPs were injected via IV bolus in the tail vein of CD-I mice.
  • Whole blood samples were collected for qPCR at 2 min, 1 hour, 3 hour and 6-hour time -points, and K2EDTA was added as an anticoagulant at 50 pL/aliquot. Body weight, mortality, and clinical observations were recorded.
  • the goal of this study was to evaluate the in vivo expression of nucleic acids formulated as LNPs with ceramides as the helper lipid, in combination with various anchored polymers such as DSPE-PEG2K-OH or DODA-PG45.
  • CD-I mice males
  • LNPs comprising various helper lipids (ceramide and DSPC) in combination with different anchored polymers (DSPE-PEG2K-OH or DODA-PG45) at 2 different doses of either 1 mg/kg or 2.0 mg/kg (0 day).
  • the LNPs used in the experiment are shown in Table 12.
  • GalNAc3 targeted LNP of the disclosure comprising different helper lipids and anchored polymers when delivered in vivo could support expression of nucleic acids without triggering any major tolerability issues and other adverse events in mice that could be clinically observed (e.g., rough hair coat, facial swelling).
  • Blood serum was collected at 6 hours post-dosing, and the levels of cytokines that are implicated in the regulation of innate immune response, i.e., IFN-alpha, IL-6, IFN-gamma, TNF- alpha, IL- 18, and IP- 10 were measured for each animal.
  • the results are shown in Figure 9, and indicate that at a dosage of 2.0 mg/kg, the blood serum levels of IFN-alpha, IL-6, IFN-gamma, TNF- alpha, and IL- 18 were lower for the C2 and Cl 8:1 ceramide -containing LNPs as compared to DSPC- containing LNPs.
  • These results also show that some cytokine levels trend lower for PG-containing LNPs, and higher for PEG-containing LNPs, especially in case with IFN-alpha.
  • helper lipid as well as the identity of the polymer in the anchor lipid-conjugated polymer, directly affects the immunogenicity of LNPs formulated as in the disclosure.
  • This example describes a method for the preparation of an LNP conjugated to a protein ligand of interest, which requires the inclusion of an additional cysteine residue not present in the native protein sequence.
  • the protein ligand of interest is initially reduced with 10 molar equivalents of TCEP for 30 minutes at 23°C. After reduction, TCEP is removed using a Zeba spin column. The reduced ligand is then incubated for 3 hours at 23 °C with LNPs formulated with DSPE-PEG5k- Maleimide using a mole percentage of 0.5%. The ratio of ligand to DSPE-PEG5k-maleimide is varied from 0.3 down to 0.02. SDS-PAGE is used to confirm whether the conjugation occurred and to what extent.
  • Example 13 Preparation of DSPE-PEG5k-DBCO-Protein
  • This example describes a method for the preparation of an LNP-conjugated to a protein ligand of interest, which requires the inclusion of an additional cysteine residue not present in the native protein sequence.
  • the protein ligand of interest is initially reduced with 10 molar equivalents of TCEP for 30 minutes at 23°C. After reduction, TCEP is removed using a Zeba spin column. The reduced ligand is then incubated with 10 molar equivalents of Sulfo DBCO-PEG4-maliemide for 3 hours at 23 °C. The excess DBCO reagent is then removed using a Zeba spin column. The extent of labelling and overall protein purity is confirmed using a UPLC-QTOF.
  • Figure 10 is a bar graph showing DiD fluorescence area normalized to area of live nuclei for the tested LNP formulations containing different amounts of poly glycerol-conjugated lipids, and formulated with DSPE-PEG5K-N3 using a mole percentage of 0.5%.
  • the formulations of the LNPs evaluated in this study are given in Table 14.

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Abstract

La présente invention concerne de nouveaux lipides conjugués à un polymère, par exemple, comprenant le DODA conjugué à un polyglycérol ou à un dérivé de polyglycérol. La présente invention concerne également une formulation de nanoparticules lipidiques (NLP) mettant en oeuvre les lipides conjugués à un polymère et des méthodes de traitement d'une maladie par administration des formulations de NLP.
EP23836699.1A 2022-12-01 2023-12-01 Nouveaux lipides conjugués à un polyglycérol et compositions de nanoparticules lipidiques les comprenant Pending EP4626400A1 (fr)

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US202263429267P 2022-12-01 2022-12-01
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US202363449617P 2023-03-03 2023-03-03
US202363452077P 2023-03-14 2023-03-14
US202363467045P 2023-05-17 2023-05-17
US202363467116P 2023-05-17 2023-05-17
US202363592852P 2023-10-24 2023-10-24
PCT/US2023/082037 WO2024119051A1 (fr) 2022-12-01 2023-12-01 Nouveaux lipides conjugués à un polyglycérol et compositions de nanoparticules lipidiques les comprenant

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WO2025090663A1 (fr) * 2023-10-24 2025-05-01 Generation Bio Co. Nouveaux lipides conjugués à un polyglycérol et compositions de nanoparticules lipidiques les comprenant
WO2026064512A1 (fr) * 2024-09-18 2026-03-26 Generation Bio Co. Lipides conjugués à un polyglycérol et compositions de nanoparticules lipidiques les comprenant
CN121064464B (zh) * 2025-11-06 2026-02-24 北京师范大学珠海校区 一种基于天然分子胆酸的抗菌牙科树脂材料

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