WO2024220807A2 - Systèmes et formulations de distribution de nanoparticules lipidiques (lnp) - Google Patents

Systèmes et formulations de distribution de nanoparticules lipidiques (lnp) Download PDF

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Publication number
WO2024220807A2
WO2024220807A2 PCT/US2024/025406 US2024025406W WO2024220807A2 WO 2024220807 A2 WO2024220807 A2 WO 2024220807A2 US 2024025406 W US2024025406 W US 2024025406W WO 2024220807 A2 WO2024220807 A2 WO 2024220807A2
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compound
independently
subscript
formula
rule
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WO2024220807A3 (fr
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Li LAI
Charitha GURUGE
Marine HATIT
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Prime Medicine Inc
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Prime Medicine Inc
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Priority to EP24731095.6A priority Critical patent/EP4698549A2/fr
Priority to CN202480040525.XA priority patent/CN121399143A/zh
Priority to KR1020257038733A priority patent/KR20260003750A/ko
Priority to MX2025012462A priority patent/MX2025012462A/es
Priority to AU2024259356A priority patent/AU2024259356A1/en
Publication of WO2024220807A2 publication Critical patent/WO2024220807A2/fr
Publication of WO2024220807A3 publication Critical patent/WO2024220807A3/fr
Priority to IL323965A priority patent/IL323965A/en
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C07H15/08—Polyoxyalkylene derivatives

Definitions

  • Sequence Listing XML is identified as follows: “272059-545516_Sequence- listing.xml” (4,562 bytes in size), which was created on April 12, 2024, at 5:45 p.m.
  • the present disclosure describes compounds, compositions, lipid nanoparticles, and methods for delivering constructs and polynucleotides.
  • GalNAc-based ligands which bind to the asialoglycoprotein receptor (ASGPR), have recently been developed in lipid nanoparticles systems to deliver nucleic acids to the liver.
  • the editing activity achieved by GalNAc-LNP system has paved the way of nucleic acid delivery and further develop as potential gene therapeutics delivery system.
  • Extensive structure-activity relationship (SAR) studies have reported that different linkers influence the GalNAc-based lipids and their delivery activity. Accordingly, there is a need to develop GalNAc-based lipids that can be incorporated into LNP compositions.
  • the present disclosure provides lipid nanoparticle (LNP) compositions.
  • the LNP compositions comprise a lipid that comprises a compound described herein.
  • the compound described herein is a GalNAc-based lipid.
  • each X 1 is independently -(CH 2 )r-, -(CEUCELCOs- -(OCEbCEb)s- -((CH 2 )m- C(O)NH)-, or -(C(O)NH-(CH 2 ) m )-, each subscript “m” is independently 1-5; each subscript “o” is independently 1-5; each subscript “r” is independently 1-10; each subscript “s” is independently 1-5; each G 1 is independently a C 1 -C 6 substituted or unsubstituted alkylene; each R a is independently H or a C 1 -C 6 substituted or unsubstituted alkyl;
  • L 1 is -NHC(O)-, -C(O)NH-, -NHC(O)O- -OC(O)NH- -NHC(O)NH-, -S-S-, or subscript “p” is independently 1-5;
  • L 2 is -O-(CH 2 CH 2 O)n-, -(CH 2 CH 2 O) n -O-, or -(OCH 2 CH 2 O) n -, each subscript “n” is independently 1-200; subscript “q 1 ” is independently 0-5; subscript “q 2 ” is independently 0-5;
  • L 3 is absent, -NHC(O)-, or -C(O)NH-; subscript “q 3 ” is independently 0-5; each R is independently a C 12 -C 20 substituted or unsubstituted alkyl; and
  • Another aspect of the disclosure provides compounds selected from the group consisting of Formula Lc-5, 1-c-6, Lc-7, and I-c-8:
  • each X 1 is independently -(CH 2 ) r - -(CH 2 CH 2 O) S - -(OCH 2 CH 2 ) 8 - -((CH 2 ) m - C(O)NH)-, or -(C(O)NH-(CH 2 ) m )-; each subscript “m” is independently 1-5; each subscript “s” is independently 1-5; each subscript “o” is independently 1-5; each subscript “r” is independently 1-10; each subscript “p” is independently 1-5; each subscript “n” is independently 1-200; each subscript “q 1 ” is independently 0-5; each subscript “q 2 ” is independently 0-5; each L 3 is independently absent, -NHC(O)-, or -C(O)NH-; and each R is independently a Ci 2 -C 2 o substituted or unsubstituted alkyl.
  • A is: each A 1 , A 2 , A 3 , A 4 , and A 5 is independently
  • each Y 1 is independently -NH-, -NHC(O)O-, -OC(O)NH-, -NHC(O)-, or -
  • M 2 is absent, -NHC(O)-, or -C(O)NH-; subscript “b 2 ” is independently 0-5; subscript “q 4 ” is independently 0-6; each R 1 is independently a C 12 -C 20 substituted or unsubstituted alkyl; and each of the bonds independently indicate an R configuration or an S configuration; with the proviso that (1) A 1 and A 2 are the same and (2) at least one of Y 2 , Y 3 and Y 4 is present.
  • a compound of Formula II is a compound of Formula Il-a:
  • a compound of Formula Il-a is a compound of Formula Il-a-
  • Another aspect of the disclosure provides a compound of Formula III or Formula
  • each A 6 , A 7 , and A 8 is independently each Y 1 is independently absent, -NHC(O)O-, or -OC(O)NH-; each Y 2 and Y 4 is independently -((CH 2 )r ; each subscript “r” is independently 1-10; each Y 3 is independently absent, -NHC(O)-, or -C(O)NH-; each subscript “d” is independently 1-5; each subscript “c” is independently 1-5; subscript “a” is 1-5; subscript “n” is independently 10-100; and each of the ” bonds independently indicate an R configuration or an S configuration; with the proviso that at least one of Y 1 or Y 3 is present.
  • a compound of Formula III is a compound Formula Ill-a or Formula Ill-b:
  • a compound of Formula IV is a compound of Formula IV-a or Formula IV-b:
  • lipid nanoparticle (LNP) composition comprising: a lipid that comprises a compound as described herein;
  • SUBSTITUTE SHEET (RULE 26) a cationic lipid; a phospholipid; a PEG-containing lipid; and a sterol.
  • Another aspect of the present disclosure provides a pharmaceutical composition comprising the LNP composition described herein.
  • Another aspect of the present disclosure provides a method for delivering polynucleotides into a target cell comprising: introducing into the target cell at least one lipid nanoparticle comprising a GalNAc- based lipid that comprises any one of the compounds described herein and one or more polynucleotides.
  • Another aspect of the present disclosure provides a method for delivering a prime editing system into a target cell comprising: introducing into the target cell at least one lipid nanoparticle comprising a GalNAc- based lipid that comprises any one of the compounds described herein and one or more components of a prime editing system.
  • FIG. 1 shows Indel% activity for compounds disclosed herein.
  • FIG. 2 shows prime editing % in a dose response study for compounds disclosed herein.
  • compositions, lipid nanoparticles, and pharmaceutical compositions provided herein can comprise the compounds described herein. Methods for delivering said compositions and lipid nanoparticles are also provided herein.
  • SUBSTITUTE SHEET (RULE 26) how the value is measured or determined, e.g., the limitations of the measurement system.
  • “about” can mean within 1 standard deviation, per the practice in the art.
  • “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value.
  • the term can mean within an order of magnitude, preferably within 5 -fold, and more preferably within 2-fold, of a value.
  • the term “substantially” as used herein may refer to a value approaching 100% of a given value. In some embodiments, the term may refer to an amount that may be at least about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of a total amount. In some embodiments, the term may refer to an amount that may be about 100% of a total amount.
  • hydroxyl or "hydroxy” refers to an -OH moiety.
  • an “alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated or unsaturated (e.g., contains one or more double and/or triple bonds), having, for example, from one to twenty- four carbon atoms (C1-C24 alkyl), one to twenty carbon atoms (C1-C20 alkyl), one to eighteen carbon atoms (Ci-C 18 alkyl), one to sixteen carbon atoms (C1-C16 alkyl), one to twelve carbon atoms (C1-C12 alkyl), one to eight carbon atoms (Ci-Cs alkyl), one to six carbon atoms (C 1 -C 6 alkyl).
  • alkyl group is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n propyl, 1 -methylethyl (iso propyl), n butyl, n pentyl, 1, 1 dimethylethyl (t butyl), 3 methylhexyl, 2 methylhexyl, ethenyl, prop 1 enyl, but-l-enyl, pent-l-enyl, penta-1, 4-dienyl,
  • SUBSTITUTE SHEET (RULE 26) ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. In some embodiments, an alkyl group is substituted.
  • Alkylene or "alkylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, which is saturated or unsaturated (e.g, contains one or more double (alkenylene) and/or triple bonds (alkynylene)), and having, for example, from one to twenty-four carbon atoms (C1-C24 alkynylene), one to twenty carbon atoms (C1-C20 alkynylene), one to eighteen carbon atoms (Ci-C 18 alkynylene), one to sixteen carbon atoms (C1-C16 alkynylene), one to twelve carbon atoms (C1-C12 alkynylene), one to eight carbon atoms (Ci-Cs alkynylene), one to six carbon atoms (C 1 -C 6 alkynylene).
  • C1-C24 alkynylene one to twenty carbon atoms (C1-C20 al
  • alkylene groups include, methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n- butynylene, and the like.
  • the alkylene is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double bond.
  • the points of attachment of the alkylene to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain.
  • an alkylene is substituted.
  • a "carbonyl” refers to -C(O)-.
  • a "cycloalkyl” refers to a stable non aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from three to fifteen carbon atoms, three to ten carbon atoms, etc. and which is saturated or unsaturated and attached to the rest of the molecule by a single bond.
  • Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • Polycyclic radicals include, for example, adamantyl, norbomyl, decalinyl, 7,7 dimethyl bicyclo[2.2.1]heptanyl, and the like.
  • substituted refers generally to the replacement of hydrogen atoms in a given structure with the radical of a specified substituent. Specific substituents are described above in the definitions and below in the description of compounds and examples thereof. Unless otherwise indicated, an optionally substituted group can have a substituent at each substitutable position of the group, and when more than one position in any given structure can be substituted with more than one substituent selected from a specified group, the substituent can be either the same or
  • a "substituted" group e.g., alkyl or alkylene
  • the substituent is a C1-C12 alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as fluoro. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.
  • structures depicted herein also are meant to include all isomeric (e.g, enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the disclosure. Unless otherwise stated, all tautomeric forms of the compounds of the disclosure are within the scope of the disclosure.
  • structures depicted herein also are meant to include compounds that differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13 C- or 14 C-enriched carbon are within the scope of this disclosure.
  • Such compounds are useful, for example, as analytical tools or probes in biological assays, or as therapeutic agents.
  • compositions disclosed herein may be “isomerically pure” compounds.
  • the term “isomerically pure” refers to an isomeric form of a compound that is substantially free from other isomeric forms of the compound (e.g., substantially free from other stereoisomers (e.g., enantiomers, diastereomers, geometric (or conformational) isomers, etc.), constitutional isomers, isotopomers, etc.).
  • stereoisomers e.g., enantiomers, diastereomers, geometric (or conformational) isomers, etc.
  • constitutional isomers isotopomers, etc.
  • SUBSTITUTE SHEET (RULE 26) compound having at least one asymmetric center of a particular configuration (e.g., R or S configuration) is substantially free from other isomeric forms of the compound having a different configuration at the at least one asymmetric center.
  • An “isomerically pure” compound comprises more than 75% by weight, more than 80% by weight, more than 85% by weight, more than 90% by weight, more than 91% by weight, more than 92% by weight, more than 93% by weight, more than 94% by weight, more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 98.5% by weight, more than 99% by weight, more than 99.2% by weight, more than 99.5% by weight, more than 99.6% by weight, more than 99.7% by weight, more than 99.8% by weight, or more than 99.9% by weight, of a single isomer of the compound based on the total weight of all isomers of the compound that are present.
  • a pharmaceutically acceptable derivative includes, but is not limited to, pharmaceutically acceptable prodrugs, salts, esters, salts of such esters, or any other adduct or derivative that upon administration to a patient in need is capable of providing, directly or indirectly, a compound as otherwise described herein, or a metabolite or residue thereof.
  • the term "pharmaceutically acceptable” means approved or approvable by a regulatory agency of the Federal or a state government or the corresponding agency in countries other than the United States, or that is listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly, in humans.
  • the term "pharmaceutically acceptable salt” refers to a salt of a compound of the disclosure that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound.
  • such salts are non-toxic may be inorganic or organic acid addition salts and base addition salts.
  • such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cy clopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3 -(4-hydroxy benzoyl) benzoic acid,
  • SUBSTITUTE SHEET (RULE 26) cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane- disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4- chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2] -oct-2-ene-l -carboxylic acid, glucoheptonic acid, 3 -phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by
  • Salts further include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkyl ammonium, and the like; and when the compound contains a basic functionality, salts of non-toxic organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.
  • pharmaceutically acceptable cation refers to an acceptable cationic counter-ion of an acidic functional group. Such cations are exemplified by sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium cations, and the like. See, e.g., Berge, et al., J. Pharm. Sci. (1977) 66(1): 1-79.
  • lipid refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are generally characterized by being poorly soluble in water, but soluble in many organic solvents.
  • a “lipid nanoparticle” may refer to particles having at least one dimension in the nanometers range (e.g., 1-1,000 nm) and include the compounds described herein.
  • lipid nanoparticles can be included in compositions that are used to deliver at least one polynucleotide and/or construct according to this disclosure.
  • the lipid nanoparticles of the disclosure comprise at least one polynucleotide and/or one or more components of a prime editing system.
  • the lipid nanoparticles can include one or more additional lipid components.
  • the at least one polynucleotide and/or construct may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle.
  • the lipid nanoparticles can have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100
  • SUBSTITUTE SHEET (RULE 26) nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm.
  • biodegradable refers to materials that, when introduced into cells, are broken down by cellular machinery (e.g., enzymatic degradation) or by hydrolysis into components that cells can either reuse or dispose of without significant toxic effect(s) on the cells.
  • components generated by breakdown of a biodegradable material do not induce inflammation and/or other adverse effects in vivo.
  • biodegradable materials are enzymatically broken down. Alternatively or additionally, in some embodiments, biodegradable materials are readily broken down and eliminated.
  • cationic lipids refer to lipids that are positively charged at the head group. Cationic lipids typically contain three domains: a hydrophilic headgroup, a hydrophobic domain, and a linker. Cationic lipids are an amphiphilic entity with the head group having hydrophilicity and lipid tails possessing hydrophobicity.
  • a “cell” can generally refer to a biological cell.
  • a cell can be the basic structural, functional and/or biological unit of a living organism.
  • a cell can originate from any organism having one or more cells. Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant, an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidanan, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate,
  • the cell is a human cell.
  • a cell may be of or derived from different tissues, organs, and/or cell types.
  • the cell is a primary cell.
  • the term primary cell means a cell isolated from an organism, e.g., a mammal, which is grown in tissue culture (e.g., in vitro) for the first time before subdivision and transfer to a subculture.
  • tissue culture e.g., in vitro
  • mammalian primary cells can be modified through introduction of one or more polynucleotides, polypeptides, and/or prime editing compositions (e.g., through transfection, transduction, electroporation and the like) and further passaged.
  • a cell is not isolated from an organism but forms
  • SUBSTITUTE SHEET part of a tissue or organ of an organism, e.g, a mammal.
  • the cell is a stem cell.
  • the cell is a human stem cell.
  • construct refers to refers to a polynucleotide or a portion of a polynucleotide, comprising one or more nucleic acid sequences encoding one or more transcriptional products and/or proteins.
  • a construct may be a recombinant nucleic acid molecule or a part thereof.
  • the one or more nucleic acid sequences of a construct are operably linked to one or more regulatory sequences, for example, transcriptional initiation regulatory sequences.
  • a construct is a vector, a plasmid, or a portion thereof.
  • a construct comprises DNA.
  • a construct comprises RNA.
  • a construct is double stranded. In some embodiments, a construct is single stranded. In some embodiments, a construct comprises an expression cassette.
  • An expression cassette means a polynucleotide comprising a nucleic acid sequence that encodes one or more transcriptional products and is operably linked to at least one transcriptional regulatory sequence, e.g., a promoter.
  • polynucleotide or “nucleic acid molecule” can be any polymeric form of nucleotides, including DNA, RNA, a hybridization thereof, or RNA-DNA chimeric molecules.
  • a polynucleotide can be a cDNA, genomic DNA, mRNA, tRNA, rRNA, microRNA, antisense DNA or RNA, plasmid DNA, microRNA inhibitors, mRNA- interfering complementary RNA (micRNA), multivalent RNA, etc.
  • a polynucleotide is double stranded, e.g., a double-stranded DNA in a gene.
  • a polynucleotide is single-stranded or substantially single-stranded, e.g., single-stranded DNA or an mRNA. In some embodiments, a polynucleotide is a cell-free nucleic acid molecule. In some embodiments, a polynucleotide circulates in blood. In some embodiments, a polynucleotide is a cellular nucleic acid molecule. In some embodiments, a polynucleotide is a cellular nucleic acid molecule in a cell circulating in blood.
  • Polynucleotides can have any three-dimensional structure.
  • a polynucleotide comprises deoxyribonucleotides, ribonucleotides or analogs thereof.
  • a polynucleotide comprises modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA.
  • the polynucleotide may comprise one or more other nucleotide bases, such as inosine (I), which is read by the translation machinery as guanine (G).
  • a polynucleotide may be modified.
  • the terms “modified” or “modification” refers to chemical modification with respect to the A, C, G, T and U nucleotides.
  • modifications may be on the nucleoside base and/or sugar portion of the nucleosides that comprise the polynucleotide.
  • the modification may be on the intemucleoside linkage (e.g, phosphate backbone).
  • multiple modifications are included in the modified nucleic acid molecule.
  • a single modification is included in the modified nucleic acid molecule.
  • protein and “polypeptide” can be used interchangeably to refer to a polymer of two or more amino acids joined by covalent bonds (e.g., an amide bond) that can adopt a three-dimensional conformation.
  • a protein or polypeptide comprises at least 10 amino acids, 15 amino acids, 20 amino acids, 30 amino acids or 50 amino acids joined by covalent bonds (e.g., amide bonds).
  • a protein comprises at least two amide bonds.
  • a protein comprises multiple amide bonds.
  • a protein comprises an enzyme, enzyme precursor proteins, regulatory protein, structural protein, receptor, nucleic acid binding protein, a biomarker, a member of a specific binding pair (e.g., a ligand or aptamer), or an antibody.
  • a protein may be a full-length protein (e.g., a fully processed protein having certain biological function).
  • a protein may be a variant or a fragment of a full-length protein.
  • a variant of a protein or enzyme, for example a variant reverse transcriptase comprises a polypeptide having an amino acid sequence that is about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95%
  • SUBSTITUTE SHEET (RULE 26) identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, about 99.5% identical, or about 99.9% identical to the amino acid sequence of a reference protein.
  • a protein comprises one or more protein domains or subdomains.
  • polypeptide domain when used in the context of a protein or polypeptide, refers to a polypeptide chain that has one or more biological functions, e.g., a catalytic function, a protein-protein binding function, or a protein-DNA function.
  • a protein comprises multiple protein domains.
  • a protein comprises multiple protein domains that are naturally occurring.
  • a protein comprises multiple protein domains from different naturally occurring proteins.
  • a protein that comprises amino acid sequences from different origins or naturally occurring proteins may be referred to as a fusion, or chimeric protein.
  • a protein or polypeptide includes naturally occurring amino acids (e.g., one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V).
  • a protein or polypeptides includes non-naturally occurring amino acids (e.g, amino acids which is not one of the twenty amino acids commonly found in peptides synthesized in nature, including synthetic amino acids, amino acid analogs, and amino acid mimetics).
  • a protein or polypeptide is modified.
  • a protein comprises an isolated polypeptide.
  • isolated means free or removed to varying degrees from components which normally accompany it as found in the natural state or environment. For example, a polypeptide naturally present in a living animal is not isolated, and the same polypeptide partially or completely separated from the coexisting materials of its natural state is isolated.
  • a protein is present within a cell, a tissue, an organ, or a virus particle.
  • a protein is present within a cell or a part of a cell (e.g., a bacteria cell, a plant cell, or an animal cell).
  • the cell is in a tissue, in a subject, or in a cell culture.
  • the cell is a microorganism (e.g., a bacterium, fungus, protozoan, or virus).
  • a protein is present in a mixture of analytes (e.g., a lysate).
  • the protein is present in a lysate from a plurality of cells or from a lysate of a single cell.
  • encode refers to a polynucleotide
  • SUBSTITUTE SHEET (RULE 26) which is said to “encode” another polynucleotide, a polypeptide, or an amino acid if, in its native state or when manipulated by methods well known to those skilled in the art, it can be used as a polynucleotide synthesis template, e.g., transcribed into an RNA, reverse transcribed into a DNA or cDNA, and/or translated to produce an amino acid, or a polypeptide or fragment thereof.
  • a polynucleotide comprising three contiguous nucleotides form a codon that encodes a specific amino acid.
  • a polynucleotide comprises one or more codons that encode a polypeptide.
  • a polynucleotide comprising one or more codons comprises a mutation in a codon compared to a wild-type reference polynucleotide.
  • the mutation in the codon encodes an amino acid substitution in a polypeptide encoded by the polynucleotide as compared to a wild-type reference polypeptide.
  • LNP compositions comprise an N-acetylgalactosamine (“GalNAc”) based lipid that comprises a compound described herein.
  • the LNP compositions comprise a GalNAc-based lipid that is or comprises a compound described herein, a cationic lipid, a phospholipid, a polyethylene gly col-lipid, and a sterol.
  • LNP compositions comprise a GalNAc-based lipid that is or comprises a compound described herein.
  • Ac refers to an acetyl -(C(O)CHs) functional group and is not to be confused with the element actinium.
  • GalNAc-based ligands which bind to the asialoglycoprotein receptor (ASGPR), have recently been developed in lipid nanoparticles systems to deliver nucleic acids to the liver.
  • the editing activity achieved by GalNAc-LNP system has paved the way of nucleic acid delivery and further develop as potential gene therapeutics delivery system.
  • Extensive SAR studies have reported that different linkers influence the GalNAc-based lipids and their delivery activity. Accordingly, there is a need to develop GalNAc-based lipids that can be incorporated into LNP composition.
  • the present disclosure provides a new series of GalNAc-based lipids which include a PEG moiety.
  • the compounds of Formula I and II disclosed herein employed three major design strategies: 1) insertion of CH 2 motif between NH 2 and a carbon atom; 2) insertion of a biodegradable carbamide motif within the linker; and/or 3) employed different amino acid as linkers.
  • PEG moieties were selected to bridge the GalNAc-based moieties with lipids.
  • a series of tri-valent-GalNAc and tetra-valvent GalNAc PEG lipids are disclosed herein, as shown in Scheme 1 below.
  • a biodegradable carbamide motif can be incorporated into to linker, basic and acidic amino acids, such as Lys and Glu can be selected to conjugate with tri-valent and tetra-valent GalNAc, and PEG-based lipits can be conjugated with alpha amine (7? or S isomer) of the amine acids.
  • each X 1 is independently -(CH 2 ) r - -(CH 2 CH 2 O)s- -(OCH 2 CH 2 )- -((CH 2 ) m -
  • L 1 is -NHC(O)-, -C(O)NH-, -NHC(O)O- -OC(O)NH-, -NHC(O)NH-, -S-S-, or subscript “p” is independently 1-5;
  • L 2 is -O-(CH 2 CH 2 O) n - -(CH 2 CH 2 O)n-O- or -(OCH 2 CH 2 O) n - each subscript “n” is independently 1-200; subscript “q 1 ” is independently 0-5; subscript “q 2 ” is independently 0-5;
  • L 3 is absent, -NHC(O)-, or -C(O)NH-; subscript “q 3 ” is independently 0-5; each R is independently a C 12 -C 20 substituted or unsubstituted alkyl; and the indicates an R configuration or an S configuration.
  • the compound of Formula I is a compound of Formula I-a:
  • each X 1 is independently -(CH 2 )I-, -(CH 2 CH 2 O) S -, - (OCH 2 CH 2 ) S -, -((CH 2 ) m -C(O)NH)-, or -(C(O)NH-(CH 2 )m)-.
  • each subscript “m” is independently 1-5.
  • each subscript “r” is independently 1-10.
  • each subscript “s” is independently 1-5.
  • each X 1 is independently -(CH 2 ) r - In some embodiments, each subscript “r” is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • each X 1 is independently -(CH 2 CH 2 O) s -. In some embodiments, each X 1 is independently - (OCH 2 CH 2 ) S -. In some embodiments, each subscript “s” is independently 1, 2, 3, 4, or 5. In some embodiments, each X 1 is independently-((CH 2 ) m -C(O)NH)-. In some embodiments, each X 1 is independently -(C(O)NH-(CH 2 ) m )-. In some embodiments, each subscript “m” is independently 1, 2, 3, 4, or 5.
  • each subscript “o” is independently 1, 2, 3, 4, or 5.
  • each G 1 is independently a C 1 -C 6 substituted or unsubstituted alkylene. In some embodiments, each G 1 is independently a C 1 -C 6 substituted alkylene. In some embodiments, each G 1 is independently a C 1 -C 6 unsubstituted alky lene.
  • each G 1 is independently a C 1 (-CH 2 -), C 2 (-CH 2 CH 2 -), C 3 (e.g, -(CH 2 )s-), C 4 (e.g, -(CH 2 )4-), C 5 (e.g., -(CH 2 ) 5 -), or C 6 (e.g., -(CH 2 ) 6 -) unsubstituted alkyl.
  • each R a is independently H or a C 1 -C 6 substituted or unsubstituted alkyl. In some embodiments, each R a is independently H. In some embodiments, each R a is independently a C 1 -C 6 substituted or unsubstituted alkyl. In some embodiments, each R a is independently a C 1 -C 6 substituted alkyl. In some embodiments, each R a is independently a C 1 -C 6 unsubstituted alkyl.
  • L 1 is -NHC(O)-, -C(O)NH-, -NHC(O)O-, -OC(O)NH-, - some embodiments, L 1 is -NHC(O)-. In some embodiments, L 1 is -C(O)NH-. In some embodiments, L 1 is -NHC(O)O- In some embodiments, L 1 is -OC(O)NH- In some embodiments, L 1 is -NHC(O)NH- In some embodiments, L 1 is -S-S-. In some embodiments,
  • subscript “p” is independently 1, 2, 3, 4, or 5.
  • L 2 is -O-(CH 2 CH 2 O) n -, -(CH 2 CH 2 O)n-O-, or - (OCH 2 CH 2 O) n -. In some embodiments, L 2 is -O-(CH 2 CH 2 O) n - In some embodiments, L 2 is -(CH 2 CH 2 O)n-O-. In some embodiments, L 2 is -(OCH 2 CH 2 O) n - In some embodiments, each subscript “n” is independently 1-200, 10-200, 10-100, 10-50, 10-40, 10-30, 10-20, or 100-200. In some embodiments, each subscript “n” is independently 10-200.
  • each subscript “n” is independently 100-200. In some embodiments, each subscript “n” is independently 10, 20, 30, 40, or 50. In some embodiments, the quantity of each subscript “n” depends on the molecular weight average of the moiety, such that the PEG-like moiety results in a molecular weight average of about 100-2000 Daltons (Da), about 1000-2000 Da, about 1000 Da, or about 2000 Da.
  • Da Daltons
  • subscript “q 1 ” is independently 0, 1, 2, 3, 4, or 5. In some embodiments, subscript “q 2 ” is independently 0, 1, 2, 3, 4, or 5. In some embodiments, subscript “q 3 ” is independently 0, 1, 2, 3, 4, or 5.
  • L 3 is absent, -NHC(O)-, or -C(O)NH- In some embodiments, L 3 is absent. In some embodiments, L 3 is -NHC(O)-. In some embodiments, L 3 is -C(O)NH-
  • each R is independently a C 12 -C 20 substituted or unsubstituted alkyl. In some embodiments, each R is independently a C 12 -C 20 substituted alkyl. In some embodiments, each R is independently a C 12 -C 20 unsubstituted alkyl. In some embodiments, each R is independently a C 14 -C 18 unsubstituted alkyl. In some embodiments, each R is independently a C 14 unsubstituted alkyl. In some embodiments, each R is independently a C 16 unsubstituted alkyl. In some embodiments, each R is independently a C 18 unsubstituted alkyl. In some embodiments, each R is a C 12 alkyl, C 14 alkyl, C 15 alkyl, C 16
  • each R is n- dodecanyl, n-tridecanyl, n-tetradecanyl, or n-pentadecanyl.
  • the ” indicates an R configuration or an 5 configuration. In some embodiments, the “• « « «- ” indicates an A configuration. In some embodiments, the “ AAA. ” indicates an S configuration.
  • the compound of Formula I is a compound of Formula I-b:
  • the compound of Formula I is a compound of Formula I-c:
  • the compound of Formula I is any one of a compound of
  • the compound of Formula I is any one of a compound of
  • the compound of Formula I is any one of a compound of
  • the compound of Formula I is any one of a compound of
  • the disclosure provides a compound selected from the group consisting of Formula I-c-5, 1-c-6, I-c-7, and I-c-8:
  • each X 1 is independently -(CH 2 ) r -, -(CH 2 CH 2 O) S -, -(OCH 2 CH 2 ) S -, -((CH 2 ) m - C(O)NH)-, or -(C(O)NH-(CH 2 ) m )-; each subscript “m” is independently 1-5; each subscript “s” is independently 1-5; each subscript “o” is independently 1-5; each subscript “r” is independently 1-10; each subscript “p” is independently 1-5; each subscript “n” is independently 1-200; each subscript “q 1 ” is independently 0-5; each subscript “q 2 ” is independently 0-5; each L 3 is independently absent, -NHC(O)-, or -C(O)NH-; and each R is independently a C 12 -C 20 substituted or unsubstituted alkyl.
  • each X 1 is independently -(CH 2 )I-, -(CH 2 CH 2 O) S - - (OCH 2 CH 2 ) S - -((CH 2 ) m -C(O)NH)-, or -(C(O)NH-(CH 2 )m)-.
  • each subscript “m” is independently 1-5.
  • each subscript “r” is independently 1-10.
  • each subscript “s” is independently 1-5.
  • each X 1 is independently -(CH 2 ) r - In some embodiments, each subscript “r” is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • each X 1 is independently -(CH 2 CH 2 O) S - In some embodiments, each X 1 is independently - (OCH 2 CH 2 ) S - In some embodiments, each subscript “s” is independently 1, 2, 3, 4, or 5. In some embodiments, each X 1 is independently-((CH 2 ) m -C(O)NH)-. In some embodiments, each X 1 is independently -(C(O)NH-(CH 2 ) m )-. In some embodiments, each subscript “m” is independently 1, 2, 3, 4, or 5.
  • each subscript “0” is independently 1, 2, 3, 4, or 5.
  • each subscript “p” is independently 1, 2, 3, 4, or 5.
  • each subscript “n” is independently 1-200. In some embodiments, each subscript “n” is independently 10-200. In some embodiments, each subscript “n” is independently 100-200. In some embodiments, each subscript “n” is independently 100. In some embodiments, each subscript “n” is independently 200.
  • each subscript “q 1 ” is independently 0, 1, 2, 3, 4, or 5. In some embodiments, each subscript “q 2 ” is independently 0, 1, 2, 3, 4, or 5. In some embodiments, each subscript “q 3 ” is independently 0, 1, 2, 3, 4, or 5.
  • each L 3 is absent, -NHC(O)-, or -C(O)NH- In some embodiments, each L 3 is absent. In some embodiments, each L 3 is -NHC(O)-. In some embodiments, each L 3 is -C(O)NH-
  • each R is independently a C 12 -C 20 substituted or unsubstituted alkyl. In some embodiments, each R is independently a C 12 -C 20 substituted alkyl. In some embodiments, each R is independently a C 12 -C 20 unsubstituted alkyl. In some embodiments, each R is independently a C 14 -C18 unsubstituted alkyl. In some embodiments, each R is independently a C 14 unsubstituted alkyl. In some embodiments, each R is independently a C 16 unsubstituted alkyl. In some embodiments, each R is independently a C 18 unsubstituted alkyl.
  • each R is a C12 alkyl, C 14 alkyl, C15 alkyl, C 16 alkyl, C17 alkyd, C 18 alkyl, C19 alkyl, or C20 alkyl. In some embodiments, each R is n- dodecanyl, n-tridecanyl, n-tetradecanyl, or n-pentadecanyl.
  • each X 1 is -((CH 2 ) m -C(O)NH)-, each subscript “m” is independently 2, and each subscript “0” is independently 1.
  • each X 1 is -(CH 2 CH 2 O) S -, each subscript “s” is independently 2, and each subscript “0” is independently 1.
  • the compound is a compound of Formula I-c-7 and, optionally, each X 1 is -(CH 2 X-, each subscript “r” is independently 5, and each subscript “0” is 1.
  • the compound is a compound of Formula I-c-7 and each X 1 is - (CH 2 )r- , each subscript “r” is independently 5, and each subscript “0” is 1.
  • the compound is a compound of Formula I-c-5 and L 2 is -0- (CH 2 CH 2 O) n - , and subscript “n” is 100 to 200.
  • the compound is a compound of Formula I-c-6, I-c-7, or I-c- 8, and each L 2 is -(OCFhCFbCOn- and each subscript “n” is 100 to 200.
  • subscript “p” is 4, subscript “q 1 ” is 0, and subscript “q 2 ” is 0.
  • each subscript “p” is 2
  • subscript “q 1 ” is 0, and subscript “q 2 ” is 0.
  • subscript “p” is 4, subscript “q 1 ” is 1, and subscript “q 2 ” is 4.
  • Scheme 4 shows a general synthetic scheme of 2,2-Bis(hydroxymethyl)propane-l,3- diol (1) based GalNAc PEG lipids.
  • M 2 is absent, -NHC(O)-, or -C(O)NH-;
  • SUBSTITUTE SHEET (RULE 26) subscript “b 2 ” is independently 0-5; subscript “q 4 ” is independently 0-6; each R 1 is independently a C 12 -C 20 substituted or unsubstituted alkyl; and each of the bonds independently indicate an R configuration or an S configuration; with the proviso that at least one of Y 2 , Y 3 and Y 4 is present.
  • A is: some embodiments, A is:
  • each subscript “c” is independently 0,1, 2, 3, 4, or 5. In some embodiments, each subscript “c” is independently 0. In some embodiments, each subscript “c” is independently 1.
  • a 1 and A 2 are the same. In some embodiments, A 1 and A 2 are different. In some embodiments, A 1 , A 2 , and A 3 are the same. In some embodiments, A 1 , A 2 , and A 3 are different. In some embodiments, A 1 , A 2 , A 4 , and A 5 are the same. In some embodiments, A 1 , A 2 , A 4 , and A 5 are different. In some embodiments, A 1 , A 4 , and A 5 are the same. In some embodiments, A 1 , A 4 , and A 5 are different.
  • a 1 and A 2 are the same and both different from A 3 .
  • a 5 are the same. In some embodiments, when A is: and A 4 are the same and A 5 is different from A 1 , A 2 , and A 4 . In some embodiments, when A is: are the same and A 4 is different from A 1 ,
  • each A 1 , A 2 , A 3 , A 4 , and A 5 is independently some embodiments, each subscript “d” is independently 0, 1, 2, 3, 4, or 5.
  • each Y 1 is independently -NH-, -NHC(O)O-, -OC(O)NH- -NHC(O)-, or -C(O)NH- In some embodiments, each Y 1 is independently -NH-. In some embodiments, each Y 1 is independently -NHC(O)O- In some embodiments, each Y 1 is independently -OC(O)NH- In some embodiments, each Y 1 is independently -NHC(O)-. In some embodiments, each Y 1 is independently -C(O)NH-
  • each Y 2 is independently absent, -(CH 2 ) r -, -(CFhCFbO) ⁇ , or -(OCH 2 CH 2 )t- In some embodiments, each Y 2 is independently absent. In some embodiments, each Y 2 is independently -(ODr- In some embodiments, each Y 2 is independently -(CH 2 )r- and each subscript “r” is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, each Y 2 is independently -(CEhCEhO))-. In some embodiments, each Y 2 is independently -(CH 2 CH 2 O)t- and each subscript “t” is independently 1, 2, 3, 4, or 5. In some embodiments, each Y 2 is independently -(OCH 2 CH 2 )t- and each subscript “t” is independently 1, 2, 3, 4, or 5.
  • each Y 3 is independently absent, -NHC(O)-, or -C(O)NH- In some embodiments, each Y 3 is independently absent. In some embodiments, each Y 3 is independently -NHC(O)-. In some embodiments, each Y 3 is independently -C(O)NH- [0192] In some embodiments, each Y 4 is independently absent or -(CH 2 ) r - In some embodiments, each Y 4 is independently absent. In some embodiments, each Y 4 is
  • each Y 4 is independently -(CH 2 )r- and each subscript “r” is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • each subscript “n” is independently 1-200, 10-200, 10-100, 10-50, 10-40, 10-30, 10-20, or 100-200. In some embodiments, each subscript “n” is independently 10-200. In some embodiments, each subscript “n” is independently 100-200. In some embodiments, each subscript “n” is independently 10, 20, 30, 40, or 50. In some embodiments, the quantity of each subscript “n” depends on the molecular weight average of the moiety, such that the PEG-like moiety results in a molecular weight average of about 100- 2000 Daltons (Da), about 1000-2000 Da, about 1000 Da, or about 2000 Da.
  • Da molecular weight average of the moiety
  • subscript “a” is independently 1, 2, 3, 4, or 5.
  • subscript “b 1 ” is independently 0, 1, 2, 3, 4, or 5. In some embodiments, subscript “b 2 ” is independently 0, 1, 2, 3, 4, or 5.
  • M 2 is absent, -NHC(O)-, or -C(O)NH- In some embodiments, M 2 is absent. In some embodiments, M 2 is -NHC(O)-. In some embodiments, M 2 is -C(O)NH-
  • subscript “q 4 ” is independently 0, 1, 2, 3, 4, 5, or 6.
  • each R 1 is independently a C 12 -C 20 substituted or unsubstituted alkyl. In some embodiments, each R 1 is independently a C 12 -C 20 substituted alkyl. In some embodiments, each R 1 is independently a C 12 -C 20 unsubstituted alkyl. In some embodiments, each R 1 is independently a unsubstituted alkyl. In some embodiments, each R 1 is independently a C 14 unsubstituted alkyl. In some embodiments, each R 1 is independently a C 16 unsubstituted alkyl. In some embodiments, each R 1 is independently a C 18 unsubstituted alkyl.
  • each R 1 is a C 12 alkyl, C 14 alkyl, C 15 alkyl, C 16 alkyl, C 17 alkyd, C 18 alkyl, C 19 alkyl, or C 20 alkyl. In some embodiments, each R 1 is n- dodecanyl, n-tridecanyl, n-tetradecanyl, or n-pentadecanyl.
  • each of the Ju ' A - ” bonds independently indicate an R configuration or an S configuration. In some embodiments, in Formula II, each of the “• « « ⁇ ” bonds independently indicate an R configuration. In some embodiments, in Formula II, each of the bonds independently indicate an S configuration. In some embodiments, in Formula II, the “• « « ⁇ ” bond next to the “A” moiety indicates an S configuration and the other ” bond indicates an R configuration. In some embodiments, in Formula II, the ” bond next to the “A” moiety indicates an R configuration and the other ” bond indicates an R configuration. In some embodiments, in Formula II, the “
  • the compound of Formula II is a compound of Formula Il-a:
  • the compound of Formula II is a compound of Formula Il-a-
  • the compound of Formula II is a compound of Formula Il-a-
  • the compound of Formula II is a compound of Formula Il-a-
  • the compound of Formula II is a compound of Formula Il-a- 4:
  • the compound of Formula II is a compound of Formula Il-a- 5:
  • the compound of Formula II is a compound of Formula Il-a- 6:
  • a 1 , A 2 , and A 3 are the same. In some embodiments, in the compound of Formula II, Formula Il-a, Formula II-a-1, Formula II-a-2, or Formula II-a-3, A 1 and A 2 are different from A 3 .
  • a 1 , A 2 , A 4 , and A 5 are the same. In some embodiments, in the compound of Formula II, Formula Il-a, Formula II-a-4, or Formula II-a-5, A 1 , A 2 , and A 4 are the same. In some embodiments, in the compound of Formula II, Formula Il-a, Formula II-a-4, or Formula II-a-5, A 1 , A 2 , and A 4 are the same. In some embodiments, in the compound of Formula II, Formula Il-a, Formula II-a-4, or Formula II-a-5, A 1 , A 2 , and A 4 are the same and A 5 is different from A 1 , A 2 , and A 4 . In some
  • each of Y 1 and Y 4 are present and each of Y 2 and Y 3 are absent.
  • each Y 1 is -C(O)NH- and each Y 4 is -(CH 2 )r-.
  • each Y 1 is -OC(O)NH- and each Y 4 is -(CFhV.
  • each subscript “r” is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • each of Y 1 , Y 2 , Y 3 , and Y 4 are present.
  • each Y 1 is independently -NHC(O)O-;
  • each Y 2 is independently -((CH 2 )r ;
  • each Y 3 is independently -C(O)NH-;
  • each Y 4 is independently -((CH 2 )r .
  • each subscript “r” is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • each of Y 1 and Y 2 are present and each of Y 3 and Y 4 are absent.
  • each Y 1 is independently -OC(O)NH- and each Y 2 is independently -(CEbCFhCOt-.
  • each subscript “t” is independently 1, 2, 3, 4, or 5.
  • each of A 1 , A 2 , A 3 , A 4 , and A 3 is selected from the group consisting of:
  • Another aspect of the disclosure provides acompound of Formula III or Formula IV:
  • each A 6 , A 7 , and A 8 is independently each Y 1 is independently absent, -NHC(0)0-, or -OC(O)NH-; each Y 2 and Y 4 is independently -(CH 2 ) r -; each subscript “r” is independently 1-10; each Y 3 is independently absent, -NHC(O)-, or -C(O)NH-; each subscript “d” is independently 1-5; each subscript “c” is independently 1-5; subscript “a” is 1-5; subscript “n” is independently 35-46; and each of the ” bonds independently indicate an R configuration or an S configuration;
  • SUBSTITUTE SHEET (RULE 26) with the proviso that at least one of Y 1 or Y 3 is present.
  • the compound of Formula III is a compound of Formula ffl-a or Formula Ill-b:
  • the compound of Formula IV is a compound of Formula IV-a or Formula IV-b:
  • each of the variables, Y 1 ,Y 2 , Y 3 , Y 4 , subscript “a”, “c”, “d” and “n” have values as described above for Formula II.
  • each of the variables A 6 , A 7 , and A 8 variables are as described above for A 1 through A 5 for Formula II.
  • a 6 , A 7 , and A 8 are the same. In some embodiments, A 6 , A 7 , and A 8 are each different. In some embodiments, A 6 and A 7 are different. In some embodiments, A 6 and A 7 are different.
  • subscript “a” is 3. In some embodiments, subscript “a” is 1.
  • subscript “n” is independently between 34-46 or in a range of 34-46.
  • subscript “n” is independently between 34-36 or in a range of 34-36. In some embodiments, subscript “n” is independently 34, 35, or 36.
  • subscript “n” is independently between 44-46 or in a range of 44-46. In some embodiments, subscript “n” is independently 44, 45, or 46.
  • subscript “c” is 1.
  • each of A 6 , A 7 , and A 8 is selected from the group consisting of
  • a 6 and A 8 are each
  • a 6 and A 7 are each independently
  • a 6 , A 7 , and A 8 are each independently
  • the compounds of Table 2 each have a subsen pt “n” of between 35-47. In some embodiments, the compounds of Table 2 each have a subscript “n” of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47. In some embodiments, the compounds of Table 2 each have a subscript “n” of between 35-37. In some embodiments, the compounds of Table 2 each have a subscript “n” of between 44-46.
  • the present disclosure provides lipid nanoparticle (LNP) compositions.
  • LNP lipid nanoparticle
  • the LNP compositions comprise a GalNAc-based lipid that comprises a compound described herein.
  • a GalNAc-based lipid is or comprises a compound described herein (e.g., a compound of Formulae I or II).
  • a lipid nanoparticle (LNP) composition comprises: GalNAc-based lipid that comprises a compound as described herein; a cationic lipid (also referred to as an ionizable lipid); a phospholipid (also referred to as a helper lipid); a PEG-containing lipid; and a sterol.
  • a lipid nanoparticle (LNP) composition comprises: about 0.001-5 mol% of a GalNAc-based lipid that comprises a compound as described herein, based on the total lipid presented in the LNP composition; about 35-60 mol% of a cationic lipid (also referred to as an ionizable lipid), based on the total lipid presented in the LNP composition; about 10-45 mol% of a phospholipid (also referred to as a helper lipid), based on the total lipid presented in the LNP composition; about 0.5-3 mol% of a PEG-containing lipid, based on the total lipid presented in the LNP composition; and about 15-50 mol% of a sterol, based on the total lipid presented in the LNP composition.
  • a lipid nanoparticle (LNP) composition comprises: about 0.001-5 mol% of a GalNAc-based lipid that comprises a compound as described herein, based on the total lipid presented in the LNP composition
  • a lipid nanoparticle (LNP) composition comprises: about 0.05-1 mol% of a GalNAc-based lipid that comprises a compound as described herein, based on the total lipid presented in the LNP composition;
  • SUBSTITUTE SHEET (RULE 26) about 35-55 mol% of a cationic lipid (also referred to as an ionizable lipid), based on the total lipid presented in the LNP composition; about 10-20 mol% of a phospholipid (also referred to as a helper lipid), based on the total lipid presented in the LNP composition; about 1-2.5 mol% of a PEG-containing lipid, based on the total lipid presented in the LNP composition; and about 30-45 mol% of a sterol, based on the total lipid presented in the LNP composition.
  • a cationic lipid also referred to as an ionizable lipid
  • a phospholipid also referred to as a helper lipid
  • a lipid nanoparticle (LNP) composition comprises about 0.0001, 0.0005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, or 5 mol% of a GalNAc-based lipid that comprises a compound as described herein, based on the total lipid presented in the LNP composition.
  • a lipid nanoparticle (LNP) composition comprises about 35, 40, 45, 50, 55, or 60 mol% of a cationic lipid (also referred to as an ionizable lipid), based on the total lipid presented in the LNP composition.
  • a lipid nanoparticle (LNP) composition comprises about 10, 12.5, 15, 17.5, 20, 25, 30, 35, 40, or 45 mol% of a phospholipid (also referred to as a helper lipid), based on the total lipid presented in the LNP composition.
  • a lipid nanoparticle (LNP) composition comprises about 0.5, 1, 1.25, 1.5, 1.75, 2, 2.5, or 3 mol% of a PEG-containing lipid, based on the total lipid presented in the LNP composition. In some embodiments, a lipid nanoparticle (LNP) composition comprises about 15, 20, 25, 30, 35, 40, 45, or 50 mol% of a sterol, based on the total lipid presented in the LNP composition.
  • a LNP composition comprises a cationic lipid.
  • a cationic lipid is referred to as an ionizable lipid.
  • a cationic lipid comprises an amino lipid.
  • a cationic lipid comprises one or more nitrogen atoms which are positively charged.
  • a cationic lipid includes, but is not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”); N-(2,3- dioleyloxy )propyl-N,N-N-triethylammonium chloride (“DOTMA”); N,N-distearyl- N,N- dimethylammonium bromide (“DDAB”); N-(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (“DOTAP”); l,2-Dioleyloxy-3-trimethylaminopropane chloride salt (“DOTAP.C1"); 3P-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (“DC- Choi”), N-(l-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)
  • DODAC
  • cationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMA and DOPE, available from GIBCO/BRL), and LIPOFECTAMINE (comprising DOSPA and DOPE, available from GIBCO/BRL).
  • LIPOFECTIN including DOTMA and DOPE, available from GIBCO/BRL
  • LIPOFECTAMINE comprising DOSPA and DOPE, available from GIBCO/BRL
  • a LNP composition comprises a phospholipid.
  • a phospholipid is referred to as a helper lipid.
  • a phospholipid includes, but is not limited to, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and sphingomyelin.
  • a fatty acid moiety includes but is not limited to lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alphalinolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
  • Non-natural species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated.
  • a phospholipid may be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond).
  • phospholipids include, but are not limited to, l,2-distearoyl-snglycero-3- phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerophosphocholine
  • SUBSTITUTE SHEET (RULE 26) (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycerophosphocholine (DUPC), 1-palmitoyl- 2- oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoy l-sn-glycero-3 - phosphocholine (OChemsPC), 1-hexadecyl snglycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn- g
  • a LNP composition comprises a polyethylene glycol-lipid (PEG-lipids).
  • PEG lipids include PEG conjugated to saturated or unsaturated C6-C20 alkyl chains.
  • polyethylene glycol (PEG) lipids include PEG-modified lipids such as PEG-modified phosphatidylethanolamines, PEG- modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG- modified diacylglycerols, and PEG-modified dialkylglycerols.
  • polyethylene glycol lipids include DMG-PEG, DLPE-PEGs, DMPE-PEGS, DPPC-PEGS, and DSPE-PEGs.
  • the polyethylene glycol lipid’s name is followed by XXX, intended to signify the molecular weight of the polyethylene glycol moiety.
  • the polyethylene glycol lipid is DMG-PEGXXX
  • the “XXX” representes the molegulcar weight, such as molecular weight of the polyethylene glycol moiety, e.g, DMG- PEG1000, DMG-PEG2000, DMG-PEG3000, or DMG-PEG5000.
  • a LNP composition comprises a sterol.
  • Sterols are known in the art and typically refer to compounds having a perhydrocyclopentanophenanthrene ring system with one or more OH substituents. Examples of sterols include, but are not limited to, cholesterol, campesterol, ergosterol, sitosterol, and the like.
  • the sterol is cholesterol.
  • a cholesterol is modified.
  • a cholesterol is an oxidized cholesterol.
  • a cholesterol is esterified cholesterol.
  • the sterol is a cholesterol-based lipid such as PEGylated cholesterol, DC-Choi (N,N-dimethyl-N-ethylcarboxamidochol esterol), l,4-bis(3-N- oleylamino-propyl)piperazine, or combinations thereof.
  • PEGylated cholesterol DC-Choi (N,N-dimethyl-N-ethylcarboxamidochol esterol), l,4-bis(3-N- oleylamino-propyl)piperazine, or combinations thereof.
  • Exemplary sterols include, but are not limited to, 25 -hydroxy cholesterol (25-OH), 20a-hydroxycholesterol (20a-OH), 27- hydroxy cholesterol, 6-keto-5a- hydroxy cholesterol, 7-ketocholesterol, 7(3- hydroxycholesterol, 7 a-hydroxy cholesterol, 7(3-25- dihydroxycholesterol, beta-sitosterol, stigmasterol, brassicasterol, campesterol, or combinations thereof.
  • the LNP composition further comprises at least one polynucleotide.
  • the at least one polynucleotide present in the compositions described herein can be a DNA, cDNA, and RNA of all types.
  • the at least one polynucleotide is a double stranded DNA, single-stranded DNA, complexed DNA, encapsulated DNA, naked RNA, encapsulated RNA, messenger RNA (mRNA), tRNA, short interfering RNA (siRNA), double stranded RNA (dsRNA), micro-RNA (miRNA), antisense RNA (asRNA), or combinations thereof.
  • the at least one polynucleotide can also be a DNA construct, such as expression vectors, expression vectors encoding a desired gene product, and the like.
  • the at least one polynucleotide is an mRNA.
  • the at least one polynucleotide is or encodes one or more components of a prime editing system.
  • the LNP composition further comprises one or more components of a prime editing system.
  • Prime editing system refers to compositions involved in the methods of prime editing.
  • a prime editing system may include a prime editor, e.g., a prime editor fusion protein, and one or more prime editing guide RNAs (PEgRNAs).
  • PEgRNAs prime editing guide RNAs
  • a prime editing system may further comprise additional elements.
  • a prime editing system may comprise a prime editor, a PEgRNA, and a second strand nick
  • a prime editing system may include one or more prime editors and one or more PEgRNAs.
  • a prime editing system may include (i) one or more polynucleotides encoding one or more prime editor polypeptides and (ii) one or more PEgRNAs, or one or more polynucleotides encoding one or more PEgRNAs.
  • Prime editing refers to programmable editing of a target DNA using a prime editor complexed with a PEgRNA to incorporate an intended nucleotide edit into the target DNA through target-primed DNA synthesis.
  • a target DNA may comprise a double stranded DNA molecule having two complementary strands.
  • the prime editing process may search specific targets and edit the endogeneous sequence of a target DNA, e.g., a target gene.
  • the spacer sequence of a PEgRNA comprises complementarity to a target strand of the target gene, and can anneal with the target strand.
  • the PEgRNA may form a complex with a prime editor, which may generate a nick in the target gene on the edit strand which is the complementary strand of the target strand.
  • the prime editing complex may then use a free 3’ end formed at the nick site of the edit strand to initiate DNA synthesis, where a primer binding site (PBS) of the PEgRNA complexes with the free 3’ end, and a single stranded DNA is synthesized using an editing template of the PEgRNA as a template.
  • the editing template may comprise one or more nucleotide edits compared to the endogenous target gene sequence. Accordingly, the newly-synthesized single stranded DNA also comprises the nucleotide edit(s) encoded by the editing template.
  • the intended nucleotide edit(s) are incorporated into the target gene.
  • Prime editor refers to the polypeptide or polypeptide components involved in prime editing.
  • a prime editor includes a polypeptide domain having DNA binding activity and a polypeptide domain having DNA polymerase activity.
  • the polypeptide domain having DNA binding activity is a polypeptide domain having programmable DNA binding activity.
  • the prime editor further comprises a polypeptide domain having nuclease activity.
  • the polypeptide domain having DNA binding activity comprises a nuclease domain or nuclease activity.
  • the polypeptide domain having nuclease activity comprises a nickase, or a fully active nuclease.
  • nickase refers to a nuclease capable of cleaving only one strand of a double-stranded DNA target.
  • the prime editor comprises a polypeptide domain that is an inactive nuclease.
  • the polypeptide domain having programmable DNA binding activity comprises a nucleic acid guided DNA binding domain, for example, a CRISPR-Cas protein, for example, a Cas9 nickase, a Cpfl nickase, or another CRISPR-Cas nuclease.
  • the polypeptide domain having DNA polymerase activity comprises a template-dependent DNA polymerase, for example, a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase.
  • the DNA polymerase is a reverse transcriptase.
  • the prime editor comprises additional polypeptides or polypeptide domains involved in prime editing, for example, a polypeptide domain having 5’ endonuclease activity, e.g, a 5' endogenous DNA flap endonucleases (e.g, FEN1), for helping to drive the prime editing process towards the edited product formation.
  • the prime editor further comprises an RNA-protein recruitment polypeptide, for example, a MS2 coat protein.
  • polypeptide domains of a prime editor may be fused or linked by a peptide linker to form a fusion protein.
  • a prime editor comprises one or more polypeptide domains provided in trans as separate proteins, which are capable of being associated to each other through non-peptide linkages or through aptamers or recruitment sequences.
  • a prime editor may comprise a DNA binding domain and a reverse transcriptase domain associated with each other by an RNA-protein recruitment aptamer, e.g., a MS2 aptamer, which may be linked to a PEgRNA.
  • Prime editor polypeptide components may be encoded by one or more polynucleotides in whole or in part.
  • a single polynucleotide, construct, or vector encodes the prime editor fusion protein.
  • multiple polynucleotides, constructs, or vectors each encode a polypeptide domain or portion of a domain of a prime editor, or a portion of a prime editor fusion protein.
  • a prime editor may comprise an N-terminal portion fused to an intein-N and a C-terminal portion fused to an intein-C, each of which is individually encoded by a vector.
  • a prime editor polypeptide is fused to one or more nuclear localization signals. In some embodiments, a prime editor polypeptide is fused to a polypeptide permeant domain to promote uptake by the cell. In some embodiments, the permeant domain is a peptide, a peptidomimetic, or a non-peptide carrier.
  • a prime editor polypeptide is produced in vitro or by host cells. In some embodiments, a prime editor polypeptide is prepared by in vitro synthesis. Various commercial synthetic apparatuses can be used. In some embodiments, a prime editor
  • SUBSTITUTE SHEET (RULE 26) polypeptide is isolated and purified in accordance with recombinant synthesis methods, for example, by expression in a host cell and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique.
  • PEgRNA refers to a guide polynucleotide that comprises one or more intended nucleotide edits for incorporation into the target double stranded DNA.
  • the PEgRNA associates with and directs a prime editor to incorporate the one or more intended nucleotide edits into the target gene via prime editing.
  • a PEgRNA comprises a spacer that is complementary or substantially complementary to a search target sequence on a target strand of the target gene.
  • the PEgRNA comprises a gRNA core that associates with a DNA binding domain, e.g., a CRISPR-Cas protein domain, of a prime editor.
  • a PEgRNA comprises an extension arm that comprises an editing template and a primer binding site (PBS).
  • the editing template comprises comprises one or more intended nucleotide edits to be incorporated in the target DNA by prime editing.
  • the editing template comprises substantial or partial complementarity to the editing target sequence except at the position of the intended nucleotide edits to be incorporated into the target gene.
  • the PBS comprises complementarity or substantially complementarity to a free 3’ end on the edit strand of the target gene at a nick site generated by the prime editor.
  • a PEgRNA consists of RNA.
  • a PEgRNA is a chimeric or hybrid PEgRNA that comprises an RNA portion (e.g., including the spacer and the gRNA core) and a DNA portion (e.g, the extension arm comprising the editing template that includes a strand of DNA).
  • a PEgRNA comprises a single polynucleotide molecule that comprises a spacer, a gRNA core, and an extension arm. In some embodiments, a PEgRNA comprises multiple polynucleotide molecules, for example, two polynucleotide molecules.
  • the prime editing system comprises a PEgRNA, a prime editor, and further comprises a second strand nick guide polynucleotide, e.g., a nick guide RNA (ngRNA).
  • ngRNA nick guide RNA
  • a ngRNA comprises a spacer (referred to as a ngRNA spacer or ng spacer) and a gRNA core, wherein the ng spacer comprises a region of complementarity to the edit strand, and wherein the gRNA core can interact with a Cas, e.g., Cas9, of a prime editor.
  • a Cas e.g., Cas9
  • SUBSTITUTE SHEET (RULE 26) may bind to the edit strand and direct a Cas nickase to generate a nick on the non-edit strand (or target strand).
  • a prime editing system comprises a first prime editing guide RNA (PEgRNA), a second PEgRNA, and one or more prime editors (a dual prime editing system).
  • dual prime editing involves two different PEgRNAs each complexed with a prime editor.
  • the prime editor is the same for each of the PEgRNA-prime editor complexes.
  • the prime editor is different for each of the PEgRNA-prime editor complexes.
  • the first PEgRNA and the second PEgRNA each comprises a spacer, a gRNA core, and an extension arm comprising a PBS and an editing template.
  • each of the two PEgRNAs comprises a spacer comprising a region of complementarity to a distinct search target sequence of the double stranded target DNA, wherein the two distinct search target sequences are on the two complementary strands of the double stranded target DNA.
  • the two PEgRNAs each can direct a prime editor to initiate the prime editing process on the two complementary strands of the double stranded target DNA.
  • the editing template of the first PEgRNA and the editing template of the second PEgRNA comprises a region of complementarity to each other.
  • the editing template of the first PEgRNA and the editing template of the second PEgRNA each comprises a region of complementarity to a distinct sequence of the double stranded target DNA.
  • a prime editing system comprises (i) a prime editor or one or more polynucleotides encoding the prime editor, and (ii) a PEgRNA or one or more polynucleotides encoding the PEgRNA.
  • a prime editing system comprises (i) a prime editor or one or more polynucleotides encoding the prime editor, (ii) a PEgRNA or one or more polynucleotides encoding the PEgRNA, and (iii) a ngRNA or one or more polynucleotides the ngRNA.
  • a prime editing system is a dual prime editing system that comprises (i) a prime editor or one or more polynucleotides encoding the prime editor, (ii) a first PEgRNA or one or more polynucleotides encoding the PEgRNA, and (iii) a second PEgRNA or one or more polynucleotides encoding the second PEgRNA.
  • the one or more polynucleotides encoding the prime editor, the PEgRNA, the ngRNA, the first PEgRNA, and/or the second PEgRNA is a part of, or encoded by, an expression cassette, a construct, or a vector.
  • the vector is a non-viral vector.
  • the one or more polynucleotides is operably linked to a regulatory element, e.g, a transcriptional control element, such as a promoter.
  • the polynucleotide is operably linked to multiple control elements.
  • any of a number of suitable transcription and translation control elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (e.g., U6 promoter, Hl promoter).
  • Prime editing system components may be encoded individually by separate polynucleotides, or two or more prime editing system components may be encoded by a single polynucleotide.
  • a prime editor polypeptide and one or more PEgRNA(s) or ngRNA are encoded by a single polynucleotide.
  • the polynucleotide encodes a prime editor fusion protein comprising a DNA binding domain and a DNA polymerase domain. In some embodiments, the polynucleotide encodes a DNA polymerase domain of a prime editor. In some embodiments, the polynucleotide encodes a DNA binding domain of a prime editor. In some embodiments, the polynucleotide encodes a portion of a prime editor protein, for example, aN-terminal portion of a prime editor fusion protein connected to an intein-N.
  • the polynucleotide encodes a portion of a prime editor protein, for example, a C-terminal portion of a prime editor fusion protein connected to an intein-C. In some embodiments, the polynucleotide encodes a PEgRNA. In some embodiments, the polynucleotide encodes two or more components of a prime editing composition, for example, a prime editor fusion protein and a PEgRNA.
  • a prime editing system comprises one or more polynucleotides that encode prime editor components and/or the first PEgRNA and/or the second PEgRNA. In some embodiments, a prime editing system comprises a polynucleotide encoding a fusion protein comprising a DNA binding domain and a DNA polymerase domain.
  • a prime editing system comprises (i) a polynucleotide encoding a fusion protein comprising a DNA binding domain and a DNA polymerase domain, (ii) a first PEgRNA or a polynucleotide encoding the first PEgRNA, and (iii) a second PEgRNA or a polynucleotide encoding the second PEgRNA.
  • a prime editing system comprises (i) a polynucleotide encoding a DNA binding domain of a prime editor, e.g., a Cas9 nickase, (ii) a polynucleotide encoding a DNA polymerase domain of a prime editor, e.g., a reverse transcriptase, (iii) a first PEgRNA or a polynucleotide encoding the first PEgRNA, and (iv) a second PEgRNA or a polynucleotide encoding the second PEgRNA.
  • a prime editing system comprises (i) a polynucleotide encoding a DNA binding domain of a prime editor, e.g., a Cas9 nickase, (ii) a polynucleotide encoding a DNA polymerase domain of a prime editor, e.g., a reverse transcriptase, (iii) a first
  • compositions comprising any of the compounds, compositions and/or nanoparticles described herein, and one or more pharmaceutically acceptable excipient.
  • the compounds and LNP compositions descnbed herein can be formulated into pharmaceutical compositions that further comprise a pharmaceutically acceptable earner, diluent, adjuvant or vehicle.
  • the present disclosure provides a pharmaceutical composition comprising a compound described herein, and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle.
  • the present disclosure provides a pharmaceutical composition comprising a LNP composition described herein, and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle.
  • a pharmaceutical composition comprises any of the compounds described herein, at least one polynucleotide, and one or more pharmaceutically acceptable excipients.
  • a pharmaceutical composition comprises any of the compounds described herein, one or more components of a prime editing system, and one or more pharmaceutically acceptable excipients. In some embodiments, a pharmaceutical composition comprises any of the compositions described herein and one or more pharmaceutically acceptable excipients. In some embodiments, a pharmaceutical composition comprises any of the lipid nanoparticles described herein and one or more pharmaceutically acceptable excipients.
  • composition refers to a composition formulated for pharmaceutical use.
  • the pharmaceutical composition comprises additional agents, e.g., for specific delivery, increasing half-life, or other therapeutic compounds.
  • a pharmaceutically-acceptable excipient comprises any vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g, lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g, the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body).
  • a pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to a tissue and/or subject (e.g, physiologically compatible, sterile, physiologic pH, etc.).
  • a pharmaceutically acceptable carrier may contain inert ingredients that do not unduly inhibit the biological activity of the compounds.
  • the pharmaceutically acceptable carrier may contain inert ingredients that do not unduly inhibit the biological activity of the compounds.
  • SUBSTITUTE SHEET (RULE 26) carriers should be biocompatible, e.g., non-toxic, non-inflammatory, non-immunogenic or devoid of other undesired reactions or side-effects upon the administration to a subject. Standard pharmaceutical formulation techniques can be employed.
  • the pharmaceutically acceptable carrier, adjuvant, or vehicle includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various earners used in formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof.
  • any conventional carrier medium is incompatible with the compounds described herein, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, the use of such conventional carrier medium is contemplated to be within the scope of this disclosure.
  • Some examples of materials that can serve as pharmaceutically acceptable earners include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as twin 80, phosphates, glycine, sorbic acid, or potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, or zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, methylcellulose, hydroxypropyl methylcellulose, wool fat, sugars such as lactose, glucose and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and
  • SUBSTITUTE SHEET (RULE 26) and antioxidants can also be present in the composition, according to the judgment of the formulator.
  • Formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology .
  • such preparatory methods include the step of bringing the active ingredient(s) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • compositions can additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • a pharmaceutically acceptable excipient includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • compositions described herein may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenteral includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrastemal, intrathecal, intraocular, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • compositions described herein may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions.
  • carriers commonly used include lactose and com starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried cornstarch.
  • aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents also may be added.
  • compositions described herein may be administered in the form of suppositories for rectal or vaginal administration.
  • suppositories for rectal or vaginal administration.
  • These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum or vaginal cavity to release the drug.
  • suitable non-irritating excipient include cocoa butter, polyethylene glycol or a suppository wax that is solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • compositions described herein also may be administered topically, especially when the target of treatment includes areas or organs readily accessible
  • SUBSTITUTE SHEET (RULE 26) by topical application, including diseases of the eye, skin, or lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.
  • Topical application for the lower intestinal tract can be effected in a rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches also may be used.
  • the pharmaceutical compositions described herein may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers.
  • Carriers for topical administration of the compounds or LNP compositions described herein include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
  • the pharmaceutical compositions described herein can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers.
  • Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • the pharmaceutical compositions described herein may be formulated, e.g., as micronized suspensions in isotonic, pH adjusted sterile saline or other aqueous solution, or, preferably, as solutions in isotonic, pH adjusted sterile saline or other aqueous solution, either with or without a preservative such as benzylalkonium chloride.
  • the pharmaceutical compositions described herein may be formulated in an ointment such as petrolatum.
  • the pharmaceutical compositions described herein also may be administered by nasal aerosol or inhalation.
  • compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
  • Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuiyl alcohol, polyethylene glycols and fatty acid esters of sorbitan,
  • the oral compositions also can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation also may be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid may be used in the preparation of injectables.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and gly
  • Solid compositions of a similar type also may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular w eight polyethylene glycols and the like.
  • excipients as lactose or milk sugar as well as high molecular w eight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric
  • Solid dosage forms optionally may contain opacifying agents. These solid dosage forms also can be of a composition such that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type also may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art.
  • the LNP composition may be admixed with at least one inert diluent such as sucrose, lactose or starch.
  • Such dosage forms also may comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose.
  • the dosage forms also may comprise buffering agents. They may optionally contain opacifying agents and also can be of a composition such that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
  • embedding compositions examples include polymeric substances and waxes.
  • the LNP compositions described herein are used to deliver a prime editing system, a DNA plasmid encoding prime editor components, a DNA plasmid encoding a PEgRNA and optionally an ngRNA, a DNA plasmid encoding a PEgRNA pair, a DNA plasmid encoding a prime editor fusion protein, or one or more DNA plasmids encoding one or more of a prime editor fusion protein, a PEgRNA, an ngRNA, or a PEgRNA pair.
  • the LNP compositions described herein are used to deliver an mRNA encoding prime editor components, an mRNA encoding prime editor fusion protein, a PEgRNA and optionally ngRNA, or a PEgRNA pair.
  • the LNP compositions described herein are used to deliver a prime editor protein, a prime editor fusion protein, a PEgRNA and optionally ngRNA, or a PEgRNA pair.
  • a method for delivering polynucleotides into a cell comprising: introducing into the cell at least one lipid nanoparticle comprising a GalNAc- based lipid that comprises any of the compounds described herein and one or more polynucleotides.
  • the polynucleotides are DNA or mRNA
  • a method for delivering a prime editing system into a cell comprising: introducing into the cell at least one lipid nanoparticle comprising a GalNAc- based lipid that comprises any of the compounds described herein and one or more components of a prime editing system.
  • the one or more components of a prime editing system comprises a construct encoding prime editor components.
  • the construct is a DNA plasmid encoding prime editor components.
  • the one or more components of a prime editing system comprises a construct encoding a prime editing guide RNA (PEgRNA), and optionally a nick guide RNA (ngRNA).
  • the one or more components of a prime editing system comprises a construct encoding a PEgRNA pair, wherein the PEgRNA comprises a first PEgRNA and/or a second PEgRNA.
  • the construct is a DNA plasmid encoding a prime editing guide RNA (PEgRNA), and optionally a nick guide RNA (ngRNA).
  • the construct is a DNA plasmid encoding a PEgRNA pair, wherein the PEgRNA comprises a first PEgRNA and/or a second PEgRNA.
  • the prime editor components comprise a prime editing guide RNA (PEgRNA), and optionally a nick guide RNA (ngRNA).
  • the prime editor components comprise a PEgRNA pair, wherein the PEgRNA comprises a first PEgRNA and/or a second PEgRNA.
  • the one or more components of a prime editing system comprises a construct encoding a prime editor fusion protein.
  • the construct is a DNA plasmid encoding a prime editor fusion protein.
  • the one or more components of a prime editing system comprises one or more constructs encoding one or more of a prime editor fusion protein
  • the one or more constructs are one or more DNA plasmids encoding one or more of a prime editor fusion protein, PEgRNA, ngRNA, or a PEgRNA pair.
  • the one or more components of a prime editing system comprises an mRNA encoding prime editor components. In some embodiments, the one or more components of a prime editing system comprises an mRNA encoding a prime editor fusion protein.
  • the prime editor components comprise a prime editing guide RNA (PEgRNA), and optionally a nick guide RNA (ngRNA).
  • the prime editor components comprise a PEgRNA pair, wherein the PEgRNA comprises a first PEgRNA and/or a second PEgRNA.
  • the one or more components of a prime editing system comprises a PEgRNA, and optionally an ngRNA. In some embodiments, the one or more components of a prime editing system comprises a PEgRNA pair. In some embodiments, the one or more components of a prime editing system comprises a prime editor protein, a prime editor fusion protein, a PEgRNA, a ngRNA, or a PEgRNA pair.
  • the LNP composition is formulated to deliver at least one polynucleotide or one or more components of the prime editing system to target cells.
  • target cells are or comprise liver cells, bone marrow cells, lung cells, eye cells, muscle cells, or cells in the central nervous system.
  • the first and second lipid nanoparticles are the same. In some embodiments, the first and second lipid nanoparticles are different.
  • the first and second lipid nanoparticles are introduced into the cell simultaneously. In some embodiments, the first and second lipid nanoparticles are introduced into the cell sequentially.
  • Delivery to the cells can be in vitro, via ex vivo administration, or via in vivo administration.
  • the target cells are or comprise human cells, mammalian cells, liver cells (e.g., hepatocytes), bone marrow cells (e.g., bone marrow monocytes), lung cells, eye cells, muscle cells, or cells in the central nervous system.
  • liver cells e.g., hepatocytes
  • bone marrow cells e.g., bone marrow monocytes
  • lung cells eye cells, muscle cells, or cells in the central nervous system.
  • Method of LCMS LC/MS (The gradient was 0%-60 B in 0-2.5 min, maintains 60% B in 2.5-3.0 min, and 60% B-0%B in 3,0-3.01min, 0% B in 3.01-3.5 min the flow rate was 1 ml/min.
  • Mobile phase A was 0.04% trifluoroacetic acid in water
  • mobile phase B was 0.02% trifluoroacetic acid in acetonitrile.
  • the column used for chromatography was a Luna LC- C18 50*2 mm (5 um particles). Detection methods are diode array (DAD) and evaporative light scattering (ELSD) detection as well as positive electrospray ionization. MS range was 100-2000.
  • DAD diode array
  • ELSD evaporative light scattering
  • Method of LC/MS LC/MS (The gradient was 0%-60 B in 0-2.5 min, maintains 60% B in 2.5-3.0 min, and 60% B-0%B in 3.0-3.01min, 0% B in 3.01-3.5 min the flow rate was 1 ml/min.
  • Mobile phase A was 0.04% trifluoroacetic acid in water
  • mobile phase B was 0.02% trifluoroacetic acid in acetonitrile.
  • the column used for chromatography was a Luna LC- C18 50*2 mm (5 um particles). Detection methods are diode array (DAD) and evaporative light scattering (ELSD) detection as well as positive electrospray ionization. MS range was 100-2000.
  • DAD diode array
  • ELSD evaporative light scattering
  • Method of LC/MS LC/MS (The gradient was 90% B in 0-0.4 mm, 90% B-l 00% B in 0.4-3.4 min, and maintains 100% B in 3.40-3.85 min, and maintains 90% B in 3.86-4.5 min, the flow rate was 0.8 ml/min.
  • Mobile phase A was 0.05% trifluoroacetic acid in water
  • mobile phase B was 0.05% trifluoroacetic acid in acetonitrile.
  • the column used for chromatography was a Luna LC- C18 50*2 mm (5 um particles). Detection methods are diode array (DAD) and evaporative light scattering (ELSD) detection as well as positive electrospray ionization. MS range was 100-2000.
  • DAD diode array
  • ELSD evaporative light scattering
  • SUBSTITUTE SHEET (RULE 26) 3.10 (m, 38H), 2.16 (s, 9H), 2.07(s, 9H), 2.02(s, 9H), 1.98(s, 9H), 1.71-1.46(m, 56H), 1.32- 1.27(s, 90H), 0.90(t, 6H).
  • the column used for chromatography was a Luna LC- C18 50*2 mm (5 urn particles). Detection methods are diode array (DAD) and evaporative light scattering (ELSD) detection as well as positive electrospray ionization. MS range was 100- 2000.
  • Method of LCMS LC/MS (The gradient was 0%-60 B in 0-2.5 min, maintains 60% B in 2.5-3.0 min, and 60% B-0%B in 3.0-3.01min, 0% B in 3.01-3.5 min the flow rate was 1 ml/min.
  • Mobile phase A was 0.04% trifluoroacetic acid in water
  • mobile phase B was 0.02% trifluoroacetic acid in acetonitrile.
  • the column used for chromatography was a Luna LC- C18 50*2 mm (5 um particles). Detection methods are diode array (DAD) and evaporative light

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Abstract

La présente divulgation décrit des compositions, des nanoparticules (telles que des nanoparticules lipidiques) et/ou des compositions de nanoparticules lipidiques et leurs procédés d'utilisation.
PCT/US2024/025406 2023-04-19 2024-04-19 Systèmes et formulations de distribution de nanoparticules lipidiques (lnp) Ceased WO2024220807A2 (fr)

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EP24731095.6A EP4698549A2 (fr) 2023-04-19 2024-04-19 Systèmes et formulations de distribution de nanoparticules lipidiques (lnp)
CN202480040525.XA CN121399143A (zh) 2023-04-19 2024-04-19 脂质纳米颗粒(lnp)递送系统和制剂
KR1020257038733A KR20260003750A (ko) 2023-04-19 2024-04-19 지질 나노입자 (lnp) 전달 시스템 및 제제
MX2025012462A MX2025012462A (es) 2023-04-19 2024-04-19 Sistemas y formulaciones de administracion de nanoparticulas lipidicas (lnp)
AU2024259356A AU2024259356A1 (en) 2023-04-19 2024-04-19 Lipid nanoparticle (lnp) delivery systems and formulations
IL323965A IL323965A (en) 2023-04-19 2025-10-15 Lipid nanoparticle (lnp) delivery systems and formulations

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KR20180094137A (ko) * 2009-05-05 2018-08-22 알닐람 파마슈티칼스 인코포레이티드 지질 조성물
MX342785B (es) * 2009-06-10 2016-10-12 Alnylam Pharmaceuticals Inc Formulacion mejorada de lipido.
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JP2024530647A (ja) * 2021-08-03 2024-08-23 ヴァーヴ・セラピューティクス，インコーポレーテッド 標的ｒｎａ送達のための組成物および方法

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