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WO2025034764A1 - Lipides pour l'administration d'acides nucléiques à des cellules eucaryotes - Google Patents

Lipides pour l'administration d'acides nucléiques à des cellules eucaryotes Download PDF

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Publication number
WO2025034764A1
WO2025034764A1 PCT/US2024/041143 US2024041143W WO2025034764A1 WO 2025034764 A1 WO2025034764 A1 WO 2025034764A1 US 2024041143 W US2024041143 W US 2024041143W WO 2025034764 A1 WO2025034764 A1 WO 2025034764A1
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chain
optionally substituted
bis
integer
branched
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Inventor
Arezki Boudif
Joel Jessee
Neha Nitin PARAYATH
Christopher BRASSARD
Gulilat Gebeyehu
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Life Technologies Corp
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Life Technologies Corp
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C215/00—Compounds containing amino and hydroxy groups bound to the same carbon skeleton
    • C07C215/02—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C215/04—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated
    • C07C215/06—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and acyclic
    • C07C215/14—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and acyclic the nitrogen atom of the amino group being further bound to hydrocarbon groups substituted by amino groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C215/00—Compounds containing amino and hydroxy groups bound to the same carbon skeleton
    • C07C215/02—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C215/22—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being unsaturated
    • C07C215/28—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being unsaturated and containing six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C217/42—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having etherified hydroxy groups and at least two amino groups bound to the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C219/06—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having the hydroxy groups esterified by carboxylic acids having the esterifying carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C219/08—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the hydroxy groups esterified by a carboxylic acid having the esterifying carboxyl group bound to an acyclic carbon atom of an acyclic unsaturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/22—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated the carbon skeleton being further substituted by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/26—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having more than one amino group bound to the carbon skeleton, e.g. lysine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/34—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • C07C229/36—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton containing six-membered aromatic rings with at least one amino group and one carboxyl group bound to the same carbon atom of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
    • C07C279/14—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C323/24—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/25—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/18—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D209/20—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals substituted additionally by nitrogen atoms, e.g. tryptophane
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars

Definitions

  • the present invention is in the field of molecular biology and cell and gene therapy, and more particularly relates to new compounds and methods for the introduction of payloads such as nucleic acids, ribonucleoproteins, etc. into eukaryotic cells.
  • payloads such as nucleic acids, ribonucleoproteins, etc. into eukaryotic cells.
  • BACKGROUND [0002] Transfection is the process of introducing nucleic acids into eukaryotic cells by non-viral methods. Transfection methods allow the introduction of negatively charged molecules (e.g. phosphate backbones of DNA and RNA) into cells having a negatively charged membrane.
  • the DNA-transfection reagent complex easily crosses the cell membrane, especially for lipids that have a “fusogenic” component, which enhances fusion with the lipid bilayer of the cell. [0003] With the recent advances in nucleic-acid based therapeutics, and continued need for transfection reagents with low toxicity, there is a continued need for novel ionizable lipids that can be used in vitro and in vivo.
  • SUMMARY [0004] Disclosed herein are compounds, compositions and methods that improve the efficiency of introducing macromolecules, such as nucleic acids, or small molecules (e.g, therapeutics), into cells.
  • Compounds are provided, together with compositions containing these compounds and methods for using these new compounds and compositions for delivery of payloads, (e.g. a nucleic acid or small molecule), to cells.
  • payloads e.g. a nucleic acid or small molecule
  • the compounds may be used alone for transfection, or they may be used in combination with additional reagents in transfection compositions.
  • the new compounds may be combined with one or more ionizable lipids and/or neutral lipids, with one or more cell surface ligands, with one or more fusion enhancing agents, and with one or more nuclear localization agents and one or more amphipathic peptides and any combinations thereof.
  • the resulting compositions may be complexed with one or more macromolecules (e.g, nucleic acids, such as DNA or RNA, proteins, ribonucleoproteins, and the like) and used to deliver these macromolecules into cells.
  • macromolecules e.g, nucleic acids, such as DNA or RNA, proteins, ribonucleoproteins, and the like
  • the disclosure provides a compound having Formula I: or pharmaceutically acceptable salts thereof, wherein: A1 is —(CH2)x—, or —(CH2)y—A4—(CH2)z—; A4 is selected from the group consisting of —(CH2)x—, —(CO)O—, —O(CO)—, R1 and R2 are independently of one another selected from the group consisting of H, optionally substituted C1-C30 straight-chain or branched-chain alkyl, optionally substituted C4- C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, or optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched- chain alkynyl group, or —(CH 2 ) 1-7 COOR; O)NHR— or —(CO)N(R)2— chain or branched-chain alkyl groups optionally substituted with -OR 7
  • the disclosure provides a compound having the structure of Formula I-a: or a pharmaceutically acceptable salt thereof, wherein: R1 and R2 are independently of one another selected from the group consisting of optionally substituted C1-C30 straight-chain or branched-chain alkyl, and optionally substituted C 4 -C 30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group, or —(CH2)1-7COOR; A 2 and A 3 are independently selected from the group consisting of C 1 -C 30 straight-chain or branched-chain alkyl groups optionally substituted with -OR 7 , -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, - (CH 2 ) n Het, optionally substituted cycloalkyl, or C4-
  • the disclosure provides compositions and methods for introducing a nucleic acid, protein, or peptide into a eukaryotic cell by contacting the cell with a compound of Formula I or a composition thereof, thereby introducing the nucleic acid, protein, or peptide into the cell.
  • the disclosure provides a composition comprising: (i) one or more compounds according to a compound of Formula I, and (ii) one or more of a structural lipid, an ionizable lipid, and a stabilizing agent; and (iii) optionally, a payload.
  • the disclosure provides a kit comprising a compound of Formula I, and one or more cationic lipids, and/or one or more neutral lipids, and/or one or more cell surface ligands, and/or one or more fusion agents, and/or one or more nuclear localization peptides or proteins and/or one or more amphipathic peptide.
  • the disclosure provides a kit comprising: (i) one or more compounds according to a compound of Formula I, and (ii) one or more of a structural lipid, an ionizable lipid, and a stabilizing agent; and (iii) optionally, a payload.
  • Figure 1 is a graph depicting size (d.nm) and polydispersity index of the lipid-mRNA formulations.
  • Figure 2 is a graph depicting luciferase activity (in bioluminescence flux, photons/second (p/s)) in the liver of mice following intravenous administration of lipid- mRNA formulations.
  • Figure 3 is a graph depicting the ratio of luciferase activity (in bioluminescence flux, photons/second (p/s)) in the liver by luciferase activity (in bioluminescence flux, photons/second (p/s)) in the spleen of mice following intravenous administration of lipid-mRNA formulations.
  • Figure 4 is a graph depicting luciferase activity (in bioluminescence flux, photons/second (p/s)) in the liver of mice following intravenous administration of lipid- mRNA formulations.
  • Figure 5 is a graph depicting the ratio of luciferase activity (in bioluminescence flux, photons/second (p/s)) in the liver by luciferase activity (in bioluminescence flux, photons/second (p/s)) in the spleen of mice following intravenous administration of lipid-mRNA formulations.
  • Figure 6 is a graph depicting transfection efficiency two days post- transfection.
  • Figure 7 is a graph depicting GFP expression level two days post- transfection.
  • Figure 8 is a graph depicting viable T cells two days post-transfection.
  • Figure 9 is a graph is a graph depicting size (d.nm) of the lipid-mRNA formulations.
  • Figure 10 is a graph depicting the polydispersity index (PDI) of the lipid-mRNA formulations.
  • Figure 11 is a graph depicting luciferase activity (in bioluminescence flux, photons/second (p/s)) in the liver of mice following intravenous administration of lipid- mRNA formulations.
  • Figure 12 is a graph depicting the ratio of luciferase activity (in bioluminescence flux, photons/second (p/s)) in the liver by luciferase activity (in bioluminescence flux, photons/second (p/s)) in the spleen of mice following intravenous administration of lipid-mRNA formulations.
  • Figure 13 is a graph depicting size (d.nm) and polydispersity index of the lipid-mRNA formulations.
  • Figure 14 is a graph depicting luciferase activity (in bioluminescence flux, photons/second (p/s)) in the liver of mice following intravenous administration of lipid- mRNA formulations.
  • Figure 15 is a graph depicting the ratio of luciferase activity (in bioluminescence flux, photons/second (p/s)) in the liver by luciferase activity (in bioluminescence flux, photons/second (p/s)) in the spleen of mice following intravenous administration of lipid-mRNA formulations.
  • Figure 16 is a graph showing GFP expression 48h post transfection using Compound 8 formulated with DOPE at a ratio of 1:4 in water. Adding peptide SEQ ID NO. 350 to the lipid increases the transfection efficiency as seen with increased GFP expression. Addition of a second peptide, SEQ ID NO.47 further increases the GFP expression compared to just 15-24.
  • Figure 17 is a graph showing Prestoblue fluorescence of lipid only formulation shows least toxicity while the formulations with one or two peptides show similar toxicity profiles as that of the Expi293 transfection reagent.
  • Figure 18 is a graph depicting size (d.nm) of the lipid-mRNA formulations.
  • Figure 19 is a graph depicting PDI of the lipid-mRNA formulations.
  • Figure 20 is a graph depicting luciferase activity (in bioluminescence flux, photons/second (p/s)) in the spleen of mice following intravenous administration of lipid- mRNA formulations.
  • Figure 21 is a graph depicting luciferase activity (in bioluminescence flux, photons/second (p/s)) in the lung of mice following intravenous administration of lipid- mRNA formulations.
  • the disclosure relates to ionizable lipids and lipid compositions (e.g., lipoplexes and lipid nanoparticle compositions) including the lipids provided herein.
  • the disclosure also provides methods of delivering a therapeutic and/or prophylactic to a mammalian cell, specifically delivering a therapeutic and/or prophylactic to a mammalian organ, producing a polypeptide of interest in a mammalian cell, and treating a disease or disorder in a mammal in need thereof.
  • a method of producing a polypeptide of interest in a cell involves contacting a nanoparticle composition comprising an mRNA with a mammalian cell, whereby the mRNA may be translated to produce the polypeptide of interest.
  • a method of delivering a therapeutic and/or prophylactic to a mammalian cell or organ may involve administration of a nanoparticle composition including the therapeutic and/or prophylactic to a subject, in which the administration involves contacting the cell or organ with the composition, whereby the therapeutic and/or prophylactic is delivered to the cell or organ.
  • Ionizable molecules are provided that are useful for improved methods of delivering macromolecules into eukaryotic cells.
  • the compositions and methods are effective in a wide variety of cells and provide a high efficiency of transfection. Specifically, it has been found that molecules based on a core of Formula I are useful for efficient delivery of macromolecules into cells.
  • These molecules advantageously can be used with one or more neutral lipids and additional components such as fusogenic or fusion-enhancing molecules, additional cationic/ionizable lipids, cell surface ligands, cell adhesion molecules, nuclear localization agents, and endosomal release agents, together with the payload (e.g, in a complex with the macromolecule or pharmaceutical agent, or nutrient, or the like).
  • additional components such as fusogenic or fusion-enhancing molecules, additional cationic/ionizable lipids, cell surface ligands, cell adhesion molecules, nuclear localization agents, and endosomal release agents, together with the payload (e.g, in a complex with the macromolecule or pharmaceutical agent, or nutrient, or the like).
  • the complex is easily prepared by straightforward methods and can be used on a wide variety of cells, are stable, and therefore are suitable for use in in vitro, ex vivo and in vivo applications, for example, delivery of therapeutic nucleic acids (e.g., siRNA therapeutics, mRNA vaccine preparations, and the like), osomes), delivery of pharmaceutical agents, nutrients and the like to cells, e.g, in for cosmetic, nutraceutical, or therapeutic applications.
  • therapeutic nucleic acids e.g., siRNA therapeutics, mRNA vaccine preparations, and the like
  • osomes delivery of pharmaceutical agents, nutrients and the like to cells, e.g, in for cosmetic, nutraceutical, or therapeutic applications.
  • the phrases “at least one of A, B, and C;” “one or more of A, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.”
  • use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible.
  • 0.2-5 mg is a disclosure of 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg etc. up to and including 5.0 mg.
  • Compounds are generally described herein using standard nomenclature. For a recited compound having asymmetric center(s), all of the stereoisomers of the compound and mixtures thereof are encompassed unless otherwise specified. Non- limiting examples of stereoisomers include enantiomers, diastereomers, and E or Z isomers. Where a recited compound exists in various tautomeric forms, the compound is intended to encompass all tautomeric forms.
  • C1-C6 alkyl refers to an alkyl substituent containing from 1 to 6 carbon atoms.
  • C3-C6 cycloalkyl means a saturated hydrocarbyl ring containing from 3 to 6 carbon ring atoms.
  • a prefix attached to a multiple-component substituent only applies to the first component that immediately follows the prefix.
  • the term "carbocyclylalkyl” contains two components: carbocyclyl and alkyl.
  • C 3 -C 6 carbocyclyl C 1 -C 6 alkyl refers to a C 3 -C 6 carbocyclyl appended to the parent molecular moiety through a C1-C6 alkyl group.
  • the chemical structure is -L S -M-L S ''- and M is -N(R B )S(O)-, then the chemical structure is -LS-N(RB)S(O)-LS''-.
  • a linking element in a depicted structure is a bond, then the element left to the linking element is joined directly to the element right to the linking element via a covalent bond.
  • a chemical structure is depicted as -LS-M-LS' and M is selected as bond, then the chemical structure will be -L S -L S ''-.
  • the dash(es) indicates the portion of the moiety that has the free valence(s).
  • the moiety may be either substituted or unsubstituted. If a moiety is described as being optionally substituted with up to a particular number of non-hydrogen radicals that moiety may be either unsubstituted or substituted by up to that particular number of non-hydrogen radicals or by up to the maximum number of substitutable positions on the moiety, whichever is less.
  • any heterocycle with less than three substitutable positions will be optionally substituted by up to only as many non-hydrogen radicals as the heterocycle has substitutable positions.
  • tetrazolyl which has only one substitutable position
  • a primary amino nitrogen will be optionally substituted with up to two non-hydrogen radicals
  • a secondary amino nitrogen will be optionally substituted with up to only one non-hydrogen radical.
  • alkyl or alkyl group or alkylene group refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds).
  • the alkyl group may have 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as “1 to 20” refers to each integer in the given range; e.g., “1 to 20 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although the present definition also covers the occurrence of the term “alkyl” where no numerical range is designated) which is optionally substituted.
  • the alkyl group may also be a medium size alkyl having 1 to 9 carbon atoms.
  • the alkyl group could also be a lower alkyl having 1 to 4 carbon atoms.
  • the alkyl group of the compounds may be designated as “C1-4 alkyl”, “C1-20 alkyl”, “C4-30 alkyl” or similar designations.
  • the notation “C 4-30 alkyl” means an optionally substituted linear or branched, saturated hydrocarbon including 4-30 carbon atoms.
  • an alkyl group described herein refers to both unsubstituted and substituted alkyl groups.
  • the alkyl groups in the straight or branched hydrocarbon chains described above may be substituted with hydroxyl groups.
  • the methylene groups are linked via disulfide bridges (—S—S—).
  • alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, n-hexyl, lauryl, palmityl, stearyl and the like.
  • alkenyl alone or in combination with any other term, refers to a straight-chain or branched-chain monounsaturated or polyunsaturated aliphatic hydrocarbon radical containing the specified number of carbon atoms, or where no number is specified, in one embodiment from 2-30 carbon atoms (i.e.
  • alkenyl or “alkenyl group” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one double bond, which is optionally substituted.
  • C2-14 alkenyl means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon double bond.
  • An alkenyl group may include one, two, three, four, or more carbon-carbon double bonds.
  • Cis alkenyl may include one or more double bonds.
  • a C5 alkenyl group including two double bonds may be a linoleyl group.
  • an alkenyl group described herein refers to both unsubstituted and substituted alkenyl groups.
  • the alkenyl groups in the straight or branched hydrocarbon chains described above may be substituted with hydroxyl groups.
  • alkenyl groups are linked via disulfide bridges (—S—S—).
  • alkenyl radicals include, but are not limited to, ethenyl, E- and Z-propenyl, isopropenyl, E- and Z-butenyl, E- and Z-isobutenyl, E- and Z-pentenyl, E- and Z-hexenyl, E,E-, E,Z-, Z,E- and Z,Z-hexadienyl, oleoyl, palmitoleoyl and the like.
  • alkynyl refers to a straight-chain or branched-chain hydrocarbon radical having one or more triple bonds containing the specified number of carbon atoms, or where no number is specified, in one embodiment from 2 to about 20 carbon atoms.
  • alkynyl or alkynyl group means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one carbon-carbon triple bond, which is optionally substituted.
  • C2-14 alkynyl means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon triple bond.
  • An alkynyl group may include one, two, three, four, or more carbon-carbon triple bonds.
  • C18 alkynyl may include one or more carbon-carbon triple bonds.
  • an alkynyl group described herein refers to both unsubstituted and substituted alkynyl groups.
  • Examples of alkynyl radicals include, but are not limited to, ethynyl, propynyl, propargyl, butynyl, pentynyl and the like.
  • basic heterocycle refers to a stable optionally substituted 5-7 membered monocyclic heterocyclic ring or an optionally substituted 8-11 membered bicyclic heterocyclic ring which is either saturated or partially unsaturated, and which may be optionally benzofused if monocyclic and which is optionally substituted on one or more carbon atoms by halogen, alkyl, alkoxy, oxo, and the like, and/or on a secondary nitrogen atom (i.e., —NH—) by alkyl, aralkoxycarbonyl, alkanoyl, phenyl or phenylalkyl or on a tertiary nitrogen atom (i.e., +N—) by oxido and which is attached via a carbon atom.
  • a secondary nitrogen atom i.e., —NH—
  • heterocycle or “heterocyclo” or “heterocyclyl” refers to a saturated (e.g., “heterocycloalkyl"), partially unsaturated (e.g., “heterocycloalkenyl” or “heterocycloalkynyl”) or completely unsaturated (e.g., “heteroaryl”) ring system where at least one of the ring atoms is a heteroatom (i.e., nitrogen, oxygen or sulfur), with the remaining ring atoms being independently selected from the group consisting of carbon, nitrogen, oxygen and sulfur.
  • a heterocycle may be, without limitation, a single ring, two fused rings, or bridged or spiro rings.
  • a heterocycle group can be linked to the parent molecular moiety via any substitutable carbon or nitrogen atom(s) in the group.
  • a heterocycle group is a divalent moiety that links two other elements in a depicted chemical structure
  • the heterocycle group can be attached to the two other elements through any two substitutable ring atoms.
  • a heterocycle group is a trivalent moiety that links three other elements in a depicted chemical structure
  • the heterocycle group can be attached to the three other elements through any three substitutable ring atoms, respectively.
  • “Het” indicates a heterocycle containing 4- 12 carbon atom, where at least one nitrogen atom is present in the ring(s).
  • a heterocyclyl may be, without limitation, a monocycle which contains a single ring.
  • monocycles include furanyl, dihydrofuranyl, tetrahydrofuranyl, pyrrolyl, isopyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, isoimidazolyl, imidazolinyl, imidazolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, triazolyl, tetrazolyl, dithiolyl, oxathiolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, thiazolinyl, isothiazolinyl, thiazolidinyl, isothiazolidinyl, thiodiazolyl, oxathiazolyl, oxadiazolyl (including 1,2,3-oxadiazolyl
  • a heterocyclyl may also be, without limitation, a bicycle containing two fused rings, such as, for example, naphthyridinyl (including [1,8]naphthyridinyl, and [1,6]naphthyridinyl), thiazolpyrimidinyl, thienopyrimidinyl, pyrimidopyrimidinyl, pyridopyrimidinyl, pyrazolopyrimidinyl, indolizinyl, pyrindinyl, pyranopyrrolyl, 4H- quinolizinyl, purinyl, pyridopyridinyl (including pyrido[3,4-b]-pyridinyl, pyrido[3,2-b]- pyridinyl, and pyrido[4,3-b]-pyridinyl), pyridopyrimidine, and pteridinyl.
  • fused-ring heterocycles include benzo-fused heterocyclyls, such as indolyl, isoindolyl, indoleninyl (also known as “pseudoindolyl”), isoindazolyl (also known as “benzpyrazolyl” or indazolyl), benzazinyl (including quinolinyl (also known as “1- benzazinyl”) and isoquinolinyl (also known as “2-benzazinyl”)), benzimidazolyl, phthalazinyl, quinoxalinyl, benzodiazinyl (including cinnolinyl (also known as “1,2- benzodiazinyl”) and quinazolinyl (also known as "1,3-benzodiazinyl”)), benzothiazolyl, 4,5,6,7-tetrahydrobenzo[d]thiazolyl, benzothiadiazolyl, benzimidazolyl,
  • a heterocyclyl may also be, without limitation, a spiro ring system, such as, for example, 1,4-dioxa-8-azaspiro[4.5]decanyl.
  • a heterocyclyl may comprise one or more sulfur atoms as ring members; and in some cases, the sulfur atom(s) is oxidized to SO or SO2.
  • the nitrogen heteroatom(s) in a heterocyclyl may or may not be quaternized and may or may not be oxidized to N-oxide. In addition, the nitrogen heteroatom(s) may or may not be N-protected.
  • a heterocycle or carbocycle may be further substituted.
  • substituted refers to substitution by independent replacement of one, two, or three or more of the hydrogen atoms with substituents including, but not limited to, -F, -Cl, -Br, -I, hydroxy, protected hydroxy, -NO 2 , -N 3 , -CN, -NH 2 , protected amino, oxo, thioxo, - NH-C2-C8-alkenyl, -NH-C2-C8-alkynyl, -NH-C3-C12-cycloalkyl, -NH-aryl, -NH-heteroaryl, - NH-heterocycloalkyl, -dialkylamino, -diarylamino, -diheteroarylamino, -O-C 1 -C 12 -alkyl, -O- C2-C8-alkenyl, alkynyl, -O-C3
  • N-protecting group or “N-protected” refers to those groups capable of protecting an amino group against undesirable reactions. Commonly used N- protecting groups are described in Greene and Wuts, Protecting Groups in Chemical Synthesis (3 rd ed., John Wiley & Sons, NY (1999)).
  • Non-limiting examples of N-protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, benzoyl, 4- chlorobenzoyl, 4-bromobenzoyl, or 4-nitrobenzoyl; sulfonyl groups such as benzenesulfonyl or p-toluenesulfonyl; sulfenyl groups such as phenylsulfenyl (phenyl-S-) or triphenylmethylsulfenyl (trityl-S-); sulfinyl groups such as p-methylphenylsulfinyl (p- methylphenyl-S(O)-)
  • N-protecting groups include formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc) and benzyloxycarbonyl (Cbz).
  • halogen means fluorine, chlorine, bromine, or iodine.
  • a “natural amino acid side chain” refers to the side- chain substituent of a naturally occurring amino acid. Naturally occurring amino acids have a substituent attached to the ⁇ –carbon.
  • Naturally occurring amino acids include Arginine, Lysine, Aspartic acid, Glutamic acid, Glutamine, Asparagine, Histidine, Serine, Threonine, Tyrosine, Cysteine, Methionine, Tryptophan, Alanine, Isoleucine, Leucine, Phenylalanine, Valine, Proline, and Glycine.
  • a “non-natural amino acid side chain” refers to the side- chain substituent of a non-naturally occurring amino acid.
  • Non-natural amino acids include ⁇ -amino acids ( ⁇ 3 and ⁇ 2 ), Homo-amino acids, Proline and Pyruvic acid derivatives, 3- substituted Alanine derivatives, Pyrrolysine, Glycine derivatives, Ring-substituted Phenylalanine and Tyrosine Derivatives, Linear core amino acids and N-methyl amino acids.
  • Exemplary non-natural amino acids are available from Sigma-Aldrich, listed under “unnatural amino acids & derivatives.” See also, Travis S. Young and Peter G. Schultz, “Beyond the Canonical 20 Amino Acids: Expanding the Genetic Lexicon,” J. Biol. Chem. 2010285: 11039-11044, which is incorporated by reference in its entirety.
  • polypeptide refers to a polymer of amino acid residues, wherein the polymer may in embodiments be conjugated to a moiety that does not consist of amino acids.
  • the terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • a “fusion protein” refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed or chemically synthesized as a single moiety.
  • alkoxy refers to an alkyl, alkenyl or alkynyl ether radical, wherein the terms “alkyl”, “alkenyl” or “alkynyl” are defined above.
  • suitable alkyl ether radicals include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, oleyloxy, palmityloxy, palmitoleoyloxy and the like.
  • aryl refers to a carbocyclic aromatic radical (such as phenyl or naphthyl) containing the specified number of carbon atoms, in one embodiment from 6-15 carbon atoms (i.e. (C 6-15 )aryl), and in another embodiment from 6-10 carbon atoms (i.e. (C 6-10 )aryl), optionally substituted with one or more substituents selected from alkyl, alkoxy, (for example methoxy), nitro, halogen, (for example chloro), amino, carboxylate and hydroxy.
  • aryl refers to a carbocyclic aromatic radical (such as phenyl or naphthyl) containing the specified number of carbon atoms, in one embodiment from 6-15 carbon atoms (i.e. (C 6-15 )aryl), and in another embodiment from 6-10 carbon atoms (i.e. (C 6-10 )aryl), optionally substituted with one or more substituents selected from alkyl, alkoxy, (for example meth
  • aryl radicals include, but are not limited to phenyl, p-tolyl, 4-hydroxyphenyl, 1-naphthyl, 2-naphthyl, indenyl, indanyl, azulenyl, fluorenyl, anthracenyl and the like.
  • aralkyl or “arylalkyl” is an aryl group connected, as a substituent, via an alkylene group, such “C7-14 aralkyl” and the like, including but not limited to benzyl, 2-phenylethyl, 3-phenylpropyl, and naphthylalkyl.
  • the alkylene group is a lower alkylene group (i.e., a C1-4 alkylene group).
  • aralkyl or arylalkyl alone or in combination, means an alkyl radical as defined above in which one hydrogen atom is phenyl, benzyl, 2-phenylethyl and the like.
  • aralkoxycarbonyl alone or in combination, means a radical of the formula -C(O)-O-aralkyl in which the term “aralkyl” has the significance given above.
  • An example of an aralkoxycarbonyl radical is benzyloxycarbonyl.
  • aryloxy alone or in combination, means a radical of the formula aryl-O- in which the term “aryl” has the significance given above.
  • alkanoyl alone or in combination, means an acyl radical derived from an alkanecarboxylic acid, examples of which include acetyl, propionyl, butyryl, valeryl, 4-methylvaleryl, and the like.
  • aryloxyalkanoyl means an acyl radical of the formula aryl- O-alkanoyl wherein aryl and alkanoyl have the significance given above.
  • aralkanoyl means an acyl radical derived from an aryl- substituted alkanecarboxylic acid such as phenylacetyl, 3-phenylpropionyl (hydrocinnamoyl), 4-phenylbutyryl, (2-naphthyl)acetyl, 4-chlorohydrocinnamoyl, 4- aminohydrocinnamoyl, 4-phenylbutyryl, (1-naphthyl)acetyl, 4-chlorohydrocinnamoyl, 4- aminohydrocinnamoyl, 4-methoxyhydrocinnamoyl, and the like.
  • aroyl means an acyl radical derived from an aromatic carboxylic acid.
  • aromatic carboxylic acids an optionally substituted benzoic or naphthoic acid such as benzoyl, 4-chlorobenzoyl, 4-carboxybenzoyl, 4-benzyloxycarbonyl)benzoyl, 1-naphthoyl, 2-naphthoyl, 6-carboxy-2-naphthoyl, 6- (benzyloxycarbonyl)-2-naphthoyl, 3-benzyloxy-2-naphthoyl, 3-hydroxy-2-naphthoyl, 3- (benzyloxyformamido)-2-naphthoyl, and the like.
  • aminocarbonyl alone or in combination, means an amino- substituted carbonyl (carbamoyl) group derived from an amino-substituted carboxylic acid wherein the amino group can be a primary, secondary or tertiary amino group continuing substituents selected from hydrogen, alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl radicals and the like.
  • aminoalkanoyl means an acyl radical derived from an amino substituted alkanecarboxylic acid wherein the amino group can be a primary, secondary or tertiary amino group containing substituents selected from hydrogen, cycloalkyl, cycloalkylalkyl radicals and the like, examples of which include N,N- dimethylaminoacetyl and N-benzylaminoacetyl.
  • carbocycle refers to a non-aromatic stable 3- to 8- membered carbon ring which may be saturated, mono-unsaturated or poly-unsaturated.
  • the carbocycle may be attached at any endocyclic carbon atom which results in a stable structure.
  • Carbocycles in one embodiment have 5-7 carbons.
  • cycloalkyl alone or in combination, means an alkyl radical which contains from about 3 to about 8 carbon atoms and is cyclic. Examples of such cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
  • cycloalkylalkyl means an alkyl radical as defined above which is substituted by a cycloalkyl radical containing from about 3 to about 8, in one embodiment from about 3 to about 6, carbon atoms.
  • cycloalkylcarbonyl means an acyl group derived from a monocyclic or bridged cycloalkanecarboxylic acid such as cyclopropanecarbonyl, cyclohexanecarbonyl, adamantanecarbonyl, and the like, or from a benz-fused monocyclic cycloalkanecarboxylic acid which is optionally substituted by, for example, alkanoylamino, such as 1,2,3,4-tetrahydro-2-naphthoyl, 2-acetamido-1,2,3,4-tetrahydro-2-naphthoyl.
  • cycloalkylalkoxycarbonyl means an acyl group derived from a cycloalkylalkoxycarboxylic acid of the formula cycloalkylalkyl-O-COOH wherein cycloalkylalkyl has the significance given above.
  • alkylation reaction may comprise a nucleophilic attack of an alkylation reactive center of a target compound to an electron deficient region of an alkylating agent according to an SN 1 or SN 2 mechanism, as known in the art (see, e.g., Jerry March, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure.293-871 (4 th ed. John Wiley & Sons 1992)).
  • an alkylating agent further comprises one or more leaving groups (LGs).
  • LGs leaving groups
  • a leaving group X in Scheme IV
  • grammatical equivalents herein are meant an atom or molecule that detaches from a molecule, e.g., an organic molecule.
  • the residual part can be the alkyl group which becomes covalently bonded to a target compound.
  • a leaving group can be an atom or group, charged or uncharged, that becomes detached from an atom or molecule in what is considered the residual or main part of the substrate in a specified reaction.
  • a leaving group can affect the intrinsic reactivity of the alkylating agent in an alkylation reaction.
  • the lower the pKa of the conjugate acid of the leaving group the better the leaving group, because, in some embodiments, the leaving group can more easily stabilize the developing negative charge that can occur in an alkylation reaction. Therefore, in some embodiments, a leaving group can be an electronegative atom or molecule.
  • Examples of leaving groups include, but are not limited to, acetate (AcO ⁇ ), p-nitrobenzoate (PNBO ⁇ ), sulfonates (e.g., methanesulfonate (Mesylate: MsO ⁇ ), p-toluenesulfonate (tosylate: TsO ⁇ ), p-bromobenzenesulfonate (Brosylate: BsO ⁇ ), p- nitrobenzenesulfonate (Nosylate: NsO ⁇ ), fluoromethanesulfonate, difluoromethanesulfonate, trifluoromethanesulfonate (Triflate: TfO ⁇ ) and ethanesulfonate), halide esters, and halogen ions (e.g., I ⁇ , Br ⁇ , Cl ⁇ ).
  • sulfonates e.g.,
  • solid-phase bound arylalkyl chloride comprises an arylalkyl chloride covalently linked to a polystyrene based resin.
  • polystyrene based resin can have various reactive functionalities, for example a chloride as a leaving group or it can have other leaving groups as discussed above. Since the pioneering work of Merrifield (Merrifield, R. B. (1963), J. Am. Chem. Soc., 85,2149- 2153) on polystyrene (2% divinylbenzene cross-linked) as solid support for peptide synthesis, several improvements on the nature of the solid support were brought about to meet special needs of new organic chemistry.
  • compositions may also include salts of one or more compounds, e.g., compounds of Formula I.
  • Salts may be pharmaceutically acceptable salts.
  • pharmaceutically acceptable salts refers to derivatives of the disclosed compounds wherein the parent compound is altered by converting an existing acid or base moiety to its salt form (e.g., by reacting a free base group with a suitable organic acid).
  • examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pam
  • alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, ethanolamine, trimethylamine, diethylamine, triethylamine, ethylamine, isopropylamine, and the like.
  • the pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • the pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.
  • such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
  • Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p.1418, Pharmaceutical Salts: Properties, Selection, and Use, P. H. Stahl and C. G.
  • surface ligand or “cell surface ligand” refers to a chemical compound or structure which will bind to a surface receptor of a cell.
  • cell surface receptor refers to a specific chemical grouping on the surface of a cell to which the ligand can attach. Cell surface receptors can be specific for a particular cell, i.e., found predominantly in one cell rather than in another type of cell (e.g., LDL and asialoglycoprotein receptors are specific for hepatocytes).
  • a cell surface receptor includes but is not limited to a folate receptor, biotin receptor, lipoic acid receptor, low-density lipoprotein receptor, asialoglycoprotein receptor, insulin-like growth factor type II/cation-independent mannose-6-phosphate receptor, calcitonin gene-related peptide receptor, insulin-like growth factor I receptor, nicotinic acetylcholine receptor, hepatocyte growth factor receptor, endothelin receptor, bile acid receptor, bone morphogenetic protein receptor, cartilage induction factor receptor or glycosylphosphatidylinositol (GPI)-anchored proteins (e.g., ⁇ - andrenargic receptor, T-cell activating protein, Thy-1 protein, GPI-anchored 5’ nucleotidase).
  • GPI glycosylphosphatidylinositol
  • a receptor is a molecule to which a ligand binds specifically and with relatively high affinity. It is usually a protein or a glycoprotein, but may also be a glycolipid, a lipidpolysaccharide, a glycosaminoglycan or a glycocalyx.
  • epitopes to which an antibody or its fragments binds is construed as a receptor since the antigen:antibody complex undergoes endocytosis.
  • surface ligand includes anything which is capable of entering the cell through cytosis (e.g. endocytosis, potocytosis, pinocytosis).
  • ligand refers to a chemical compound or structure which will bind to a receptor. This includes but is not limited to ligands such as asialoorosomucoid, asialoglycoprotein, lipoic acid, biotin, apolipoprotein E sequence, insulin-like growth factor II, calcitonin gene-related peptide, thymopoietin, hepatocyte growth factor, endothelin-1, atrial natriuretic factor, RGD-containing cell adhesion peptides and the like. [0089] One skilled in the art will readily recognize that the ligand chosen will depend on which receptor is being bound.
  • nuclear localization agent refers to a ligand, such as a peptide, which will cause an agent covalently or non-covalently linked to it to localize at the cell nucleus, typically by binding a nuclear receptor.
  • nuclear receptor refers to a chemical grouping on the nuclear membrane which will bind a specific ligand and help transport the ligand, and accompanying linked moieties, through the nuclear membrane.
  • Nuclear receptors can be but are not limited to those receptors which bind nuclear localization sequences.
  • Nonlimiting examples of nuclear ligands include GYSTPPKKKRKVEDP (SEQ ID NO:1), GYSTPPKTRRRP (SEQ ID NO:2), GYSTPGRKKR (SEQ ID NO:3), GYSTPRRNRRRRW (SEQ ID NO:4), PDEVKRKKKPPTSYG (SEQ ID NO:5), PRRRTKPPTSYG (SEQ ID NO:6), RKKRGPTSYG (SEQ ID NO:7), WRRRRNRRPTSYG (SEQ ID NO:8), and GYGPPKKKRKVEAPYKA(K)8-40K (SEQ ID NO:9), may be used to transport nucleic acid to the nucleus.
  • GYSTPPKKKRKVEDP SEQ ID NO:1
  • GYSTPPKTRRRP SEQ ID NO:2
  • GYSTPGRKKR SEQ ID NO:3
  • GYSTPRRNRRRRW SEQ ID NO:4
  • lysis agent refers to a molecule, compound, protein or peptide which is capable of breaking down an endosomal membrane and freeing the DNA transporter into the cytoplasm of the cell. This term includes but is not limited to viruses, synthetic compounds, lytic peptides, or derivatives thereof.
  • lytic peptide refers to a chemical grouping which penetrates a membrane such that the structural organization and integrity of the membrane is lost. As a result of the presence of the lysis agent, the membrane undergoes lysis, fusion or both. Examples of lysis agents/endosomal release agents include choroquine, polyamines and polyamidoamines.
  • polycationic nucleic acid binding moiety refers to a moiety containing multiple positive charges at physiological pH that allow the moiety to bind a negatively charged nucleic acid.
  • a polycationic nucleic acid binding moiety may be linked to, for example, a cell surface ligand, a fusion agent, and/or a nuclear localization peptide. The linkage may be covalent.
  • Suitable polycationic nucleic acid binding moieties include polyamines such as PEI, spermine, spermidine, carboxyspermine and polybasic peptides containing, for example, multiple lysine, ornithine, histidine, or arginine residues.
  • Nucleic acid refers to deoxyribonucleotides or ribonucleotides and mixtures and polymers thereof in single- or double-stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as a reference nucleic acid, and which are metabolized in a manner similar to a reference nucleotides.
  • Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), 5 methyl cytosine, pseudouridine, N 1 -methyl-pseudouridine, 5 methoxyuridine, and the like.
  • PNAs peptide-nucleic acids
  • the payloads described herein can include mRNAs modified with one or more nucleotides selected from the group consisting of: pseudouridine (abbreviated by the Greek letter “psi” or “ ⁇ ”), 5-methylcytosine (m 5 C), 5- methyluridine (m 5 U), 2′-O-methyluridine (Um or m 2′-O U), 2-thiouridine (s 2 U), and N 6 - methyladenosine (m 6 A)) in place of at least a portion of the corresponding unmodified canonical nucleoside (e.g., in place of substantially all of the corresponding unmodified A, C, G, or T canonical nucleoside.
  • pseudouridine abbreviated by the Greek letter “psi” or “ ⁇ ”
  • the payloads described herein can include ribonucleoprotein complexes (e.g., Cas9/guide RNA) which are delivered into a cell at high efficiencies.
  • ribonucleoprotein complex or “ribonucleoprotein particle” as provided herein refers to a complex or particle including a nucleoprotein and a ribonucleic acid.
  • guide RNA or “gRNA” as provided herein refers to a ribonucleotide sequence capable of binding a nucleoprotein, thereby forming ribonucleoprotein complex.
  • the guide RNA includes one or more RNA molecules.
  • nucleoprotein refers to a protein capable of binding a nucleic acid (e.g., RNA, DNA). Where the nucleoprotein binds a ribonucleic acid, it is referred to as “ribonucleoprotein.”
  • the interaction between the ribonucleoprotein and the ribonucleic acid may be direct, e.g., by covalent bond, or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g.
  • ribonucleoproteins include ribosomes, telomerase, RNAseP, hnRNP, CRISPR associated protein 9 (Cas9) and small nuclear RNPs (snRNPs).
  • the ribonucleoprotein may be an enzyme.
  • the ribonucleoprotein is an endonuclease.
  • the ribonucleoprotein complex includes an endonuclease and a ribonucleic acid.
  • the endonuclease is a CRISPR associated protein 9.
  • Nucleic acids may be in the form of an antisense molecule, for example a “gap-mer” containing an RNA-DNA-RNA structure that activates RNAseH.
  • the nucleic acid can be, for example, DNA or RNA, or RNA-DNA hybrid, and can be an oligonucleotide, plasmid, parts of a plasmid DNA, pre-condensed DNA, product of a polymerase chain reaction (PCR), vectors, expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups or other form of nucleic acid molecule.
  • the nucleic acid may be a double-stranded RNA molecule of the type used for inhibiting gene expression by RNA interference.
  • the nucleic acid may be a short interfering double stranded RNA molecule (siRNA).
  • the nucleic acid molecule can also be a Stealth TM RNAi molecule (Invitrogen Corporation/Life Technologies Corporation, Carlsbad, CA).
  • an “RNA” refers to a ribonucleic acid that may be naturally or non-naturally occurring.
  • an RNA may include modified and/or non- naturally occurring components such as one or more nucleobases, nucleosides, nucleotides, or linkers.
  • An RNA may include a cap structure, a chain terminating nucleoside, a stem loop, a polyA sequence, and/or a polyadenylation signal.
  • RNA may have a nucleotide sequence encoding a polypeptide of interest.
  • an RNA may be a messenger RNA (mRNA).
  • mRNA messenger RNA
  • RNAs may be selected from the non-limiting group consisting of small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, single-guide RNA (sgRNA), self-replicating RNA (srRNA), self-amplifying RNA, stRNA, cas9 mRNA, or combinations thereof.
  • siRNA small interfering RNA
  • aiRNA asymmetrical interfering RNA
  • miRNA microRNA
  • dsRNA Dicer-substrate RNA
  • shRNA small hairpin RNA
  • mRNA single-guide RNA
  • srRNA self-replicating RNA
  • self-amplifying RNA stRNA, cas9 mRNA, or combinations thereof.
  • amphipathic peptide refers to a peptide whose secondary structure places hydrophobic and hydrophilic amino acid residue
  • amphipathic peptide may also function as a fusion agent.
  • amphipathic peptides suitable for use in the compositions described herein include, but are not limited to, peptides comprising a sequence selected from the group consisting of FEAALAEALAEALA (SEQ ID No.: 10), Ac-LARLLPRLLARL-NHCH3 (SEQ ID No.: 11), GLLEELLELLEELWEELLEG (SEQ ID No.: 12), GWEGLIEGIEGGWEGLIEG(SEQ ID No.: 13), GLFEALAEFIEGGWEGLIEG (SEQ ID No.: 14), GLFEALLELLESLWELLLEA (SEQ ID No.: 15), GGYCLEKWMIVASELKCFGNTA (SEQ ID No.: 16), GGYCLTRWMLIEAELKCFGNTAV (SEQ ID No.: 17), and WEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID No.: 18).
  • the amphipathic peptide may optionally be linked to a polycationic nucleic acid binding moiety, for example via a covalent linkage.
  • Exosomes, exosome lipids [0099] The term “exosome” refers to the small membrane vesicles secreted by most cells that contain cell specific payloads of proteins, lipids and, genetic material and other biomolecules that are transported to other cells in different location of the tissue. Exosomes can be considered liposomal particles. Exosomes or lipid mixtures obtained therefrom, can be used in combination with other transfection agents or helper lipid mixtures.
  • Exosomes are also referred to as microvesicles, epididimosomes, argosomes, exosome-like vesicles, microparticles, promininosomes, prostasomes, dexosomes, texosomes, archeosomes and oncosomes.
  • Exosomes useful in the compositions and methods described herein also include synthetic exosomes.
  • Non-limiting examples of synthetic exosomes useful in the embodiments described herein are described, e.g., in Li, YJ., Wu, JY., Liu, J. et al. Artificial exosomes for translational nanomedicine. J Nanobiotechnol 19, 242 (2021), US Patent No. 11938219, US Patent Application Publication No.
  • lipid compositions can also be combined with one or more exosomes, or biological materials (e.g., lipids, proteins, nucleic acids, or the like) derived or purified from exosomes.
  • Structural Lipids [0103] The lipid component of a lipid nanoparticle composition may include one or more structural lipids.
  • Structural lipids can be selected from the group consisting of, but are not limited to a sterol, e.g., cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof.
  • the structural lipid is cholesterol.
  • the structural lipid includes a sterol, (e.g., cholesterol) and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.
  • the lipid compositions provided herein can also include a stabilizing agent, such as a stabilizing lipid.
  • Stabilizing lipids can be neutral lipids, or they can be charged.
  • Stabilizing lipids that can advantageously be used in the formulations provided herein include, but are not limited to, polyethylene glycol (PEG)-modified lipids.
  • PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG- ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines.
  • lipids are also referred to as PEGylated lipids.
  • a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • Other stabilizing lipids useful in the compositions disclosed herein include, e.g., polyglycol lipids, polyoxyethylene alkyl ethers, diblock polyoxyethylene ether co-polymers, triblock polyoxyethylene alkyl ethers co-polymers, and amphiphilic branched polymers.
  • the stabilizing agent is present at about 0.1 - 5 mol% of the lipid composition.
  • the stabilizing agent is present at about 0.5 mol%, 1 mol%, 1.5 mol%, 2 mol%, 2.5 mol %, 3 mol%, 3.5 mol %, 4 mol %, 4.5 mol%, 5 mol%, or any value in between, of the lipid component of the compositions provided herein.
  • the stabilizing agent is present at about 0.5 mol% to about 5 mol% of the lipid component of the compositions provided herein.
  • the stabilizing agent is present at about 0.5 mol% to about 4 mol% of the lipid component of the compositions provided herein.
  • the stabilizing agent is present at about 0.5 mol% to about 3 mol% of the lipid component of the compositions provided herein. In other examples, the stabilizing agent is present at about 0.5 mol% to about 2 mol% of the lipid component of the compositions provided herein. In other examples, the stabilizing agent is present at about 0.5 mol% to about 1 mol% of the lipid component of the compositions provided herein. In other examples, the stabilizing agent is present at about 1 mol% to about 5 mol% of the lipid component of the compositions provided herein. In other examples, the stabilizing agent is present at about 1 mol% to about 4 mol% of the lipid component of the compositions provided herein.
  • the stabilizing agent is present at about 1 mol% to about 3 mol% of the lipid component of the compositions. In other examples, the stabilizing agent is present at about 1 mol% to about 2 mol% of the lipid component of the compositions provided herein.
  • Complexation of Payloads [0105] The disclosure herein provided compositions for the delivery of payloads, including but not limited to nucleic acids, to cells. Nucleic acids can be complexed to the exterior of the lipid complexes provided herein (e.g., liposomes, lipid nanoparticles). In some embodiments, the compositions have from about 20% to about 50% of the nucleic acid complexed to the exterior of the lipid complex.
  • the compositions have about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% of the nucleic acid complexed to the exterior of the lipid complex. Exterior complexation of a nucleic acid can be measured by methods know in the art, such as in Blakney et al. (2019) Gene Therapy 26:363-372.
  • Compounds of Formula I [0106] It has been found that compounds based on a core structure of Formula I are useful for the efficient delivery of macromolecules into eukaryotic cells. The compositions and methods are effective in a wide variety of cells and provide a high efficiency of transfection.
  • the disclosure provides a compound having Formula I: or pharmaceutically acceptable salts thereof, wherein: A1 is —(CH2)x—, or —(CH2)y—A4—(CH2)z—; A 4 is selected from the group consisting of —(CH 2 ) x —, —(CO)O—, —O(CO)—,
  • R1 and R2 are independently of one another selected from the group consisting of H, optionally substituted C 1 -C 30 straight-chain or branched-chain alkyl, optionally substituted C 4 - C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, or optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched- chain alkynyl group, or —(CH 2 ) 1-7 COOR;
  • R3 is —COR, —(CH2)nCOR, —(CH2)nCOOR, —(CO)NHR—, —(CO)N(R)2—, C 1 -C 30 straight-chain or branched-chain alkyl groups optionally substituted with -OR 7 , -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nA
  • R9 is H
  • R 10 is selected from the group consisting of H; C1-C30 straight-chain or branched-chain alkyl groups optionally substituted with -OR7, -N(R 7 ) 2 , -SR 7 , -(CH 2 ) n OR 7 , -(CH 2 ) n SR 7 , -(CH 2 ) n N(R 7 ) 2 , -(CH 2 ) n Aryl, -(CH 2 ) n Arylalkyl, - (CH2)nHet, optionally substituted cycloalkyl; C 1 -C 30 straight-chain or branched-chain alkyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -NR7-, aryl groups; C 3 -C 6 cycloalkyl alkyl groups , optionally substituted with -OR 7 , -N(R 7 ) 2 ,
  • A1 is —(CH2)x—, or —(CH2)y—A4—(CH2)z—;
  • a 4 is selected from the group consisting of —(CH 2 ) x —,—(CO)O—, —O(CO)—, Q1 is N;
  • Q2 is N;
  • R 1 and R 2 are independently of one another selected from the group consisting of optionally substituted C 1 -C 30 straight-chain or branched-chain alkyl, optionally substituted C 4 - C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, or optionally substituted C 4 -C 30 monounsaturated or polyunsaturated straight-chain or branched- chain alkynyl group;
  • R 3 is —COR, —(CH 2 ) n COR, —(CH 2 ) n COOR, —(CO)NHR—, —(CO)N(R) 2 —
  • the disclosure provides a compound having the structure of Formula I-a: or a pharmaceutically acceptable salt thereof, wherein: R1 and R2 are independently of one another selected from the group consisting of optionally substituted C1-C30 straight-chain or branched-chain alkyl, and optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group, or —(CH 2 ) 1-7 COOR; A 2 and A 3 are independently selected from the group consisting of C 1 -C 30 straight-chain or branched-chain alkyl groups optionally substituted with -OR 7 , -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, - (CH 2 ) n Het, optionally substituted cycloalkyl, or C4
  • R 3 is —COR or —(CH2)nCOOR.
  • R is selected from the group consisting of oleyl, oleoyl, linoleyl, linoleoyl, palmitoleyl, palmitoleoyl, palmityl, palmitoyl, myristyl, myristoyl, lauryl and lauroyl group.
  • n5 is 1 or n7 is 1 or n9 is 1.
  • x is 4.
  • the disclosure provides a compound having the structure of Formula I-b: or a pharmaceutically acceptable salt thereof, wherein: R1 and R2 are independently of one another selected from the group consisting of optionally substituted C1-C30 straight-chain or branched-chain alkyl, and optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; R3 is —COR, —(CH2)nCOR, —(CH2)nCOOR, —(CO)NHR—, —(CO)N(R)2—, A 2 and A 3 are independently selected from the group consisting of R 4 is H or optionally substituted C 1 -C 20 straight-chain or branched-chain alkyl, optionally substituted C1-C20 monounsaturated or polyunsaturated straight-chain or branched- chain alkenyl; R
  • R3 is —COR or —(CH 2 ) n COOR.
  • R is selected from the group consisting of oleyl, oleoyl, linoleyl, linoleoyl, palmitoleyl, palmitoleoyl, palmityl, palmitoyl, myristyl, myristoyl, lauryl and lauroyl group.
  • n5 is 1 or m2 is 2 or n9 is 1.
  • y and z are independently an integer from 1 to 2.
  • the disclosure provides a compound having the structure of Formula I-c: or a pharmaceutically acceptable salt thereof, wherein: R 1 and R 2 are independently of one another selected from the group consisting of H, optionally substituted C1-C30 straight-chain or branched-chain alkyl, and optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; R3 is —COR, —(CH2)nCOR, —(CH2)nCOOR, —(CO)NHR—, —(CO)N(R)2—.
  • a 4 is selected from the group consisting of —(CO)O—, —O(CO)—, —S-S—, R4 is H or optionally substituted C1-C20 straight-chain or branched-chain alkyl, optionally substituted C 1 -C 20 monounsaturated or polyunsaturated straight-chain or branched- chain alkenyl; R is selected from the group consisting of optionally substituted C 4 -C 30 straight-chain or branched-chain alkyl, optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl
  • R3 is —COR or —(CH 2 ) n COOR.
  • R is selected from the group consisting of oleyl, oleoyl, linoleyl, linoleoyl, palmitoleyl, palmitoleoyl, palmityl, palmitoyl, myristyl, myristoyl, lauryl and lauroyl group.
  • n1, n2, n3, and n 4 are independently an integer from 1 to 2.
  • the disclosure provides a compound having the structure of Formula I-d: or a pharmaceutically acceptable salt thereof, wherein: A2 and A3 are independently selected from the group consisting of H, R 1 and R 2 are independently of one another selected from the group consisting of H, optionally substituted C1-C30 straight-chain or branched-chain alkyl, and optionally substituted C 4 -C 30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; R 3 is —COR, —(CH 2 ) n COR, —(CH 2 ) n COOR, —(CO)NHR—, —(CO)N(R) 2 —, straight-chain or branched-chain alkyl, or optional
  • the disclosure provides a compound having the structure of Formula I-k: or a pharmaceutically acceptable salt thereof, wherein: A 1 is —(CH 2 ) x —, or —(CH 2 ) y —S—S—(CH 2 ) z —; R1 and R2 are independently of one another selected from the group consisting of optionally substituted C 1 -C 30 straight-chain or branched-chain alkyl, and optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; wherein R10 is selected from the group consisting of H; or C1-C30 straight-chain or branched-chain alkyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalky
  • A1 is —(CH 2 ) 4 — or —(CH 2 ) 2 —S—S—(CH 2 ) 2 —.
  • n5 is 1.
  • a and b are independently an integer from 1 to 3.
  • R 10 is a functional group selected from but not limited to H,
  • the disclosure provides a compound having the structure of Formula I-n: or a pharmaceutically acceptable salt thereof, wherein: A1 is —(CH2)y—S—S—(CH2)z—; R 1 and R 2 are independently of one another selected from the group consisting of optionally substituted C1-C30 straight-chain or branched-chain alkyl, and optionally substituted C 4 -C 30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; wherein AA represents any natural or non-natural amino acid side chain; and a is an integer from 1 to 6; b is an integer from 1 to 6; y is an integer from 1 to 4; z is an integer from 1 to 4; and n 5 is an integer from 1 to 5.
  • AA is a functional group selected from but not limited to:
  • the disclosure provides a compound having the structure of Formula I-y: or a pharmaceutically acceptable salt thereof, wherein: R 1 and R 2 are independently of one another selected from the group consisting of optionally substituted C1-C30 straight-chain or branched-chain alkyl, and optionally substituted C 4 -C 30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; y is an integer from 1 to 4; and z is an integer from 1 to 4; [0136] In some embodiments of the compounds of Formula I-y; y and z are independently an integer from 1 to 3.
  • the disclosure provides a compound having the structure of Formula I-z: or a pharmaceutically acceptable salt thereof, wherein: R1 and R2 are independently of one another selected from the group consisting of optionally substituted C 1 -C 30 straight-chain or branched-chain alkyl, and optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; R3 is optionally substituted C1-C30 straight-chain or branched-chain alkyl, or optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group or optionally substituted C4-C30 monounsaturated or polyunsaturated straight- chain or branched-chain alkynyl group; y is an integer from 1 to 4; and z is an integer from 1 to 4; [0138] In some embodiments of the compounds of Formula I-z; y and z are independently an integer
  • R 3 is —COR or —(CH2)nCOOR.
  • R is selected from the group consisting of oleyl, oleoyl, linoleyl, linoleoyl, palmitoleyl, palmitoleoyl, palmityl, palmitoyl, myristyl, myristoyl, lauryl and lauroyl group.
  • a is 1 and b is an integer from 0-1.
  • y and z are independently an integer from 1 to 2.
  • y and z are independently an integer from 1 to 2.
  • a 2 is A 3 is H;
  • a 4 is R1 and R5 are CH3;
  • R 2 and R 6 are H;
  • R3 is —COR, —(CH2)nCOOR, —(CO)NHR—, —(CO)N(R)2—,
  • R 7 is H;
  • b is 0; and a pharmaceutically acceptable salt thereof.
  • A2 and A3 are independently selected from the group consisting of R 7 is H; a and b are 1; and pharmaceutically acceptable salt thereof.
  • R1 and R2 are —(CH2)13CH3.
  • A1 is —(CH2)4— or —(CH2)—A4—(CH2)—.
  • AA is glycine, histidine, serine, tryptophan, arginine, aspartic acid, or pyrrolysine, tyrosine.
  • the AA is histidine.
  • R9 is a peptide chain that includes RGD or RYD tripeptide units.
  • R9 is a peptide chain that includes histidine or an RGD or RYD tripeptide unit preceded by spacer such as a glycine or serine spacer (e.g., one or multiple glycine residues, one or more serine residues, or combination of glycine and serine residues).
  • spacer such as a glycine or serine spacer (e.g., one or multiple glycine residues, one or more serine residues, or combination of glycine and serine residues).
  • R9 is a peptide chain that includes repeat RGD or RYD tripeptide units preceded by spacer such as a glycine or serine spacer (e.g., one or multiple glycine residues, one or more serine residues, or combination of glycine and serine residues).
  • spacer such as a glycine or serine spacer (e.g., one or multiple glycine residues, one or more serine residues, or combination of glycine and serine residues).
  • R9 is a peptide chain that includes repeat GLF or WYG tripeptide units preceded by spacer such as a glycine or serine spacer (e.g., one or multiple glycine residues, one or more serine residues, or combination of glycine and serine residues).
  • spacer such as a glycine or serine spacer (e.g., one or multiple glycine residues, one or more serine residues, or combination of glycine and serine residues).
  • R 9 is a peptide chain that includes repeat Poly-Arg or Poly-His units preceded by spacer such as a glycine or serine spacer (e.g., one or multiple glycine residues, one or more serine residues, or combination of glycine and serine residues).
  • spacer such as a glycine or serine spacer (e.g., one or multiple glycine residues, one or more serine residues, or combination of glycine and serine residues).
  • R 9 is a peptide chain selected from any of the peptide chains listed in Table 1.
  • R 7 is a neurotransmitter-based functional group selected from but not limited [0155] In some embodiments of Formulas (I), (I-a), (I-b), (I-c), (I-d), (I-g), or (II-o-2).
  • R is selected from the group consisting of oleyl, oleoyl, linoleyl, linoleoyl, palmitoleyl, palmitoleoyl, palmityl, palmitoyl, myristyl, myristoyl, lauryl and lauroyl groups.
  • the 5-7 membered monocyclic basic heterocycle is selected from the group consisting of
  • Some embodiments include a compound selected from the group consisting of:
  • Some embodiments include a compound selected from the group consisting of:
  • Some embodiments include a compound selected from the group consisting of:
  • Some embodiments include a compound selected from the group consisting of:
  • Some embodiments include a compound selected from the group consisting of:
  • Some embodiments include a compound selected from the group consisting of:
  • Some embodiments include a compound selected from the group consisting of: wherein:
  • Some embodiments include a compound selected from the group consisting of: w n6 is 0 or 1; R10 is H;
  • Some embodiments include a compound selected from the group consisting of: wherein: and pharmaceutically acceptable salts thereof. [0166] Some embodiments include a compound selected from the group consisting of:
  • Some embodiments include a compound selected from the group consisting of:
  • compositions comprising (i) one or more compounds according to Formula I (ii) one or more of a structural lipid, an ionizable lipid, and a stabilizing agent; and (iii) optionally, a payload.
  • compositions comprising (i) a compound according to Formula I (ii) one or more structural lipids (iii) one or more of stabilizing agents; and (iv) optionally, a payload.
  • compositions comprising (i) a compound according to Formula I (ii) one or more structural lipids (iii) one or more of stabilizing agents; (iv) one or more transfection enhancing agents, and (v) optionally, a payload. [0171] Also provided are compositions comprising (i) one or more compounds according to Formula I and (ii) a payload. [0172] Also provided are compositions comprising one or more compounds according to Formula I at 10 to 80 mol% (excluding any payload), or mol% of the total lipid present in the composition.
  • compositions comprising one or more compounds according to Formula I and wherein a structural lipid is present at 14-50 mol% (excluding any payload) of the composition, or mol% of the total lipid present in the composition.
  • compositions comprising one or more compounds according to Formula I and wherein a stabilizing agent is present at 0.1-10 mol% (excluding any payload) of the composition, or mol% of the total lipid present in the composition.
  • compositions comprising one or more compounds according to Formula I and further comprising an exosome or a biological material derived or purified from an exosome.
  • compositions comprising one or more compounds according to Formula I and further comprising a polymer.
  • compositions comprising one or more compounds according to Formula I and wherein the polymer is selected from the group consisting of: a dense star dendrimer, a PAMAM dendrimer, an NH3 core dendrimer, an ethylenediamine core dendrimer, a dendrimer of generation 5 or higher, a dendrimer with a substituted group, a dendrimer comprising one or more amino acids, a grafted dendrimer, an activated dendrimer, polyethylenimine (PEI), polyethylenimine conjugates, polylysine, polyarginine, polyornithine, histone, and any combination thereof.
  • PAMAM dendrimer an NH3 core dendrimer
  • an ethylenediamine core dendrimer a dendrimer of generation 5 or higher
  • a dendrimer with a substituted group a dendrimer comprising one or more amino acids
  • a grafted dendrimer an activated dend
  • compositions can include one or more compounds according to Formula I and a linear or branched PEI.
  • compositions comprising one or more compounds according to Formula I, and a stabilizing agent selected from the group consisting of: a surfactant, a neutral lipid, a polymer-conjugated lipid, polyethylene glycol, a phospholipid, and any combination thereof.
  • a stabilizing agent selected from the group consisting of: a surfactant, a neutral lipid, a polymer-conjugated lipid, polyethylene glycol, a phospholipid, and any combination thereof.
  • compositions that include one or more compositions of Formula I, and a stabilizing agent that is a PEG-modified lipid.
  • compositions including one or more compounds according to Formula I and one or more transfection enhancing agents, such as a polycationic nucleic acid binding moiety, or a transfection enhancing agent selected from the group consisting of: an endosomal release agent, a cell surface ligand, a nuclear localization agent, a cell-penetrating peptide, a fusogenic peptide, an amphipathic peptide, and any combination thereof.
  • transfection enhancing agents such as a polycationic nucleic acid binding moiety, or a transfection enhancing agent selected from the group consisting of: an endosomal release agent, a cell surface ligand, a nuclear localization agent, a cell-penetrating peptide, a fusogenic peptide, an amphipathic peptide, and any combination thereof.
  • compositions comprising one or more compounds according to Formula I and a payload.
  • the disclosure provides compositions comprising one or more compounds according to Formula I and wherein the payload comprises a nucle
  • compositions comprising one or more compounds according to Formula I and wherein the compound comprises a charge N and the nucleic acid molecule comprises a charge P and wherein the combination of the compound and the nucleic acid contacting the cell comprises an N/P ratio from about 1 to 20.
  • the disclosure provides compositions comprising one or more compounds according to Formula I and wherein the payload comprises a nucleic acid and wherein the nucleic acid is an RNA.
  • compositions comprising one or more compounds according to Formula I and further comprises a nucleic acid wherein the nucleic acid is an RNA and wherein the RNA is mRNA, siRNA, shRNA, self-replicating RNA (srRNA), an o-RNA, self-amplifying RNA, stRNA, trRNA, crRNA, sgRNA, RNAi molecule, an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), or any combination thereof.
  • aiRNA asymmetrical interfering RNA
  • miRNA microRNA
  • dsRNA Dicer-substrate RNA
  • shRNA small hairpin RNA
  • compositions comprising one or more compounds according to Formula I and wherein the payload comprises a nucleic acid and wherein the nucleic acid is an DNA.
  • the disclosure provides compositions comprising one or more compounds according to Formula I and wherein the payload further comprises one or more peptides, and optionally a nucleic acid.
  • the disclosure provides compositions comprising one or more compounds according to Formula I and wherein the payload comprises a nucleic acid wherein the nucleic acid is an RNA and further wherein the RNA is mRNA.
  • compositions comprising one or more compounds according to Formula I and wherein the payload comprises a nucleic acid wherein the nucleic acid is an RNA, and the RNA encodes an immunogen.
  • the disclosure provides compositions comprising one or more compounds according to Formula I and wherein the payload comprises a nucleic acid wherein the nucleic acid is an RNA, and the RNA encodes a cancer antigen.
  • compositions comprising one or more compounds according to Formula I and wherein the structural lipid is selected from the group consisting of: cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and any combination thereof.
  • structural lipid is selected from the group consisting of: cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and any combination thereof.
  • compositions of a compound of Formula I and one or more of stabilizing agent wherein the stabilizing agent comprises one or more phospholipids selected from the group consisting of: 1,2-dilinoleoyl- sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), 1,2-di-O-octadecenyl-sn
  • DLPC 1,2-dilino
  • the formulations may also include one or more lipids derived from viral capsids, e.g, from enveloped viruses.
  • the disclosure provides compositions comprising one or more compounds according to Formula I further comprising a transfection enhancing agent selected from the group consisting of an endosomal release agent, a cell surface ligand, a nuclear localization agent, a cell-penetrating peptide, a fusogenic peptide, and any combination thereof.
  • compositions of a compound of Formula I and at least one or more neutral lipids wherein the one or more neutral lipids is selected from DOPE, DPhPE, cholesterol, sterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, DOPC, Lyso-PE (1-acyl-2-hydroxy-sn-glycero-3-phospho-ethanolamine), Lyso-PC (1-acyl-3- hydroxy-sn-glycero-3-phosphocholine), and 3-alkoxy-2-hydroxy-1-acetamidopropane, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palm
  • DOPE distearoylphosphati
  • the disclosure provides compositions of a compound of Formula I, and at least one or more cationic lipids, and/or at least one or more neutral lipids, and/or at least one or more PEG-modified lipids, wherein the PEG-modified lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, a PEG-ceramide conjugate, a PEG-modified 1,2-diacyloxypropan-3-amine, or any combinations thereof.
  • the PEG-modified lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-
  • compositions of a compound of Formula I and wherein the one or more PEG-modified lipids is selected from the group consisting of: PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, and any combination thereof.
  • the disclosure provides compositions of a compound of Formula I, and at least one or more cationic lipids, and/or at least one or more neutral lipids, wherein the alkoxy is selected form oleyl, palmityl, palmitoleoyl, myristoleyl, myristyl, and lauryl.
  • the disclosure provides compositions of a compound of Formula I, and one or more polyamine transfection agents. [0202] In another embodiment, the disclosure provides compositions of a compound of Formula I, and one or more fusion agents. [0203] In another embodiment, the disclosure provides compositions of a compound of Formula I, and one or more amphipathic peptides. [0204] In another aspect, the disclosure provides compositions of a compound of Formula I, and one or more amphipathic peptides, wherein the one or more amphipathic peptide functions as a fusion agent.
  • the disclosure provides method of delivering a payload to a cell, comprising: (i) providing a composition comprising a compound of Formula I; (ii) providing a cell; and (iii) contacting the cell with the composition.
  • the payload includes a nucleic acid that encodes a therapeutic protein, such as an antibody, growth factor, cytokine, enzyme, or the like.
  • the disclosure provides methods for introducing a nucleic acid, protein, or peptide into a eukaryotic cell, comprising contacting the cell with a composition of any of the disclosed compositions, thereby introducing the nucleic acid, protein, or peptide into the cell.
  • the disclosure provides methods for introducing a nucleic acid, protein, or peptide into a eukaryotic cell, wherein the cell is a human cell, comprising contacting the cell with a composition of any of the disclosed compositions, thereby introducing the nucleic acid, protein, or peptide into the cell.
  • the disclosure provides methods for introducing a nucleic acid, protein, or peptide into a eukaryotic cell, wherein the cell is a mammalian cell, comprising contacting the cell with a composition of any of the disclosed compositions, thereby introducing the nucleic acid, protein, or peptide into the cell.
  • the disclosure provides method for delivering a composition to a subject, comprising administering the composition according to a compound of Formula I to the subject.
  • the disclosure provides methods of delivering a payload to a cell, wherein contacting the cell is in vitro.
  • the disclosure provides methods of delivering a payload to a cell, wherein contacting the cell is in vivo.
  • the disclosure provides methods of delivering a payload to a cell, wherein contacting the cell is ex vivo.
  • the disclosure provides methods for administering any of the disclosed compositions, to subject, wherein the administration is systemic.
  • the disclosure provides methods for administering any of the disclosed compositions, to subject, wherein the administration is selected from the group consisting of subcutaneous administration, intramuscular administration, intranasal administration, intra-tumoral administration, administration to the brain, administration to the spinal cord, administration to the eye, administration to the lymph node of a subject, and any combination thereof.
  • the disclosure provides kits including a compound of Formula I, and one or more structural lipids, ionizable lipid, and a stabilizing agent.
  • the disclosure provides kits including a compound of Formula I, and one or more structural lipids, and/or one or more stabilizing agents, and/or optionally a payload.
  • kits including a compound of Formula I, and one or more structural lipids, and/or one or more stabilizing agents, and/or one or more fusion agents, and/or optionally a payload.
  • the disclosure provides methods for inhibiting expression of a protein in a cell, comprising contacting the cell with an RNAi molecule and a compound of Formula I, as described herein, or the composition of any one of the disclosed compositions.
  • any of the processes for preparation of the compounds disclosed herein it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups, such as those described in Protective Groups in Organic Chemistry (ed. J.F.W. McOmie, Plenum Press, 1973); and P.G.M. Green, T.W. Wutts, Protecting Groups in Organic Synthesis (3rd ed.) Wiley, New York (1999), which are both hereby incorporated herein by reference in their entirety.
  • the protecting groups may be removed at a convenient subsequent stage using methods known from the art. Synthetic chemistry transformations useful in synthesizing applicable compounds are known in the art and include e.g. those described in R.
  • protecting groups for oxygen atoms are selected for their compatibility with the requisite synthetic steps as well as compatibility of the introduction and deprotection steps with the overall synthetic schemes (P.G.M. Green, T.W. Wutts, Protecting Groups in Organic Synthesis (3rd ed.) Wiley, New York (1999)).
  • the compounds of the present technology contain one or more chiral centers, such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or d(l) stereoisomers, or as stereoisomer-enriched mixtures. All such stereoisomers (and enriched mixtures) are included within the scope of the present technology, unless otherwise indicated.
  • Pure stereoisomers may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents and the like.
  • the starting materials for the following reactions are generally known compounds or can be prepared by known procedures or obvious modifications thereof. For example, many of the starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wisconsin, USA), Bachem (Torrance, California , USA), Emka-Chemce or Sigma (St. Louis, Missouri, USA).
  • the method involves treatment of intermediate I-e with BOC-anhydride in presence of a base to obtain the bis-BOC derivative I-h which is subsequently treated with an acid chloride II-h to obtain the intermediate I-f. This is then subjected to BOC-removal in presence of an acid to obtain compounds of formula I-n.
  • the bis-BOC derivative I-h is treated with a BOC-protected amino acid (II-m) in presence of an appropriate base and coupling reagent to obtain the derivative I-m which is then subjected to BOC- removal in presence of an acid to yield the compounds of formula I-n.
  • a BOC-protected amino acid II-m
  • SCHEME III In one embodiment, for example, compounds 13-21 are synthesized following reactions conditions as shown in Scheme III.
  • the bis-BOC derivative I-h is subjected to alkylation reaction of the hydroxyl group with an alkylating agent R3-X (II-j; X is a leaving group) followed by BOC-removal in presence of an acid to yield the compounds of formula I-o.
  • compound 24 is synthesized following reactions conditions as shown in Scheme V using N 1 ,N 4 -ditetradecylbutane-1,4- diamine as the amine I-x to couple with 2-(2-(chloromethyl)allyl)isoindoline-1,3-dione and 2-aminoethan-1-ol as the amine I-k to couple with the corresponding intermediate I-q.
  • solid-phase bound arylalkyl chloride II-a is reacted with amino-thiol II-b to yield the solid-phase bound amine II-c.
  • the amine II-c is then subjected to reductive amination in presence of aldehyde II-n to yield intermediate II-d which is then treated with 2-(oxiran-2-ylmethyl)isoindoline-1,3-dione in the presence of a base and heated in an appropriate solvent to yield the adduct II-e.
  • This adduct II-e is then subjected to release from the solid-phase followed by oxidation to yield the disulfide I-s.
  • disulfide II-f is subjected to reductive amination with aldehyde II-n to yield intermediate II-g which is subjected to treatment with 2-(oxiran-2-ylmethyl)isoindoline-1,3-dione in the presence of a base and heated in an appropriate solvent to yield the adduct I-t.
  • the adduct I-t is then subjected to treatment with hydrazine in ethanol followed by treatment with (BOC) 2 O to obtain the derivative I-u.
  • the bis-BOC derivative I-u is treated with a BOC-protected amino acid (II-m) in presence of an appropriate base and coupling reagent followed by subjecting the resulting product to BOC-removal in presence of an acid to yield the compounds of formula I-v.
  • the bis-BOC derivative I-u is treated with mesyl chloride in presence of a base to yield the bis-mesylate intermediate which is then treated with amines represented by formula II-k in presence of a base followed by BOC-removal in presence of an acid to yield the compounds of formula I- w.
  • compounds 25-37 are synthesized following reactions conditions as shown in Scheme VII using 2,2'-disulfanediylbis(ethan-1- amine) as the diamine II-f and tetradecanal as the aldehyde II-n to yield the corresponding intermediate II-g.
  • the corresponding intermediate II-g was elaborated to yield the intermediate I-u. Then this intermediate was further converted to compounds 25-37 using reactions shown in Scheme VII.
  • N 1 ,N 4 -ditetradecylbutane-1,4-diamine (7A) is treated with 2-ethyloxirane in 2,2,2-trifluoroethanol in presence of DIPEA at 80 °C to obtain the compound 7.
  • compound 7 was treated with oleoyl chloride to obtain the intermediate 6A.
  • This was subsequently treated with HCl to obtain the bis-oleoyl derivative 6 as a hydrochloride salt.
  • compound 5 was also synthesized using procedures as described above by treating compound 7 with linoleoyl chloride.
  • Compounds 8-12 were synthesized using schemes as shown below in Scheme IIA.
  • diamine II-u is subjected to reductive amination conditions using the aldehyde II-q and NaBH(OAc) 3 to yield the intermediate II-r.
  • reductive amination conditions using the aldehyde II-q and NaBH(OAc) 3 to yield the intermediate II-r.
  • the skilled artisan will appreciate that there are many other reductive amination conditions and reagents which are within the scope of this disclosure to carry out the reaction.
  • the resulting product II-r is then treated with 2-(oxiran-2-ylmethyl)isoindoline- 1,3-dione in the presence of a base followed by treatment with hydrazine in ethanol to yield the product II-s.
  • the intermediate II-s is then treated with (BOC) 2 O followed by treatment with 2,2-dimethoxypropane in the presence of a Lewis acid (for example BF 3 .Et 2 O) to obtain the derivative II-s-1.
  • Intermediate II-s-1 is then subjected to indium catalyzed transesterification with alcohol II-p followed by acetonide-removal and BOC-removal under acid-catalyzed conditions to yield the compounds of formula II-t.
  • a Lewis acid for example BF 3 .Et 2 O
  • the lipids of formula (I) can be combined with a nucleic acid and/or protein payload in a to produce a lipid nanoparticle formulation.
  • the lipids of formula (I) can be combined with a payload selected from one or more of the following: an siRNA, an miRNA, an mRNA, a shRNA, a self-amplifying RNA, an oRNA (or non-naturally occurring circular RNA), an anti-sense oligonucleotide (ASO), a gRNA, a ribonucleoprotein (e.g., a CRISPR complex), a dsDNA, a plasmid DNA, or the like.
  • a payload selected from one or more of the following: an siRNA, an miRNA, an mRNA, a shRNA, a self-amplifying RNA, an oRNA (or non-naturally occurring circular RNA), an anti-sense oligonucleotide (ASO), a gRNA
  • the amount of nucleic acid (e.g., mRNA, self-amplifying RNA or the like) in lipid nanoparticle formulation may depend on the size, sequence, and other characteristics of the nucleic acid.
  • the amount of nucleic acid in a lipid nanoparticle formulation may also depend on the size, composition, desired target, and other characteristics of lipid nanoparticle formulation.
  • the relative amounts of mRNA and other elements (e.g., lipids) may also vary.
  • the wt/wt ratio of the lipid component to a nucleic acid, such as an mRNA, in a nanoparticle composition may be from about 5:1 to about 50:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, and 50:1.
  • the wt/wt ratio of the lipid component to a nucleic acid, such as an mRNA may be from about 10:1 to about 40:1.
  • the amount of nucleic acid in a nanoparticle composition may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible (UV-vis) spectroscopy).
  • the lipid nanoparticle formulations can comprise a nucleic acid in a concentration from approximately 0.1 mg/ml to 2 mg/ml such as, but not limited to, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2.0 mg/ml or greater than 2.0 mg/ml.
  • the one or more nucleic acids e.g. mRNAs
  • lipids, and amounts thereof may be selected to provide a specific N:P ratio.
  • the N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in a nucleic acid (e.g., an mRNA). In general, a lower N:P ratio is preferred.
  • the one or more mRNA, lipids, and amounts thereof may be selected to provide an N:P ratio from about 2:1 to about 8:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, and 8:1. In certain embodiments, the N:P ratio may be from about 2:1 to about 5:1.
  • the N:P ratio may be about 4:1. In other embodiments, the N:P ratio is from about 5:1 to about 8:1. For example, the N:P ratio may be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1.
  • Additional lipid components [0254]
  • the lipid nanoparticle formulations include one or more co-lipids, most advantageously neutral co- lipids, although the skilled artisan will recognize that other lipids, including cationic/ionizable lipids, may be used. Some formulations, however, include just the lipids of formula (I), in combination with a nucleic acid.
  • Ionizable lipids described herein refer to lipids that have at least one protonatable or deprotonatable group.
  • ionizable lipids can be positively charged at a pH at or below physiological pH (e.g., pH 7.4) and neutral at a second pH, e.g., at or above physiological pH.
  • the ionizable lipids provided herein can have a pKa of the protonatable group in the range of about 4 to about 11, e.g., about 4 to about 7, e.g., between about 5 and 7, such as between about 5.5 and 6.9, when incorporated into lipid nanoparticles.
  • the lipid nanoparticle formulations can include neutral lipids such as phospholipids.
  • exemplary phospholipids that can be used in the lipid nanoparticle formulations provided herein include, but are not limited to, Phospholipids useful in the compositions disclosed herein include, but are not limited to, DOPE, DPhPE, DOPC, Lyso-PE ( 1-acyl-2-hydroxy-sn-glycero-3- phosphoethanolamine), Lyso-PC (1-acyl-3-hydroxy-sn-glycero-3-phosphocholine), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl
  • DOPE distearoylphosphatidylcholine
  • Phospholipids useful in the compositions provided herein can be present, for example at about 5 mol% to about 20 mol% of the lipid nanoparticle formulation.
  • phopsolipids are present at a ragne from about 1 mol % to about 40 mol%, e.g., from 1 mol% to about 25 mol %.
  • the amount of the phospholipid in the lipid nanoparticle formulations disclosed herein is at least about 0.5 mol %, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol%, 10 mol%, 12 mol%, 14 mol%, 16 mol %, 18 mol%, or 20 mol %, or any amount in between, of the overall lipid nanoparticle formulations.
  • Other neutral lipids that can be advantageously included in the lipid nanoparticle formulations provided herein include sterols, or lipids containing sterol moieties (“sterol derivatives”).
  • sterols are a subgroup of steroids consisting of steroid alcohols.
  • exemplary sterols and lipids containing sterol moieties useful in the lipid nanoparticle formulations provided herein include, but are not limited to holesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof.
  • the structural lipid is a sterol.
  • Some lipid nanoparticle formulations provided herein include a sterol or sterol derivative.
  • the sterols or sterol derivatives can be present at about 5-60 mol% of the overall lipid nanoparticle formulation.
  • the sterol or sterol derivatives are present from about 15-50 mol%, e.g., 25-40 mol %.
  • the amount of the sterol (such as cholesterol) or sterol derivative in the lipid composition disclosed herein is at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol % of the overall lipid formulation.
  • the lipid nanoparticle formulations provided herein do not include a sterol or sterol derivative.
  • the lipid nanoparticle formulations provided herein can also include a stabilizing agent, such as a surfactant, a neutral lipid, a polymer-conjugated lipid, polyethylene glycol, a phospholipid, and any combination thereof
  • a stabilizing agent such as a surfactant, a neutral lipid, a polymer-conjugated lipid, polyethylene glycol, a phospholipid, and any combination thereof
  • non-ionic stabilizing agents include: Polysorbates (Tweens), BrijTM S20 (polyoxyethylene (20) stearyl ether), BrijTM35 (Polyoxyethylene lauryl ether, Polyethyleneglycol lauryl ether), BrijTMS10 (Polyethylene glycol octadecyl ether, Polyoxyethylene (10) stearyl ether), and MyrjTM52 (polyoxyethylene (40) stearate
  • exemplary stabilizing agents useful in the embodiments provided herein include TPGS 1000 (D- ⁇ -Tocopherol polyethylene glycol 1000 succinate); or Tween 20/Polysorbate 80/ Tridecyl-D-maltoside in equal ratios (called Lipid H in Table 15).
  • exemplary stabilizing agents useful in the embodiments provided herein include TPGS 1000 (D- ⁇ -Tocopherol polyethylene glycol 1000 succinate); or Tween 20/Polysorbate 80/ Tridecyl-D-maltoside in equal ratios (called Lipid H in Table 15).
  • Yet other stabilizing lipids that can advantageously be used in the formulations provided herein include, but are not limited to, polyethylene glycol (PEG)-modified lipids.
  • Non-limiting examples of PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines.
  • PEGylated lipids are also referred to as PEGylated lipids.
  • a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG- DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • stabilizing lipids useful in the compositions disclosed herein include, e.g., polyglycol lipids, yoxyethylene alkyl ethers, diblock polyoxyethylene ether co-polymers, triblock polyoxyethylene alkyl ethers co- polymers, and amphiphilic branched polymers.
  • the stabilizing agent is present at about 0.1 - 5 mol% of the lipid nanoparticle formulation.
  • the stabilizing agent is present at about 0.5 mol%, 1 mol%, 1.5 mol%, 2 mol%, 2.5 mol %, 3 mol%, 3.5 mol %, 4 mol %, 4.5 mol%, 5 mol%, or any value in between, of the lipid nanoparticle formulation.
  • Lipid nanoparticle formulations can include one or more cationic/ionizable lipids, in addition to the lipid of Formula (I).
  • some lipid nanoparticle formulations include a cationic/ionizable lipid selected from the group consisting of DOTMA, DOTAP, DMRIE, DC-Chol, DDAB, DOSPA, DOSPER, DOGS, TMTPS, TMTOS, TMTLS, TMTMS, TMDOS, N-1-dimethyl-N-1-(2,3-diaoleoyloxypropyl)-2- hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-diamyristyloxypropyl)-2- hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-diapalmityloxypropyl)-2- hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-diaoleoyloxypropyl)-2-(3-amino-2- hydroxypropyloxy)propane
  • the lipid of Formula (I), or the combination of the lipid of Formula (I) with one or more cationic/ionizable lipids is present at about 5-80 mol% of the lipid nanoparticle formulation (excluding payload), e.g., about 5-80 mol% of the lipid component of the lipid nanoparticle formualtion.
  • some lipid nanoparticle formulations include less than 50 mol% Formula (I), or combination of Formula (I) and one or more additional cationic/ionizable lipids.
  • Other lipid nanoparticle formulations include more than 50 mol% Formula (I), or combination of Formula (I) and one or more additional cationic/ionizable lipids.
  • some lipid nanoparticle formulations include a lipid of Formula (I), or a combination of Formula (I) and one or more cationic/ionizable lipids at 15- 80 mol%, a sterol at 20-60 mol%, a stabilizing agent at 0.5-5 mol%, and a phospholipid at 1- 40 mol% of the lipid nanoparticle formulation.
  • An exemplary lipid nanoparticle formulation can include about 20-60 mol % Formula (I) lipid or combination of Formula (I) lipid and one or more additional cationic/ionizable lipids, about 5-25 mol % phospholipid, about 25-55 mol% sterol or sterol derivative; and about 0.5-15 mol% stabilizing agent.
  • Another exemplary lipid nanoparticle formulation includes a about 50 mol % lipid of Formula (I) or combination of lipid of Formula (I) and one or more cationic/ionizable lipids, about 1.5 mol% stabilizing agent, about 38.5 mol% sterol or sterol derivative, and about 10 mol% phospholipid.
  • Another exemplary lipid nanoparticle formulation comprises about 55% lipid of Formula (I) or combination of lipid of Formula (I) and one or more cationic/ionizable lipids, about 2.5 mol % stabilizing agent, about 32.5 mol % sterol or sterol derivative, and about 10 mol % phospholipid.
  • polyamine transfection agents such as dense star dendrimers, PAMAM dendrimers, NH 3 core dendrimers, ethylenediamine core dendrimers, dendrimers of generation 5 or higher, dendrimers with substituted groups, dendrimers comprising one or more amino acids, grafted dendrimers, activated dendrimers, polyethylenimine (PEI), and/or polyethylenimine conjugates.
  • polyamine transfection agents such as dense star dendrimers, PAMAM dendrimers, NH 3 core dendrimers, ethylenediamine core dendrimers, dendrimers of generation 5 or higher, dendrimers with substituted groups, dendrimers comprising one or more amino acids, grafted dendrimers, activated dendrimers, polyethylenimine (PEI), and/or polyethylenimine conjugates.
  • PEI polyethylenimine
  • transfection Enhancing Agents such as a fusion agent (such as an endosomal release agent), a cell surface ligand and/or a nuclear localization agent such as a nuclear receptor ligand peptide
  • transfection enhancing agents include, but are not limited to, reovirus-related fusogenic peptides (see WO07/130073, which is hereby incorporated by reference in its entirety), insulin, a transferrin, epidermal growth factor, fibroblast growth factor, a cell targeting antibody, a lactoferrin, a fibronectin, an adenovirus penton base, Knob, a hexon protein, a vesicular stomatitis virus glycoprotein, a Semliki Forest Virus core protein, a influenza hemagglutinin, a hepatitis B core protein, an HIV Tat protein, a herpes simplex virus VP22 protein, a histone protein,
  • lipid nanoparticle compositions provided herein can also be combined with one or more exosomes, or biological materials (e.g., lipids, proteins, nucleic acids, or the like) derived or purified from exosomes.
  • biological materials e.g., lipids, proteins, nucleic acids, or the like
  • compositions can include, for example, a lipid of Formula (I) and one or more exosomes; a lipid of Formula (I), and one or more exosomes, and one or more neutral lipids; a lipid of Formula (I), and one or more exosomes, one or more neutral lipids, and one or more stabilizing agents; a lipid of Formula (I), and one or more exosomes, and one or more neutral lipids, optionally one or more stabilizing agents, and optionally one or more cell penetrating peptides.
  • compositions include, for example; a lipid of Formula (I), and one or more biological materials derived or purified from exosomes; a lipid of Formula (I), and one or more biological materials derived or purified from exosomes, and one or more neutral lipids; a lipid of Formula (I), and one or more biological materials derived or purified from exosomes, one or more neutral lipids, and or more stabilizing agents; a lipid of Formula (I), and one or more biological materials derived or purified from exosomes, and one or more neutral lipids, optionally one or more stabilizing agents, and optionally one or more cell penetrating peptides.
  • the lipids described above may be formulated by various methods to be used in transfection.
  • One of the simplest methods for formulation is reverse evaporation, as described in U.S. Pat. No. 9,259,475, which is hereby incorporated by reference in its entirety.
  • Other methods for formulation that can be used are sonication and microfluidization.
  • the lipids are formulated as lipid nanoparticles using microfluidic mixing as described, for example, in Roces et al., Pharmaceutics, 12:1095 (2020). Suitable microfluidic mixing devices are commercially available from, for example, Precision Nanosystems (Vancouver, BC).
  • microfluidic mixing combines two fluid streams, one containing the nucleic acid(s) and one containing the lipid of Formula (I) and other components, such as the peptide, ligand and other lipid components as described below.
  • lipid nanoparticle compositions including an RNA solutions of the RNA at concentrations of 0.1 mg/ml in deionized water are diluted in 50 mM sodium citrate buffer at a pH between 3 and 4 to form a stock solution. Nanoparticle compositions can be processed by dialysis to remove ethanol and achieve buffer exchange.
  • Formulations may be dialyzed against phosphate buffered saline (PBS), pH 7.4, using a desired molecular weight cutoff, e.g.10 kD.
  • PBS phosphate buffered saline
  • the resulting nanoparticle suspension may be filtered through a 0.2 ⁇ m sterile filters (Sarstedt, Numbrecht, Germany) into glass vials and sealed.
  • Methods of determining particle size in nanoparticles formulations are well-known in the art. For example, a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK) can be used to determine the particle size, the polydispersity index (PDI) and the zeta potential of the nanoparticle compositions.
  • PDI polydispersity index
  • UV-visible spectroscopy can be used to determine the concentration of payload such as nucleic acid (e.g., mRNA) in nanoparticle compositions.
  • a quantity of the composition is diluted in a suitable solvent and the absorbance spectrum of the solution is recorded, for example, between 230 nm and 330 nm on a spectrophotometer.
  • the concentration of therapeutic and/or prophylactic in the nanoparticle composition can be calculated based on the extinction coefficient of the therapeutic and/or prophylactic used in the composition and on the difference between the absorbance at a wavelength of, for example, 260 nm and the baseline value at a wavelength of, for example, 330 nm.
  • a QUANT-ITTM RIBOGREEN ® RNA assay (Invitrogen Corporation, Carlsbad, CA) can be used to evaluate the encapsulation of an RNA by the nanoparticle composition using methods provided by the manufacturer.
  • the fluorescence intensity generated after addition of the RIBOGREEN reagent can be measured using a fluorescence plate reader at an excitation wavelength of, for example, about 480 nm and an emission wavelength of, for example, about 520 nm.
  • the fluorescence values of the reagent blank are subtracted from that of each of the samples and the percentage of free RNA is determined by dividing the fluorescence intensity of the intact sample (without addition of Triton X-100) by the fluorescence value of the disrupted sample (caused by the addition of Triton X-100).
  • the lipids of Formula (I) can be formulated with one or more co-lipids, most advantageously neutral co-lipids, although the skilled artisan will recognize that other lipids, including cationic lipids described above, may be used.
  • lipids of Formula (I) can be formulated with one or more ionizable/cationic lipids and/or one or more neutral lipids.
  • Exemplary ionizable/cationic lipids useful in the compositions provided herein include, but are not limited to GeneIn TM , LipofectAmine TM 2000, LipofectAmine TM , Lipofectin®, DMRIE-C, CellFectin®(Invitrogen), Oligofectamine® (Invitrogen), LipofectAce® (Invitrogen), Fugene® (Roche, Basel, Switzerland), Fugene® HD (Roche), Transfectam® (Tranfectam, Promega, Madison, WI), Tfx-10® (Promega), Tfx-20® (Promega), Tfx-50® (Promega), Transfectin TM (BioRad, Hercules, CA), SilentFect TM (Bio-Rad), Effectene® (Qiagen, Valencia, CA), DC-chol (Avanti Polar Lipids), GenePorter® (Gene Therapy Systems, San Diego, CA), DharmaFect 1® (
  • transfection enhancing agents such as a fusion agent, a cell surface ligand and/or a nuclear localization agent such as a nuclear receptor ligand peptide
  • transfection enhancing agents include, but are not limited to, reovirus-related fusogenic peptides, insulin, a transferrin, epidermal growth factor, fibroblast growth factor, a cell targeting antibody, a lactoferrin, a fibronectin, an adenovirus penton base, Knob, a hexon protein, a vesicular stomatitis virus glycoprotein, a Semliki Forest Virus core protein, a influenza hemagglutinin, a hepatitis B core protein, an HIV Tat protein, a herpes simplex virus VP22 protein, a histone protein, a arginine rich cell permeability protein, a high mobility group protein, and invasin protein, and internalin
  • compositions provided herein can advantageously include, e.g., one or more peptide sequences that enhance transfection efficiency, such as linkers, spacers, or nuclear targeting sequences.
  • the peptides provided herein can be included in the compositions herein independently (i.e., not covalently attached to another molecule) or alternatively, can be covalently linked to one or more molecules of the compositions provided herein (e.g., covalently linked to an ionizable or other lipid as described herein, covalently linked to another transfection enhancer as described herein, or covalently linked to a payload as described herein, or the like).
  • the covalent linkage can be via a spacer.
  • spacer refers to a chemical structure that links two molecules to each other.
  • the spacer binds each molecule on a different part of the spacer molecule.
  • the spacer is a hydrophilic moiety and comprises about 6 to 30 carbon atoms.
  • the spacer comprises a polyether, for example -CH 2 -0-(CH 2 -CH 2 - 0-)iCH 2 -.
  • the spacer comprises a hydrophilic polymer, for example [(gly)i(ser)j]k (SEQ ID NO: 585).
  • the spacer is a peptide of sequence APYKAWK (SEQ ID NO:505). In other embodiments, the spacer is a sequence that is degraded in vivo by a peptidase.
  • a liposomal preparation of the lipid, with or without co-lipid is prepared, and is then mixed with the payload(e.g., a nucleic acid such as DNA or RNA) to form a transfection complex.
  • the payload e.g., a nucleic acid such as DNA or RNA
  • the complex is then added to a cell culture and transfection is monitored using well known methods. Additional components such as cell surface ligands, fusion agents, nuclear localization agents and the like may be added to the nucleic acid prior to admixture with the liposome, or may be added to the liposome prior to addition of nucleic acid.
  • Cells which can be transfected according to these methods include, but are not limited to, virtually any eukaryotic cell including primary cells, cells in culture, a passaged cell culture or a cell line, and cells in cultured tissue. Suitable cells include human cell lines and animal cell lines. The cell may be a fibroblast. The cells can be attached cells or cells in suspension (suspension cells). In certain illustrative aspects, the cells are suspension CHO-S cells and suspension 293-F cells.
  • Other cells include, without limitation, 293, 293-S, CHO, Cos, 3T3, Hela, primary fibroblasts, A549, Be2C, SW480, CHOK1, Griptite 293, HepG2, Jurkat, LNCap, MCF-7, NIH-3T3, PC12, C6, Caco-2, COS-7, HL60, HT-1080, IMR-90, K-562, SK-BR3, PHP1, HUVEC, MJ90, NHFF, NDFF and primary neurons.
  • the formulations are used in a method for producing a protein which includes contacting a cell with a lipid-nucleic acid complex as described above, where the nucleic acid encodes the protein.
  • compositions which can be used for protein production are described above.
  • any composition which includes a lipid of Formula I can be used for transfection of cells.
  • Such compositions are further discussed herein, and include, but are not limited to compositions comprising lipids of Formula I, a co-lipid and an optional transfection enhancing agent such as a fusogenic peptide or protein.
  • the lipids formulated into Lipid nanoparticle (LNP) formulations were screened, and assessed by in vivo functional testing using the RNA payload of the complex. Performance and transfection efficiency analyses included payload delivery, biodistribution, and expression of the payload-encoded protein.
  • lipids formulated in this manner were used in transfection.
  • the transfection of was carried out using the formulations described below, which contain Compound 1, which is a lipid of Formula (I). These results are shown in Figures 1-5.
  • Reagent Kits [0279] Components of the transfection compositions described above can be provided in a reagent kit.
  • the kits contain the lipid of Formula (I), together with additional components, such as a neutral lipid, a cationic lipid, cell surface ligands, fusion agents, amphipathic peptide and/or nuclear localization agents and the like.
  • the kit components may be separate or may be premixed in any manner.
  • the lipid of Formula I may be admixed with one or more neutral lipid.
  • kits typically include vessels, such as vials and/or tubes, which are packaged together, for example in a cardboard box.
  • the kits can be shipped from a supplier to a customer.
  • a kit that includes a vial that includes a liposomal formulation as described above and, optionally, a transfection agent and a transfection enhancing peptide.
  • the kit can also include, for example, a separate vessel that includes a transfection enhancing agent, such as a transfection enhancing peptide, for example Plus Reagent TM (Invitrogen Corp., Carlsbad, CA).
  • kits can also include in separate containers, cells, cell culture medium, and a reporter nucleic acid sequence, such as a plasmid that expresses a reporter gene.
  • the culture medium can be reduced-serum medium and/or protein expression medium.
  • a kit comprises individual portions of, or a mixture of, ionizable lipid, such as a lipid of Formula I, and peptide, protein, or fragment thereof or modified peptide, protein or fragment thereof.
  • a kit comprises individual portions of, or a mixture of, polycationic polymers and peptide, protein or fragments thereof or modified peptide, protein or fragments thereof.
  • Cationic lipid transfection kits can optionally include neutral lipid as well as other transfection-enhancing agents or other additives, and the relative amounts of components in the kit may be adjusted to facilitate preparation of transfection compositions.
  • Kit components can include appropriate medium or solvents for other kit components.
  • Payloads that can be delivered by the methods of this invention include nucleic acids, proteins, ribonucloeproteins, and the like, including DNA and RNA (including RNAi/siRNA) of any size from any source comprising natural bases or non- natural bases, and include those encoding and capable of expressing therapeutic or otherwise useful proteins in cells, those which inhibit undesired expression of nucleic acids in cells, those which inhibit undesired enzymatic activity or activate desired enzymes, those which catalyze reactions (ribozymes), and those which function in diagnostic assays (e.g., diagnostic nucleic acids).
  • DNA and RNA including RNAi/siRNA of any size from any source comprising natural bases or non- natural bases
  • RNAi/siRNA include those encoding and capable of expressing therapeutic or otherwise useful proteins in cells, those which inhibit undesired expression of nucleic acids in cells, those which inhibit undesired enzymatic activity or activate desired enzymes, those which catalyze reactions (ribozymes
  • Therapeutic nucleic acids include those nucleic acids that encode or can express therapeutically useful proteins, peptides or polypeptides in cells, those which inhibit undesired expression of nucleic acids in cells, and those which inhibit undesired enzymatic activity or activate desired enzymes in cells.
  • the payload comprises an RNA molecule.
  • the compositions can be used to deliver RNA payloads such as mRNA, siRNA, shRNA, miRNA, self-replicating RNA (srRNA), self-amplifying RNA, stRNA, sgRNA, or combinations thereof.
  • the RNA molecule comprises more than one RNA molecule, e.g., more than one mRNA.
  • compositions and methods provided herein can also be readily adapted in view of the disclosure herein to introduce biologically active macromolecules other than nucleic acids including, among others, polyamines, polyamine acids, polypeptides and proteins into eukaryotic cells.
  • Other materials useful for example as therapeutic agents, diagnostic materials, research reagents, which can be bound to the peptides and modified peptides and introduced into eukaryotic cells by the methods of this invention.
  • the compositions provided herein can be delivered to cells via in vivo administration.
  • the pharmaceutical compositions are preferably administered parenterally (e.g., intraarticularly, intravenously, intraperitoneally, subcutaneously, intrathecally, intradermally, intratracheally, intraosseous, intramuscularly or intratumorally).
  • parenterally e.g., intraarticularly, intravenously, intraperitoneally, subcutaneously, intrathecally, intradermally, intratracheally, intraosseous, intramuscularly or intratumorally.
  • the pharmaceutical compositions are administered intravenously, intrathecally, or intraperitoneally by a bolus injection.
  • Other routes of administration include topical (skin, eyes, mucus membranes), oral, pulmonary, intranasal, sublingual, rectal, and vaginal administration.
  • Typical applications include using well known procedures to provide intracellular delivery of siRNA to knock down or silence specific cellular targets in vitro and in vivo.
  • applications include delivery of DNA or mRNA sequences that code for therapeutically useful polypeptides.
  • therapy is provided for genetic diseases by supplying deficient or absent gene products.
  • Methods of the present invention may be practiced in vitro, ex vivo, or in vivo.
  • the compositions of the present invention can also be used for delivery of payloads to cells in vivo, using methods which are known to those of skill in the art.
  • the compositions of the invention can be used for delivery of a payload to a sample of patient cells that are ex vivo, then are returned to the patient.
  • the pharmaceutical compositions are preferably administered parenterally (e.g., intraarticularly, intravenously, intraperitoneally, subcutaneously, intrathecally, intradermally, intratracheally, intraosseous, intramuscularly or intratumorally).
  • parenterally e.g., intraarticularly, intravenously, intraperitoneally, subcutaneously, intrathecally, intradermally, intratracheally, intraosseous, intramuscularly or intratumorally.
  • the compositions provided herein are administered intravenously, intrathecally, or intraperitoneally by a bolus injection.
  • Other routes of administration include topical (skin, eyes, mucus membranes), oral, pulmonary, intranasal, sublingual, rectal, and vaginal.
  • compositions provided herein are preferably administered to biological samples that have been removed from the organism, then the cells are washed and restored to the organism.
  • the organism may be a mammal, and in particular may be a mammal (e.g., a primate), such as a human. This process is used for cell reprogramming, genetic restoration, immunotherapy, for example.
  • the present invention provides a method of modulating the expression of a target polynucleotide or polypeptide.
  • These methods generally include contacting a cell with a composition of the present invention that is associated with a payload (e.g., a nucleic acid) that is capable of modulating the expression of a target polynucleotide or polypeptide.
  • a payload e.g., a nucleic acid
  • modulating refers to altering the expression of a target polynucleotide or polypeptide. Modulating can mean increasing or enhancing, or it can mean decreasing or reducing.
  • compositions provided herein, wherein the composition includes a payload that is a therapeutic agent selected from an siRNA, a microRNA, an antisense oligonucleotide, and a plasmid capable of expressing an siRNA, a microRNA, or an antisense oligonucleotide, and wherein the siRNA, microRNA, or antisense RNA includes a polynucleotide that specifically binds to a polynucleotide that encodes the polypeptide, or a complement thereof.
  • a therapeutic agent selected from an siRNA, a microRNA, an antisense oligonucleotide, and a plasmid capable of expressing an siRNA, a microRNA, or an antisense oligonucleotide
  • the siRNA, microRNA, or antisense RNA includes a polynucleotide that specifically binds to a polynucleotide that encodes the polypeptide, or a complement thereof.
  • kits for treating a disease or disorder characterized by under-expression of a polypeptide in a subject can include providing to the subject a composition as provided herein, wherein the composition includes a payload that is a therapeutic agent selected from an mRNA, a self- amplifying RNA (SAM), a self-replicating DNA, or a plasmid, comprises a nucleic acid therapeutic that specifically encodes or expresses the under-expressed polypeptide, or a complement thereof.
  • SAM self- amplifying RNA
  • plasmid comprises a nucleic acid therapeutic that specifically encodes or expresses the under-expressed polypeptide, or a complement thereof.
  • the compounds, compositions, and methods and uses of the described herein are for delivering a biologically active agent to liver cells (e.g. hepatocytes).
  • the compounds, compositions, and methods and uses of the invention are for delivering a biologically active agent to a tumor or to tumor cells (e.g. a primary tumor or metastatic cancer cells).
  • the compounds, compositions, and methods and uses are for delivering a biologically active agent to the skin adipose, muscle and lymph nodes (subcutaneous dosing).
  • a composition of the invention is contacted with the liver or liver cells of the via parenteral administration (e.g. intravenous, intramuscular, subcutaneous administration) or local administration (e.g. direct injection, portal vein injection, catheterization, stenting), to facilitate delivery.
  • a composition of the invention is contacted with the kidney or kidney cells of the patient via parenteral administration (e.g. intravenous, intramuscular, subcutaneous administration) or local administration (e.g. direct injection, catheterization, stenting), to facilitate delivery.
  • parenteral administration e.g. intravenous, intramuscular, subcutaneous administration
  • local administration e.g. direct injection, catheterization, stenting
  • a composition of the invention is contacted with the tumor or tumor cells of the patient via parenteral administration (e.g. intravenous, intramuscular, subcutaneous administration) or local administration (e.g. direct injection, catheterization, stenting), to facilitate delivery.
  • compositions described herein can be contacted with the CNS or CNS cells (e.g. brain cells and/or spinal cord cells) of the patient via parenteral administration (e.g. intravenous, intramuscular, subcutaneous administration) or local administration (e.g. direct injection, catheterization, stenting, osmotic pump administration (e.g. intrathecal or ventricular)), to facilitate delivery.
  • parenteral administration e.g. intravenous, intramuscular, subcutaneous administration
  • local administration e.g. direct injection, catheterization, stenting, osmotic pump administration (e.g. intrathecal or ventricular)
  • PNS Peripheral Nervous System
  • parenteral administration e.g.
  • compositions provided herein can be contacted with the lung or lung cells of the patient via parenteral administration (e.g. intravenous, intramuscular, subcutaneous administration) or local administration (e.g. pulmonary administration directly to lung tissues and cells), to facilitate delivery.
  • parenteral administration e.g. intravenous, intramuscular, subcutaneous administration
  • local administration e.g. pulmonary administration directly to lung tissues and cells
  • the compositions include lipid molecules functionalized with neurotransmitter-based functional groups allowing for the delivery of the payload to the brain via the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • compositions provided herein can be contacted with the vasculature or vascular cells of the patient via parenteral administration (e.g. intravenous, intramuscular, subcutaneous administration) or local administration (e.g. clamping, catheterization, stenting), to facilitate delivery.
  • parenteral administration e.g. intravenous, intramuscular, subcutaneous administration
  • local administration e.g. clamping, catheterization, stenting
  • compositions described herein can be contacted with the skin or skin cells (e.g. dermis cells and/or follicular cells) of the patient via parenteral administration (e.g. intravenous, intramuscular, subcutaneous administration) or local administration (e.g.
  • a composition of the invention is contacted with the eye or ocular cells (e.g. macula, fovea, cornea, retina) of the patient via parenteral administration (e.g. intravenous, intramuscular, subcutaneous administration) or local administration (e.g. direct injection, intraocular injection, periocular injection, subretinal, iontophoresis, use of eyedrops, implants), to facilitate delivery.
  • parenteral administration e.g. intravenous, intramuscular, subcutaneous administration
  • local administration e.g. direct injection, intraocular injection, periocular injection, subretinal, iontophoresis, use of eyedrops, implants
  • ear or cells of the ear e.g.
  • compositions provided herein can be contacted with the ear or cells of the ear (e.g. cells of the inner ear, middle ear and/or outer ear) of the patient as is generally known in the art, such as via parenteral administration (e.g. intravenous, intramuscular, subcutaneous administration) or local administration (e.g. direct injection), to facilitate delivery.
  • parenteral administration e.g. intravenous, intramuscular, subcutaneous administration
  • local administration e.g. direct injection
  • a payload e.g. RNA encoding an immunogen
  • antigen-presenting cells including professional antigen presenting cells
  • compositions provided herein can be delivered intramuscularly, after which immune cells can infiltrate the delivery site and process delivered RNA and/or process encoded antigen produced by non- immune cells, such as muscle cells.
  • immune cells can include macrophages (e.g. bone marrow derived macrophages), dendritic cells (e.g. bone marrow derived plasmacytoid dendritic cells and/or bone marrow derived myeloid dendritic cells), monocytes (e.g. human peripheral blood monocytes), etc. (for example, see WO2012/006372 by Geall, Andy et al.).
  • macrophages e.g. bone marrow derived macrophages
  • dendritic cells e.g. bone marrow derived plasmacytoid dendritic cells and/or bone marrow derived myeloid dendritic cells
  • monocytes e.g. human peripheral blood monocytes
  • compositions provided herein can be prepared as an injectable, a pulmonary or nasal aerosol, or in a delivery device (e.g. syringe, nebulizer, sprayer, inhaler, dermal patch, etc.).
  • This delivery device can be used to administer a pharmaceutical composition to a subject, e.g. to a human, for immunization.
  • the embodiments provided herein include delivering one or more RNAs that encode(s) an immunogen.
  • the immunogen can elicit an immune response which recognizes the immunogen, to provide immunity against a pathogen, or against an allergen, or against a tumor antigen.
  • nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications or as commonly accomplished in the art or as described herein.
  • the nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.
  • Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
  • Preparation of the Compounds of Formula I [0299]
  • the compounds disclosed herein may be synthesized by methods described below, or by modification of these methods. Ways of modifying the methodology include, among others, temperature, solvent, reagents etc., known to those skilled in the art.
  • the compounds of the present technology contain one or more chiral centers, such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or d(l) stereoisomers, or as stereoisomer-enriched mixtures. All such stereoisomers (and enriched mixtures) are included within the scope of the present technology, unless otherwise indicated. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents and the like.
  • the starting materials for the following reactions are generally known compounds or can be prepared by known procedures or obvious modifications thereof.
  • many of the starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wisconsin, USA), Bachem (Torrance, California , USA), Emka-Chemce or Sigma (St. Louis, Missouri, USA).
  • oleoyl chloride (2.8 g, 9.3 mmol, 8.7 eq.) was added and allowed to stir at room temperature for 10 minutes after which the solution was heated and stirred for 16 h at 60 °C.
  • the reaction mixture was then concentrated under reduced pressure to produce a yellow oil.
  • the resulting oil was then diluted with DCM (100 mL) and washed with water (3 x 50 mL).
  • the combined aqueous layers were then back extracted with DCM (2 x 50 mL), and the combined organic layers were dried over Na2SO4, filtered, and concentrated to produce a yellow oil.
  • Example 4 Lipid Nanoparticle (LNP) formulations were screened and assessed by invivo functional testing using the RNA payload of the complex. Performance and transfection efficiency analyses included payload delivery, biodistribution, and expression of the payload- encoded protein. Compositions including Compound 1-9 and helper lipids were made and complexed with mRNA. As shown in Table 2, the formulations examined varied in molar ratios of Compound 1 and helper lipids. Table 2: Exemplary LNP formulations [0335] All the LNP formulations contained the lipid compound, DOPE, Cholesterol, and DMG-PEG. Some formulations included the peptide SEQ ID NO: 47.
  • lipids were weighed and solubilized in ethanol at the desired molar ratio.
  • This lipid mix and firefly luciferase (fLuc) mRNA were complexed into LNPs using a microfluidic device.
  • the LNPs were dialyzed in phosphate buffer (LNP1 - LNP5, LNP13 – LNP60) or TRIS buffer (LNP6 - LNP10), and particle size and homogeneity were measured using dynamic light scattering.
  • the LNPs were injected in mice the next day.
  • Female BALB/c mice aged 6-10 weeks old were purchased from the Jackson Laboratory and were acclimatized for 7 days before the study.
  • mice were injected with LNPs equivalent to 10 ⁇ g fLuc mRNA using intravenous tail vein injection in a total volume of 200 ⁇ l.
  • mice were anesthetized with isofluorane anesthesia and imaged 10 min after intraperitoneal injection of 100 ⁇ L Rediject D-Luciferin (Perkin Elmer).
  • Bioluminescence imaging was quantified invivo (whole body) and exvivo (organ) using an IVIS Lumina III imaging system (Perkin Elmer) and analyzed using Living Image software.
  • All the LNP formulations with Compound 1 had particle size >100nm and polydispersity index ⁇ 0.3 ( Figure 1).
  • LNP formulations with Compound 1 resulted in mRNA delivery and luciferase expression in the liver ( Figure 2) and in the spleen ( Figure 3) of the injected mice.
  • LNPs with Compound 1 show higher liver expression compared to spleen indicating that LNPs with Compound 1 are efficient mRNA delivery systems for liver delivery.
  • Example 5 LNP formulations were screened and assessed by in vivo functional testing using the RNA payload of the complex. Performance and transfection efficiency analyses included payload delivery, biodistribution, and expression of the payload-encoded protein.
  • Compositions including Compound 1-9 and helper lipids were made and complexed with mRNA.
  • Table 3 Exemplary LNP formulations [0339] All the LNP formulations contained the lipid compound, either DOPE or DSPC or both, Cholesterol, and either DMG-PEG or C16-PEG. Some formulations included the peptide SEQ ID NO: 47. All the lipids were weighed and solubilized in ethanol at the desired molar ratio. This lipid mix and firefly luciferase (fLuc) mRNA were complexed into LNPs using a microfluidic device. The LNPs were dialyzed in phosphate buffer and particle size and homogeneity were measured using dynamic light scattering.
  • fLuc firefly luciferase
  • mice Female BALB/c mice aged 6-10 weeks old were purchased from the Jackson Laboratory and were acclimatized for 7 days before the study. Mice were injected with LNPs equivalent to 10 ⁇ g fLuc mRNA using intravenous tail vein injection in a total volume of 200 ⁇ l. At 4 h post-injection, mice were anesthetized with isofluorane anesthesia and imaged 10 min after intraperitoneal injection of 100 ⁇ L Rediject D-Luciferin (Perkin Elmer). Bioluminescence imaging was quantified in vivo (whole body) and ex vivo (organ) using an IVIS Lumina III imaging system (Perkin Elmer) and analyzed using Living Image software.
  • All the LNP formulations with Compound 1-9 had particle size >400nm and polydispersity index ⁇ 0.3 ( Figure 9, 10, 13, 18, 19).
  • Intravenous administration of the LNP formulations with Compound 1-9 resulted in mRNA delivery and luciferase expression in the liver ( Figure 4, 11, 14) and in the spleen ( Figure 5, 12, 15, 20) and in the lung ( Figure 21) of the injected mice.
  • LNPs with Compound 1-4 show higher liver expression compared to spleen indicating that LNPs with Compound 1-4 are efficient mRNA delivery systems for liver delivery.
  • LNPs with Compound 5-9 show higher spleen expression compared to liver indicating that LNPs with Compound 5-9 are efficient mRNA delivery systems for liver delivery.
  • Example 6 Human Primary T Cell Transfection Protocol [0342] Compound 1 was dissolved in ethanol to obtain a final concentration at 25 mg/mL. Formulation was prepared by mixing Compound 1 with DOPE, 25 mg/mL, at molar ratio 1:1 and 1:2. LNP was prepared by adding 4 mL formulated lipids to 90 mL diluted 5-methoxyuridine (5moU) modified enhanced green fluorescent protein (eGFP) mRNA, 1 mg mRNA in 100 mM sodium acetate pH ⁇ 5.2 buffer, and vortexed three times. After 10 minutes incubation at room temperature, appropriate amount of mRNA was added to activated human primary T cells. Transfection efficiency was evaluated by flow cytometry two days post- transfection.
  • 5moU 5-methoxyuridine
  • eGFP enhanced green fluorescent protein
  • EP Electroporation as control/reference, 100 ng 5moU modified eGFP mRNA mixed with 2 x 10 5 T cells in 10 ⁇ L, electroporation conditions, 1400V, 20ms and 1 pulse.
  • Example 7 Compound 8 Formulation and Screening: [0344] Compound 8 was dissolved in chloroform to make a stock of 25 mg/mL. Similarly, DOPE and cholesterol as co-lipids were dissolved in chloroform at 25 mg/mL. Cationic lipid, Compound 8 with DOPE or Cholesterol were mixed at different molar ratios (2:1, 1:1, 1:2, 1:4, and 1:8) in a glass vial. Chloroform from these vials was removed and the lipids dried using a rotary evaporator. The vials were left overnight in a vacuum desiccator to completely remove the chloroform.
  • Liposome preparation [0345]
  • Lipids in chloroform were added to a vial along with the desired volume of water needed for a final lipid concentration of 2 mg/mL followed by removal of chloroform using a rotary evaporator.
  • VPC 1.0 cells Supension HEK 293 cells
  • VPC 1.0 cells Supension HEK 293 cells
  • ExpiFectamine 293 Expi293 expression media by splitting every 3-4 days to maintain a cell density less than 6 x 10 6 cells/mL.
  • the cells are split to a density of 0.3 x 10 6 to 0.5 x 10 6 cells/mL for regular maintenance.
  • a day before transfection the cells are split at 3 x 10 6 cells/mL and the day of transfection, cells are re-diluted to 3 x 10 6 cells/mL and aliquoted into a 96-deep well plate.
  • About 800 ⁇ L of cells were aliquoted into 2-mL 96- deep well plates (inner 60 wells only) and incubated on a shaker set at 900 rpm until ready for transfection.
  • Transfection complexes were made in 100 ⁇ L of Opti-MEM containing DNA (0.8 ⁇ g/mL) aliquoted in a 96-well plate.
  • Transfection reagent was added to the DNA containing wells and mixed by pipetting up and down followed by incubation for 20 min for the lipoplex/lipid nanoparticle to form. After 20 min, 100 ⁇ L of the DNA-transfection reagent complex was added to the cells and transfection efficiency and toxicity determined at 24 and 48h post-transfection. Transfection efficiency and toxicity [0347] Plate reader-based assays were used at 24 and 48h post transfection to determine the efficacy in comparison with existing catalog products like the ExpiFectamine 293 (Expi293) reagent (2.5 ⁇ L). GFP fluorescence of the cells was also measured using a plate reader. See Figure 16.

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Abstract

L'invention concerne des lipides ionisables qui sont utiles pour administrer des macromolécules, telles que des acides nucléiques, dans des cellules eucaryotes. Les lipides peuvent être utilisés seuls, en combinaison avec d'autres lipides et/ou en combinaison avec d'autres réactifs améliorant la transfection pour préparer des complexes de transfection.
PCT/US2024/041143 2023-08-07 2024-08-06 Lipides pour l'administration d'acides nucléiques à des cellules eucaryotes Pending WO2025034764A1 (fr)

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WO2025209467A1 (fr) * 2024-04-01 2025-10-09 Longuide Biopharma Corporation Lipides et formulations de nanoparticules lipidiques

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